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Multicellular organisms evolved efficient host-defense mechanisms to sense viruses and to block their replication and spread. Invertebrates and plants mainly rely on RNA interference (RNAi) for antiviral defense. In mammals, the initiation of antiviral defense mechanisms is largely based on the detection of viral nucleic acids by innate receptors: retinoic acid–inducible gene I (RIG-I)–like helicases (RLHs) and Toll-like receptors (TLRs). RLHs play a pivotal role in sensing viral RNA and DNA in the cytoplasm of cells. RLHs, like Dicer of the RNAi pathway, belong to the phylogenetically conserved DExD/H-box family of helicases. Unlike TLRs, RLHs are functional in all somatic cells. Activation of RIG-I triggers antiviral responses including type I interferon (IFN), inflammasome activation and proapoptotic signaling. Here, we provide a comprehensive overview of the current literature on the ligand structures detected by RIG-I, and conclude with the molecular definition of the RIG-I ligand: short double-stranded blunt-end 5′-triphosphate RNA. The recent information on the RIG-I ligand now allows the design of short double-stranded RNA (dsRNA) oligonucleotides that are ideally suited alone or in combination with small-interfering RNA (siRNA) for the treatment of viral infection and cancer. [ABSTRACT FROM AUTHOR]

Abstract: Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for humoral immune responses but data on cellular immune responses in primates are scarce. Rhesus macaque blood contained similar numbers of plasmacytoid dendritic cells and B cells, the key sensors of CpG ODN, as human blood, and these cells were activated by CpG-A and CpG-B in vitro. In vivo, both ODNs induced equal plasma levels of interferon-inducible protein 10 and similarly enhanced antibody responses following i.m. injections of the ODNs, protein antigen, and aluminium hydroxide into rhesus macaques, whereas antigen-specific CD4+ T cell responses were only slightly increased by CpG ODN. [Copyright &y& Elsevier]

The authors discuss the role of macrophage colony-stimulating factor 1 (CSF1) Fc conjugate in inducing tissue regeneration in the liver. They explore the paper by Gow and colleagues in the September 1, 2014 issue of the journal "Molecular Therapy," which describes the development of Fc conjugate of CSF1 for tissue regeneration. They say that the study by Gow and colleagues suggests that CSF1 can also be beneficial in the treatment of heart diseases, spinal cord injury, and fertility issues.

With the recent success of mRNA vaccines and the approval of several RNA oligonucleotide therapeutics, RNA holds great promise for future drug development. The rise of RNA therapeutics has been enabled by the tremendous progress in our understanding of the sophisticated cellular mechanisms that disarm potentially dangerous exogenous RNA and safeguard RNA homeostasis. Exogenous RNA, such as an mRNA vaccine when injected, faces an intricate system of immune-sensing receptors, restriction factors, and nucleases referred to as nucleic acid immunity. A careful analysis of the functional interaction between the innate response to mRNA, the efficacy to translate the encoded protein antigen, and the quality of the resulting adaptive immunity bears great potential for further improvement of mRNA vaccines and RNA therapeutics for various clinical applications. In this review, we summarize the most recent efforts to advance mRNA vaccines by capitalizing on recent insight in innate RNA sensing. • Overview how insight in nucleic acid immunity has prepared the ground for successful mRNA vaccines. • Summary of most recent efforts to understand mRNA vaccines at the interface to innate immunity. • Points out important hurdles and promising future directions. [ABSTRACT FROM AUTHOR]

Abstract: Malignant melanoma is a malignant neoplasm originating from the melanocyte lineage. Microphthalmia-associated transcription factor (Mitf) is crucially involved in the melanin synthesis as well as proliferation and survival of melanocyte and melanoma. We previously showed that short interfering RNA (siRNA) that is specific for the Mitf gene (Mitf-siRNA) significantly inhibited growth of B16 melanoma after electro-transfected in vivo into preestablished tumor in mice. Here we assessed efficacy of electroporation-mediated co-transfection of Mitf-siRNA and IL-12 gene in the treatment of murine melanoma. As results, the tumor growth was more strongly inhibited by intratumor co-transfection with Mitf-siRNA and IL-12-encoding plasmid DNA than by transfection with either of the molecules alone. The co-transfection induced intratumor infiltration of CD4+ and CD8+ T cells, and hampered neoangiogenesis in the tumor. The findings suggest that the RNAi/cytokine gene combination therapy by means of electroporation may become a novel and efficacious therapeutic modality to treat neoplasms including melanoma. [Copyright &y& Elsevier]

