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PubMed2004

Methods for delivery of double-stranded RNA into Caenorhabditis elegans.

The nematode Caenorhabditis elegans is often employed in investigations of diverse aspects of biology, including behavior, development, basic cellular processes, and disease states. The ability to utilize double-stranded RNA (dsRNA) to inhibit specific gene function in this organism has dramatically increased its value for these kinds of studies and has provided more flexibility in experimental design that include procedures. Here, we have collected a set of protocols from the C. elegans community for propagation of C. elegans, techniques for dsRNA preparation, four basic methods for delivery of dsRNA to C. elegans (injection, soaking, feeding, and in vivo delivery), and we suggest schemes that should facilitate detection of specific gene silencing.

Animals↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Animals↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. The method of choice today to examine gene function in these organisms is RNA interference (RNAi). Messenger RNA (mRNA) degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. This chapter describes some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA, and analysis of dsRNA and target mRNA.

Animals↗
PubMed2001

Effects of double-stranded RNA viruses on the reproduction of Phaffia rhodozyma.

DsRNA viruses were transferred from a virus-containing strain to a virus-free strain of Phaffia rhodozyma by protoplast fusion. The resulting new strain carried all three types of dsRNA of the virus-containing strain and had the electrophoretic karyotype of the virus-free strain. The effects of the dsRNA viruses on the host fitness were checked by following the asexual and the sexual reproductivity. The results demonstrated that viruses have no effect on the growth rate during the lag and log phases of the vegetative reproduction, but the maximum cell numbers in the stationary phase differ significantly. Inconclusive results were obtained as concerns the effects of viruses on the sexual reproduction.

Basidiomycota↗
PubMed2005

RNA interference-based gene silencing in mice: the development of a novel therapeutical strategy.

RNAi (RNA interference) was originally detected in Caenorhabditis elegans as biological response to exogenous double-stranded RNA (dsRNA), which induces very effective sequence-specific silencing of gene expression. Further investigations revealed that RNAi can occur in many eukaryotic species. Increasing understanding of the biochemical components of RNAi indicates the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer processes the dsRNA to small interfering RNAs (siRNAs) 21-23 nt in length. These siRNAs enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induce destruction of the corresponding mRNAs. Since RNAi has become an excellent strategy for gene silencing, it is tempting to apply this technology to 'knock-down' gene expression in living animals. The generation of transgenic mice from embryonic stem cells expressing small hairpin RNAs (shRNAs) has provided evidence for in vivo application of RNAi. Furthermore, different experimental strategies have been developed to analyze the influence of chemically synthesized siRNAs and of vector-based shRNAs on the expression of different transgenes and endogenous genes in vivo. Recent studies describe the in vivo delivery of siRNAs to inhibit transgene expression in certain organs of adult mice, predominately murine liver. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor-bearing animals with siRNAs are beginning to emerge. They are of utmost interest for systemic diseases such as cancer. In addition, several groups have shown that RNAi can also be used to block the infectivity or suppress the replication of different RNA viruses relevant to human diseases including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). In summary, multiple lines of evidence indicate that RNAi seems to become a powerful tool for the fight against undesirable gene expression in human diseases.

Animals↗
PubMed2004

[Virus-induced expression of type I interferon genes].

Intracellular double-stranded (ds) RNA is a major sign of replication for many viruses. Host mechanisms detect the dsRNA and provoke antiviral responses. Recently, we identified retinoic acid inducible gene-I (RIG-I), which encodes a DExD/H box RNA helicase containing the caspase recruitment domain (CARD) as a critical regulator for dsRNA-induced signaling. The helicase domain with intact ATPase activity is responsible for recognition of dsRNA, and the CARD transmits downstream signals, resulting in the activation of genes including type I interferons. In this review, we discuss the function of RIG-I in antiviral innate immunity.

Adenosine Triphosphatases↗
PubMed2000

Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.

The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-kappaB, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-alpha. E-selectin mRNA and protein expression was inducible by both pIC and TNF-alpha in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-kappaB DNA binding activity were substantially reduced in pIC- or TNF-alpha-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-alpha induction of E-selectin via an NF-kappaB-dependent pathway. In PKR-null cells, pIC-mediated degradation of IkappaBbeta is deficient. Activation of this pathway requires the PKR-dependent degradation of the IkappaBbeta protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-alpha in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.

