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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)-β. In parallel, human MG thymuses also display an overexpression of TLR3, PKR, and IFN-β. 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., (Copyright © 2012 American Neurological Association.)

Efficacy of arbidol and ridostin in cupping postvaccinal complications due to variolation was studied by the clinico-virological, hematological and biochemical indices and it was shown that arbidol was efficient in cupping development of dermal complications, lowered the severity of the postvaccinal reaction and stimulated the cellular and humoral immune response. Ridostin, a high molecular interferon inductor, was highly efficient in cupping all the forms of the postvaccinal complications, including the neurological and cutaneous ones.

Natural killer (NK) cells are key players of innate immunity. Besides their major cytotoxic function, NK cells can also produce inflammatory cytokines such as interferon (IFN)-gamma. In this way, NK cells can shape adaptive immune responses through activation of dendritic cells (DC), thereby promoting the bidirectional cross-talk between NK cells and DC. Including this helper function of NK cells in cancer vaccination might be important for the induction of effective T cell responses. Here, we explored the capacity of purified human NK cells to produce IFN-gamma upon two-signal stimulation using different types of acute myeloid leukemia (AML) cells and type I IFN. Based on our previous findings that AML cells produce IFN-alpha upon electroporation with the synthetic double-stranded (ds)RNA polyriboinosinic polyribocytidylic acid (poly(I:C)), we hypothesized that dsRNA-loaded tumor cells provide both signals to elicit an NK cell-driven IFN-gamma production. Our results show that in vitro, NK cells become strong IFN-gamma-secreting cells upon stimulation with specific AML cells and IFN-alpha, with a variable responsiveness against different AML cell lines. Importantly, loading of AML cells with poly(I:C) is an elegant method to provide NK cells with both signals, a feature that could have important clinical implications because it obviates the side effects of systemic cytokine administration. Moreover, in addition to our previous findings that DC become activated upon phagocytosis of poly(I:C)-electroporated AML cells, these data strongly encourage future research on the potential of dsRNA-transfected AML cells and their effect to favor NK-DC cross-talk for the design of leukemia vaccines.

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.

Background: Thymic stromal lymphopoietin (TSLP) plays a key role in allergic diseases, such as atopic dermatitis (AD) and asthma. TSLP is highly expressed by keratinocytes in skin lesions of patients with AD, but environmental triggers for its release from keratinocytes with endogenous factors are not well understood. Patients with AD, in whom allergic sensitization is already established, are susceptible to viral dissemination., Objectives: We investigated TSLP's release from primary human keratinocytes stimulated with a Toll-like receptor (TLR) 3 ligand, polyinosinic-polycytidylic acid, which mimics viral double-stranded RNA (dsRNA), and its modulation by cytokines., Methods: Primary human keratinocytes were stimulated with TLR ligands, cytokines, or both. TSLP released into culture supernatants was measured by means of ELISA., Results: Stimulation of keratinocytes with dsRNA induced release of TSLP and upregulated gene expression of TSLP and other cytokines and chemokines. The release of TSLP was enhanced by the addition of IL-4, IL-13, and/or TNF-alpha. With or without the T(H)2/TNF cytokines, the dsRNA-induced release of TSLP was upregulated by IFN-alpha and IFN-beta and suppressed by IFN-gamma, TGF-beta, or IL-17., Conclusions: The effect of the TLR3 ligand on keratinocytes suggests contribution of viral dsRNA to skin inflammations under the influence of a cytokine milieu. The results imply that viral dsRNA and a T(H)2 cytokine milieu might promote T(H)2-type inflammation through an induction of TSLP expression, suggesting that a vicious cycle exists between AD with T(H)2-type inflammation and viral infections and a possible blockade of this cycle by other cytokine milieus provided by cells, such as T(H)1, regulatory T, and T(H)17 cells.