RNA interference mediated by small interfering RNAs (siRNA) has emerged as a powerful tool to target specific knockdown of gene expression in cell culture. siRNA is now the gold standard technique to study gene function, and expectations for the development of new target-specific drugs are high. In addition to the gene-silencing activity of siRNA, a number of recent studies have pointed to immunological effects of siRNAs, including the induction of proinflammatory cytokines and type I interferon. There is good evidence that gene silencing and immunostimulation are two independent functional characteristics of RNA oligonucleotides. Immunorecognition of RNA depends on certain molecular features such as length, double- versus single-strand configuration, sequence motifs, and nucleoside modifications such as triphosphate residues. RNA-sensing immunoreceptors include three members of the Toll-like receptor (TLR) family (TLR3, TLR7, TLR8) and cytosolic RNA-binding proteins like PKR and the helicases RIG-I and Mda5. Detection of RNA molecules occurs during viral infection and triggers antiviral innate defense mechanisms including the induction of type I interferons (IFN-α, IFN-β) and downregulation of gene expression. Type I interferon induction by synthetic siRNAs requires TLR7 and is sequence dependent, similar to the detection of CpG motifs in DNA by TLR9. Identification of the exact molecular mechanisms of immunorecognition of RNA will allow the development of methods to avoid immunostimulation of siRNA and the design of potent immunostimulatory RNA (isRNA) oligonucleotides, depending on the aim. Furthermore, the combination of both gene-silencing and immunostimulation in one RNA molecule may lead to novel drugs that use both functional activities of RNA as two edges of one sword for effective treatment of viral infection and cancer.Molecular Therapy (2006) 14, 463–470; doi: 10.1016/j.ymthe.2006.06.001 [ABSTRACT FROM AUTHOR]

Infections of the Coronavirus SARS-CoV-2 continue to spread around the globe, causing Coronavirus Disease (COVID)-19. Infected people are at risk of developing acute interstitial pneumonia, which can result in lethal complications, particularly in patients with pre-existing co-morbidities. Novel prophylactic and therapeutic interventions are urgently needed to limit the infection-associated health risk for the population and to contain the pandemic. Animal models are indispensable to assessing the efficacy and safety of potential new antivirals, vaccines, and other innovative therapies, such as nucleic acid agonists of innate immune sensing receptors. In this review, we provide an overview of the commonly used animal models to study SARS-CoV-2 and COVID-19, including a summary of their susceptibility to infection, the spectrum of symptoms elicited, and the potential for drug development in each model. We hope that this review will help researchers to decide on the right model organism to quickly address their specific scientific questions. [ABSTRACT FROM AUTHOR]

Abstract: Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current research, we illustrate here why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids. [Copyright &y& Elsevier]

Abstract: Enteroviral infections are associated with type I diabetes. The mechanisms by which viruses or viral products such as double-stranded RNA (dsRNA) affect pancreatic beta cell function and survival remain unclear. We have shown that extracellular dsRNA induces beta cell death via Toll-like receptor-3 (TLR3) signaling whereas cytosolic dsRNA triggers the production of type I interferons and apoptosis via a TLR3-independent process. We presently examined expression of the intracellular viral RNA sensors, the RNA helicases RIG-I and MDA5, and documented the functionality of RIG-I in pancreatic beta cells. FACS-purified rat beta cells and islet cells from wild-type or TLR3−/− mice were cultured with or without the RIG-I-specific ligand 5′-triphosphate single-stranded RNA (5′triP-ssRNA), the synthetic dsRNA polyI:C (PIC) or 5′OH-ssRNA (negative control); the RNA compounds were added in the medium or transfected in the cells using lipofectamine. RIG-I and MDA5 expression were determined by real-time RT-PCR. NF-κB and IFN-β promoter activation were studied in the presence or absence of a dominant-negative form of RIG-I (DN-RIG-I). Both extracellular (PICex) and intracellular (PICin) PIC increased expression of RIG-I and MDA5 in pancreatic beta cells. TLR3 deletion abolished PICex-induced up-regulation of the helicases in beta cells but not in dendritic cells. PICin-induced NF-κB and IFN-β promoter activation were prevented by the DN-RIG-I. The RIG-I-specific ligand 5′triP-ssRNA induced IFN-β promoter activation and beta cell apoptosis. Our results suggest that the RIG-I pathway is present and active in beta cells and could contribute to the induction of insulitis by viral RNA intermediates. [Copyright &y& Elsevier]

Short synthetic CpG oligodesoxynucleotides (CpG ODN) that activate plasmacytoid dendritic cells (PDC) and B-cells via the endosomal Toll-like receptor (TLR) 9 are the prototypical immunostimulatory nucleic acids (NAs), and their therapeutic potential is currently evaluated in numerous clinical trials. In recent years, NA recognition has emerged as a general means of virus detection by the innate immune system that involves at least seven receptors located in either the endosome or the cytosol. Endosomal TLRs are expressed selectively, and predominantly by immune cell types. By contrast, the expression of cytosolic NA receptors is ubiquitous, significantly widening the range of cell types that can be stimulated therapeutically by NAs to include even tumor cells. Here we discuss unique properties of each of these receptors, and argue that an understanding of the molecular basis of receptor–ligand interactions, and the development of chemically defined, selective ligands is required in order to fully realize the promise that NA immunetherapeutics hold. [Copyright &y& Elsevier]

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-α and IFN-β) upon viral infection. PDC employ a member of the family of tell-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-α (IFN-α) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5′ and 3′ end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-α (CpG-B) gained the potency of CpG-A to induce IFN-α. Higher ODN uptake in PDC was not responsible for the higher IFN-α-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-α in PDC. [ABSTRACT FROM AUTHOR]

