Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection.

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-ß promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

Safety and antibody immune response of CHP-NY-ESO-1 vaccine combined with poly-ICLC in advanced or recurrent esophageal cancer patients.

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8+ and CD4+ T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (µg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials.

Impact of rearing temperature on the innate antiviral immune response of growth hormone transgenic female triploid Atlantic salmon (Salmo salar).

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.

Clec9A+ Dendritic Cells Are Not Essential for Antitumor CD8+ T Cell Responses Induced by Poly I:C Immunotherapy.

In the steady state, tumors harbor several populations of dendritic cells (DCs) and myeloid cells that are key regulators of the intratumoral immune environment. Among these cells, migratory CD103+ cross-presenting DCs are thought to be critical for tumor-specific CTL responses and tumor resistance. However, it is unclear whether this prominent role also extends to immunotherapy. We used a murine orthotopic mammary tumor model, as well as Clec9A-diphtheria toxin receptor mice that can be depleted of the specialized cross-presenting CD8α+ and CD103+ DC1 subsets, to investigate the role of these DCs in immunotherapy. Treatment with monosodium urate crystals and mycobacteria at the tumor site delayed tumor growth and required DC1s for efficacy. In contrast, treatment with poly I:C was equally effective regardless of DC1 depletion. Neither treatment affected myeloid-derived suppressor cell numbers in the spleen or tumor. Similar experiments using subcutaneous B16 melanoma tumors in BATF3-knockout mice confirmed that CD103+ DCs were not necessary for successful poly I:C immunotherapy. Nevertheless, adaptive immune responses were essential for the response to poly I:C, because mice depleted of CD8+ T cells or all DC subsets were unable to delay tumor growth. In vivo experiments showed that DC1 and DC2 subsets were able to take up tumor Ags, with DC2s making up the larger proportion of lymph node DCs carrying tumor material. Both DC subsets were able to cross-present OVA to OT-I T cells in vitro. Thus, immunotherapy with poly I:C enables multiple DC subsets to cross-present tumor Ag for effective antitumor immune responses.

House dust mite impairs antiviral response in asthma exacerbation models through its effects on TLR3.

BACKGROUND: Impaired antiviral interferon expression may be involved in asthma exacerbations commonly caused by rhinovirus infections. Allergy is a known risk factor for viral-induced asthma exacerbation, but little is known whether allergens may affect interferon responses. OBJECTIVE: Our hypothesis is that house dust mite (HDM) impairs viral stimulus-induced antiviral signalling. METHODS: Experimental asthma exacerbations were produced in vitro in human bronchial epithelial cells (HBECs) and in mice using sequential challenges with HDM and a viral infection mimic, Poly(I:C). We examined rhinovirus pattern recognition receptors (PRRs) signalling pathways and potential mechanisms of impaired interferon response. RESULTS: HBECs and mice exposed to HDM prior to Poly(I:C) exhibited a reduced antiviral response compared to Poly(I:C) alone, including reduced IFN-ß, IFN-λ, TLR3, RIG-I, MDA5, IRF-3 and IRF-7. Heat inactivation of HDM partially restored the TLR3-induced interferon response in vitro and in vivo. Our HBEC-data further showed that HDM directly affects TLR3 signalling by targeting the receptor glycosylation level. CONCLUSIONS: Direct effects of allergens such as HDM on PRRs can present as potential mechanism for defective antiviral airway responses. Accordingly, therapeutic measures targeting inhibitory effects of allergens on antiviral PRRs may find use as a strategy to boost antiviral response and ameliorate exacerbations in asthmatic patients.

NY-ESO-1 Vaccination in Combination with Decitabine Induces Antigen-Specific T-lymphocyte Responses in Patients with Myelodysplastic Syndrome.

Purpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1-specific MDS-directed cytotoxic T-cell immune response.Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine.Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1-specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1-specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses.Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019-29. ©2017 AACRSee related commentary by Fuchs, p. 991.

PolyI:C and CpG Synergize with Anti-ErbB2 mAb for Treatment of Breast Tumors Resistant to Immune Checkpoint Inhibitors.