TLR3 and cytoplasmic RIG-I-like receptor (RLR) recognize virus-derived dsRNA and induce type I IFN production in a distinct manner. Human TLR3 localizes to the endosomal compartments in myeloid dendritic cells (mDCs), while it localizes to both the cell surface and interior in fibroblasts and epithelial cells. TLR3 signaling arises in the intracellular compartment in both cell types and requires endosomal maturation. The mechanisms by which extracellular dsRNA is delivered to the TLR3-containing organelle remain largely unknown. Among various synthetic dsRNAs, poly(I:C) is preferentially internalized and activates TLR3 in mDCs. In vitro transcribed dsRNAs hardly induce IFN-beta production in mDCs. In this study, we demonstrate that the clathrin-dependent endocytic pathway mediates cell entry of poly(I:C) to induce IFN-beta gene transcription. Furthermore, poly(I:C)-induced IFN-beta production is inhibited by pretreatment of cells with B- and C-type oligodeoxynucleotides (ODNs) but not with TLR7/8 ligands. The binding and internalization of B-type ODNs by mDCs was reduced in the presence of poly(I:C), suggesting that poly(I:C) shares the uptake receptor with B- and C-type ODNs. Hence, foreign dsRNA is recognized by differently categorized receptors, cytoplasmic RIG-I-like receptor, membrane-bound TLR3 and cell-surface RNA capture. The endocytic pathway is critical for dsRNA-induced TLR3-mediated cell activation.

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.

Physico-chemical and biological properties of an interferon inducer, a natural double-stranded RNA (dsRNA) obtained from RNA-genome bacteriophage-infected E. coli are described. Three independent methods i.e. sedimentation analysis, gel chromatography, and direct measurement of electronograms, were used to determine an average length of dsRNA molecules which was found to correspond to about 432 nucleotide pairs. dsRNA melting temperature in 0.15 M NaCl is 103 degrees C, and the resistance to RNase A is above 85%. Additional purification of the preparation by the methods eliminating bacterial endotoxin and other possible admixtures does not decrease acute toxicity of dsRNA. The influence of dsRNA on the cytolytic activity of natural killers (NK) in normal subjects and patients with viral diseases of the liver was demonstrated. dsRNA enhances the activity of NK in normal subjects as well as in patients with acute virus hepatitis B in the abatement stage of the disease and in patients with chronic persisting hepatitis. At the peak of acute virus hepatitis B NK stimulation was detected in only 40 +/- 16% of the patients. In patients with chronic active liver diseases dsRNA enhanced NK activity in 50 +/- 22% of cases. A similar effect on NK cell activity is exerted by human leukocyte interferon.

Experimental mice treated with an interferon inducer (double-stranded RNA) and vaccine, vaccine alone, and gamma-globulin plus vaccine were used to study the time course of development of immunity to rabies by intramuscular and intracerebral challenge with street rabies virus. Combined use of the interferon inducer and specific vaccine was shown to produce an earlier and more intensive immunity than vaccine alone or gamma-globulin and vaccine, and also a more marked humoral immune response. It is concluded that the new method for prevention of hydrophobia is effective and promising, particularly in cases of severe bites with a short incubation period.

Influence of dsRNA isolated from killer yeast of S. cerevisiae on humoral and cellular immune responses in mice CBA/CaY and C57Bl/6Y with opposing reaction to the antigen was studied. It was shown that after administration of the yeast dsRNA preparation to the animals simultaneously with the antigen there was an increase in the number of the antibody forming cells in the spleen and the titer of hemolytic antibodies in blood serum of the animals with high and low reactions to the antigen. After sensitization with different doses of sheep red blood cells (10(7) and 10(8)) the preparation had immunomodulating action on development of DTH in mice CBA/CaY. The effect of the dsRNA preparation on the immunity system depended on the preparation dose, antigen loading and animal genotype and was the most marked in mice CBA/CaY with interferon levels in blood serum 2-3 times higher than those in mice C57Bl/6Y.