Abstract: In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood–brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. This study analyzed the expression and function of Toll-like receptors (TLRs) in cultured human astrocytes. Quantitative PCR for TLRs 1 to 10 showed a basal expression of TLR3 that could be enhanced by IFN-γ, IL-1β, and IFN-β. The other TLRs were barely detectable and not inducible by the same cytokines. IFN-γ-activated astrocytes responded to TLR3 ligand poly (I:C) engagement with IL-6 production, while ligands of other TLRs, like LPS, lipoteichoic acid, peptidoglycan, flagellin, and CpG, had no effect. Poly (I:C) also triggered astrocyte production of TNF-α and the chemokines CCL2/MCP-1, CCL5/RANTES, CCL20/MIP-3α, and CXCL10/IP-10. The adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF, which are required for TLR signaling, were all expressed by astrocytes. Thus, resting and activated human astrocytes express preferentially TLR3 and, upon TLR3 engagement, produce IL-6 and chemokines active on T cells, B cells, monocytes, and dendritic cells. These data indicate that astrocytes function as sentinels for viral infections in the CNS. [Copyright &y& Elsevier]

For patients with advanced malignant melanoma, the 5 y survival rate with current treatment modalities is low. There is an urgent need for more effective therapeutic concepts. One approach with great potential is to stimulate the body's own immune defense to reject cancer cells using CpG oligonucleotides. Distinct oligonucleotides containing nonmethylated cytidine residues in cytidine-guanosine dinucleotides with particular flanking bases (CpG motifs) are capable of eliciting powerful immune stimulation by mimicking infectious disease. We evaluated the in vivo antitumoral effects of CpG oligonucleotides against human malignant melanoma xenografts in NOD/SCID mice. CpG oligonucleotides administered in single peritumoral subcutaneous injections three times per week resulted in elevated plasma levels of interleukin-12 and significant inhibition of the growth of established tumor xenografts by 60% (p<0.016) compared to the saline control. In addition to this a significant invasion of macrophages into tumor xenografts and increased numbers of Langerhans-cell-derived dendritic cells in draining lymph nodes could be observed. Our findings demonstrate the antitumor activity of oligonucleotides containing immune-stimulatory CpG motifs in a xenotransplantation model with absent B, T cells and a lack of natural killer cell function. [ABSTRACT FROM AUTHOR]

The vertebrate immune system has established TLR9 to detect microbial DNA based on unmethylated CG dinucleotides within certain sequence contexts (CpG motifs). In humans, the expression of toll-like receptor 9 (TLR9) is restricted to B cells and plasmacytoid dendritic cells (PDC). The PDC is characterized by the ability to rapidly synthesize large amounts of type I IFN (IFN-α and IFN-β) in response to viral infection. In contrast to other dendritic cell subsets which express a broad profile of TLRs, the TLR profile in PDC is restricted to TLR7 and TLR9. So far, CpG DNA is the only defined microbial molecule recognized by PDC. An intriguing feature of PDC is its ability to simultaneously produce the two major Th1-inducing cytokines in humans, IFN-α and IL-12, both at high levels. The ratio of IFN-α versus IL-12 and the quantity of these cytokines are regulated by T helper cell-mediated costimulation via CD40 ligation. The ratio also depends on the differentiation stage of the PDC at the time of stimulation and the type of CpG ODN used. We propose a model in which the establishment of Th1 responses in vivo is improved by appropriately stimulated PDC that otherwise – in the absence of CpG DNA – support Th2 or Th0 responses and thus have been called DC2. [Copyright &y& Elsevier]

Epidermal dendritic cells found in inflamed skin include Langerhans cells and the recently identified population of inflammatory dendritic epidermal cells. Another subset of dendritic cells in humans is the plasmacytoid dendritic cell in peripheral blood, which is characterized by the production of large amounts of type I interferon (interferon-α and interferon-β) upon viral infection. We hypothesized that plasmacytoid dendritic cells might be involved in anti-viral defense mechanisms of the skin. Here we investigated plasmacytoid dendritic cells, inflammatory dendritic epidermal cells, and Langerhans cells in epidermal single cell suspensions of normal looking skin from healthy volunteers and of lesional skin from patients with different inflammatory skin diseases. Langerhans cells were found in normal and in inflamed skin samples. In normal skin, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells were low or absent. Lesional skin samples from patients with psoriasis vulgaris and contact dermatitis contained relatively high numbers of both inflammatory dendritic epidermal cells and plasmacytoid dendritic cells. In contrast, many inflammatory dendritic epidermal cells but only very few plasmacytoid dendritic cells could be detected in atopic dermatitis lesions. Lupus erythematosus was characterized by high numbers of plasmacytoid dendritic cells but low numbers of inflammatory dendritic epidermal cells. These results demonstrate that in addition to resident Langerhans cells, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells are selectively recruited to the skin lesions depending on the type of skin disease. The lack of plasmacytoid dendritic cells in atopic dermatitis may predispose atopic dermatitis patients to viral infections such as eczema herpeticum, a secondary infection of atopic dermatitis lesions with herpes simplex virus. The composition of dendritic cell subsets may help to clarify the etiology of... [ABSTRACT FROM AUTHOR]