Innate and adaptive immune cells play an important role in the therapeutic activity of anti-ErbB2 mAbs, such as trastuzumab. In the clinic, breast tumors poorly infiltrated with immune cells are more resistant to trastuzumab, and patients have a worse prognosis. Because type I and II IFNs are critical to the immune-mediated activity of anti-ErbB2 mAb, we investigated the effect of combining polyI:C and CpG with trastuzumab-like therapy in immunocompetent mouse models of ErbB2+ breast cancer. We demonstrated that in situ delivery of polyI:C and CpG combined to systemic anti-ErbB2 mAb triggered a potent inflammatory response in breast tumors able to induce long-lasting CD8+ T cell-dependent antitumor immunity. Remarkably, polyI:C and CpG was superior to combined PD-1/CTLA-4 blockade in sensitizing tumors to anti-ErbB2 mAb therapy. Local injection of CpG and polyI:C in a primary tumor significantly enhanced the activity of systemic anti-ErbB2 mAb against a distant untreated tumor. Type I and II IFNs, as well as natural killer cells and CD8+ T cells, were indispensible to the synergistic activity of the combination treatment. Because synthetic RNA analogues and CpG oligodeoxynucleotides have been safely used in clinical trials, our study supports combination treatments with anti-ErbB2 mAbs. Cancer Res; 77(2); 312-9. ©2016 AACR.

Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity.

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.

Immune responses and outcome after vaccination with glioma-associated antigen peptides and poly-ICLC in a pilot study for pediatric recurrent low-grade gliomas.

BACKGROUND: Low-grade gliomas (LGGs) are the most common brain tumors of childhood. Although surgical resection is curative for well-circumscribed superficial lesions, tumors that are infiltrative or arise from deep structures are therapeutically challenging, and new treatment approaches are needed. Having identified a panel of glioma-associated antigens (GAAs) overexpressed in these tumors, we initiated a pilot trial of vaccinations with peptides for GAA epitopes in human leukocyte antigen-A2+ children with recurrent LGG that had progressed after at least 2 prior regimens. METHODS: Peptide epitopes for 3 GAAs (EphA2, IL-13Rα2, and survivin) were emulsified in Montanide-ISA-51 and administered subcutaneously adjacent to intramuscular injections of polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-lymphocyte responses against GAA epitopes. Treatment response was evaluated clinically and by MRI. RESULTS: Fourteen children were enrolled. Other than grade 3 urticaria in one child, no regimen-limiting toxicity was encountered. Vaccination induced immunoreactivity to at least one vaccine-targeted GAA in all 12 evaluable patients: to IL-13Rα2 in 3, EphA2 in 11, and survivin in 3. One child with a metastatic LGG had asymptomatic pseudoprogression noted 6 weeks after starting vaccination, followed by dramatic disease regression with >75% shrinkage of primary tumor and regression of metastatic disease, persisting >57 months. Three other children had sustained partial responses, lasting >10, >31, and >45 months, and one had a transient response. CONCLUSIONS: GAA peptide vaccination in children with recurrent LGGs is generally well tolerated, with preliminary evidence of immunological and clinical activity.

Interferon response of the cystic fibrosis bronchial epithelium to major and minor group rhinovirus infection.

Rhinoviruses (RVs) are associated with exacerbations of cystic fibrosis (CF), asthma and COPD. There is growing evidence suggesting the involvement of the interferon (IFN) pathway in RV-associated morbidity in asthma and COPD. The mechanisms of RV-triggered exacerbations in CF are poorly understood. In a pilot study, we assessed the antiviral response of CF and healthy bronchial epithelial cells (BECs) to RV infection, we measured the levels of IFNs, pattern recognition receptors (PRRs) and IFN-stimulated genes (ISGs) upon infection with major and minor group RVs and poly(IC) stimulation. Major group RV infection of CF BECs resulted in a trend towards a diminished IFN response at the level of IFNs, PRRs and ISGs in comparison to healthy BECs. Contrary to major group RV, the IFN pathway induction upon minor group RV infection was significantly increased at the level of IFNs and PRRs in CF BECs compared to healthy BECs.

An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction.

Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation.

The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freund's adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines.

Mislocalization of the interferon inducible protein IFI16 by environmental insults: implications in autoimmunity.

The nuclear DNA sensor IFI16, a member of PYHIN family of proteins, was previously studied for its role in cell cycle regulation, tumor suppression, apoptosis and DNA damage signaling. Autoantibodies against IFI16 are prevalent in the sera of patients with systemic autoimmunity, thus depicting physiological significance as an autoantigen. At present, the nuclear IFI16 protein has been thoroughly investigated for its role as an innate immune sensor involved in inflammasome signaling and viral restriction. While the sub-cellular localization of IFI16 during such events has been known, very little knowledge about its presence and significance in the extracellular space is available. Recently our group has discovered the presence of circulating IFI16 in the sera from systemic autoimmune patients indicating that in this setting it may be mislocalized form its nuclear site and secreted in the extracellular milieu. In this review, we will discuss the leakage of endogenous IFI16 that has been experimentally proved using in vivo and in vitro models. Also we will comment on the significance of mislocalized inflammasome components in the extracellular space and how it can be responsible for chronic inflammation.

Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication.

UNLABELLED: Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-ß, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. IMPORTANCE: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

Antigen-specific immune responses and clinical outcome after vaccination with glioma-associated antigen peptides and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in children with newly diagnosed malignant brainstem and nonbrainstem gliomas.

PURPOSE: Diffuse brainstem gliomas (BSGs) and other high-grade gliomas (HGGs) of childhood carry a dismal prognosis despite current treatments, and new therapies are needed. Having identified a series of glioma-associated antigens (GAAs) commonly overexpressed in pediatric gliomas, we initiated a pilot study of subcutaneous vaccinations with GAA epitope peptides in HLA-A2-positive children with newly diagnosed BSG and HGG. PATIENTS AND METHODS: GAAs were EphA2, interleukin-13 receptor alpha 2 (IL-13Rα2), and survivin, and their peptide epitopes were emulsified in Montanide-ISA-51 and given every 3 weeks with intramuscular polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for eight courses, followed by booster vaccinations every 6 weeks. Primary end points were safety and T-cell responses against vaccine-targeted GAA epitopes. Treatment response was evaluated clinically and by magnetic resonance imaging. RESULTS: Twenty-six children were enrolled, 14 with newly diagnosed BSG treated with irradiation and 12 with newly diagnosed BSG or HGG treated with irradiation and concurrent chemotherapy. No dose-limiting non-CNS toxicity was encountered. Five children had symptomatic pseudoprogression, which responded to dexamethasone and was associated with prolonged survival. Only two patients had progressive disease during the first two vaccine courses; 19 had stable disease, two had partial responses, one had a minor response, and two had prolonged disease-free status after surgery. Enzyme-linked immunosorbent spot analysis in 21 children showed positive anti-GAA immune responses in 13: to IL-13Rα2 in 10, EphA2 in 11, and survivin in three. CONCLUSION: GAA peptide vaccination in children with gliomas is generally well tolerated and has preliminary evidence of immunologic and clinical responses. Careful monitoring and management of pseudoprogression is essential.

Implication of double-stranded RNA signaling in the etiology of autoimmune myasthenia gravis.

OBJECTIVE: Myasthenia gravis (MG) is an autoimmune disease mediated mainly by anti-acetylcholine receptor (AChR) antibodies. The thymus plays a primary role in MG pathogenesis. As we recently showed an inflammatory and antiviral signature in MG thymuses, we investigated whether pathogen-sensing molecules could contribute to an anti-AChR response. METHODS: We studied the effects of toll-like receptor agonists on the expression of α-AChR and various tissue-specific antigens (TSAs) in human thymic epithelial cell (TEC) cultures. As polyinosinic-polycytidylic acid (poly[I:C]), which mimics double-stranded RNA (dsRNA), stimulated specifically α-AChR expression, the signaling pathways involved were investigated. In parallel, we analyzed the expression of dsRNA-signaling components in the thymus of MG patients, and the relevance of our data was investigated in vivo in poly(I:C)-injected mice. RESULTS: We demonstrate that dsRNA signaling induced by poly(I:C) specifically triggers the overexpression of α-AChR in TECs and not of other TSAs. A poly(I:C) effect was also observed on MG TECs. This induction is mediated through toll-like receptor 3 (TLR3) and protein kinase R (PKR), and by the release of interferon (IFN)-ß. In parallel, human MG thymuses also display an overexpression of TLR3, PKR, and IFN-ß. In addition, poly(I:C) injections specifically increase thymic expression of α-AChR in wild-type mice, but not in IFN-I receptor knockout mice. These injections also lead to an anti-AChR autoimmune response characterized by a significant production of serum anti-AChR antibodies and a specific proliferation of B cells. INTERPRETATION: Because anti-AChR antibodies are highly specific for MG and are pathogenic, dsRNA-signaling activation could contribute to the etiology of MG.

The broad-spectrum antiviral functions of IFIT and IFITM proteins.

Over the past few years, several groups have identified new genes that are transcriptionally induced downstream of type I interferon (IFN) signalling and that inhibit infection by individual or multiple families of viruses. Among these IFN-stimulated genes with antiviral activity are two genetically and functionally distinct families--the IFN-induced protein with tetratricopeptide repeats (IFIT) family and the IFN-induced transmembrane protein (IFITM) family. This Review focuses on recent advances in identifying the unique mechanisms of action of IFIT and IFITM proteins, which explain their broad-spectrum activity against the replication, spread and pathogenesis of a range of human viruses.

[The role of interferons in the socially important human viral diseases].

The role of interferon in influenza and herpes infections, general patterns of the interferon system in these diseases, the identification of interferon deficiency, the possibility of their correction with the immune active drugs, including interferon inducers combining antiviral immunomodulatory interferon effects with etiopathogenic corrective mode of action, are discussed. Clinical values of faster recovery confirm the suitability of their application in the immunocompromised patients.

TNFα enhances TLR3-dependent effects on MMP-9 expression in human mesangial cells.

The role of MMPs (matrix metalloproteinases) in kidney diseases has been widely accepted, where they can regulate inflammatory response because of their effects on both recruitment and survival of inflammatory cells. TNFα (tumour necrosis factor α) has also been implicated in the pathogenesis of inflammatory kidney diseases, including forms of glomerulonephritis associated with viral diseases. Previously, we established the functional linkage between viral receptors of the innate immune system, the TLRs (Toll-like receptors) and control of MMP activity in human MC (mesangial cells). Expression levels of MMP-2, MMP-7, MMP-9, TIMP-1 (tissue inhibitor of metalloproteinase 1) and TIMP-2 in human MC in culture were analysed by RT-PCR (reverse transcription-PCR). TNFα significantly enhanced the TLR3-dependent induction of MMP-9 in human MC. Expression levels of MMP-2, TIMP-1 and TIMP-2 were not significantly affected by the activation of TLR3 or TNFα stimulation. No significant MMP-7 expression was found. We conclude that the role of MMP-9 in chemotaxis, activation and proliferation of inflammatory cells is amplified by TNFα originating from infiltrating cells, especially monocytes, producing a regulatory loop that potentially leads to a self-propagating inflammation.

Daily intake of heat-killed Lactobacillus plantarum L-137 enhances type I interferon production in healthy humans and pigs.

We have previously reported that oral administration of heat-killed Lactobacillus plantarum L-137 (HK L-137) stimulates innate immunity for production of type I interferon (IFN) which subsequently augments host defense against influenza A virus infection in mice. We here examined the effect of HK L-137 intake on type I IFN in humans. Sixteen subjects were randomly assigned to receive a tablet containing 10 mg of HK L-137 or a matching tablet for 8 weeks and the serum levels of type I IFN were examined before and after the first or second dose of the trivalent inactivated influenza vaccine. There were no differences in the seroresponse rate, the seroprotection rate and the geometric mean Ab titers after either the first or second dose of vaccine between the HK L-137 group and the control group. On the other hand, the levels of IFN-ß were significantly higher in the HK L-137 group than in the control group before vaccination although the vaccination conferred little additional induction of IFN-ß. We further examined IFN-ß gene expression in the whole blood cells of pigs fed on a diet containing HK L-137 and found that the IFN-ß mRNA levels were significantly higher in the HK L-137 group than in the control group. The finding that daily intake of HK L-137 enhances type I IFN production and host defense against influenza A virus infection in mice may be applied to at least two additional species.