Animals↗
PubMed2003

Subcellular localization of Toll-like receptor 3 in human dendritic cells.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Animals↗
PubMed2003

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway.

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.

Bronchi↗
PubMed2004

Viral activation of macrophages through TLR-dependent and -independent pathways.

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.

Animals↗
PubMed2005

Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus.

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.

Adjuvants, Immunologic↗
PubMed2005

Regulation of the polymeric Ig receptor by signaling through TLRs 3 and 4: linking innate and adaptive immune responses.

IgA Abs help to maintain homeostasis at mucosal surfaces by promoting defense mechanisms that protect against pathogens while suppressing inflammatory responses to commensal organisms and food Ags. The polymeric Ig receptor (pIgR) mediates transport of IgA across mucosal epithelial cells. We hypothesized that signaling through TLRs may up-regulate pIgR expression by intestinal epithelial cells and thus enhance IgA-mediated homeostasis. To test this hypothesis we treated the HT29 human intestinal epithelial cell line with dsRNA, a ligand for TLR3, or LPS, a ligand for TLR4. Both dsRNA and LPS up-regulated levels of pIgR mRNA and cell surface pIgR protein. By contrast, dsRNA but not LPS up-regulated expression of TLR3 and TLR4 mRNA. However, cell surface expression of both TLR3 and TLR4 was enhanced by treatment of HT29 cells with their respective ligands. Transfection of HT29 cells with wild-type and mutated promoter/enhancer plasmids suggested that TLR3 and TLR4 signal primarily through NF-kappaB to enhance transcription of pIgR mRNA. TLR3 signaling resulted in a more pronounced inflammatory response than did TLR4, as evidenced by up-regulation of the transcription factor IFN regulatory factor-1, chemokines IL-8 and RANTES, and the proinflammatory cytokine TNF. Signaling through LPS/TLR4 appears to up-regulate pIgR expression while minimizing proinflammatory responses, a mechanism that could promote IgA-mediated homeostasis in the presence of commensal Gram-negative bacteria.

Adaptation, Physiological↗
PubMed2006

Immunologic control of tumors by in vivo Fc gamma receptor-targeted antigen loading in conjunction with double-stranded RNA-mediated immune modulation.

Despite the expression of non-self or neo-epitopes, many tumors such as lymphoid malignancies or cancers induced by oncogenic viruses are able to gradually overcome the immune defense mechanisms and spread. Using a preclinical model of hematological malignancy, we show that Ig-associated idiotypic determinants are recognized by the immune system in a fashion that results in immune deviation, allowing tumor progression and establishment of metastases. Using gene-targeted mice, we show that anti-idiotypic MHC class I-restricted immunity is promoted by ITAM motif (ITAM+) FcgammaR, but kept in check by ITIM motif (ITIM+) FcgammaRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcgammaR, effective anti-idiotypic and antitumoral immunity can be achieved by FcgammaR-targeted delivery of epitope in conjunction with administration of stimulatory motifs such as dsRNA, correcting the ineffective response to idiotypic epitopes. The immune process initiated by FcgammaR-mediated targeting of epitope together with dsRNA, resulted in control of tumor growth, establishment of immune memory and protection against tumors bearing antigenic variants. In summary, targeted delivery of MHC class I-restricted epitopes via ITAM+ FcgammaR, in conjunction with use of TLR-binding immune stimulatory motifs such as dsRNA, overcomes suboptimal responses to idiotypic determinants and may constitute a novel approach for the treatment of a broad range of malignancies. Finally, the results shed light on the mechanisms regulating the idiotypic network and managing the diversity associated with immune receptors.

Animals↗
PubMed2006

Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells.

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.

Cell Line↗
PubMed1999

A fast, sensitive and specific method for rice dwarf virus detection by northern blot hybridization.

Based on stability of double-stranded (ds) RNA, a new, fast, sensitive, and specific method for detection of genomic rice dwarf virus (RDV) dsRNA by molecular hybridization was developed. In contrary to the commonly used, standard Northern blot analysis, dsRNA is denatured in the immobilized state on the blot. Therefore, risk of degradation of single-stranded (ss) RNA by ribonuclease (RNase) during sample preparation, electrophoresis and blotting is eliminated. This method overcomes disadvantage of incomplete denaturation of dsRNA in Northern blot analysis. In conclusion, the newly developed method is reliable, sensitive, very specific, and gives a low background. The entire procedure is also less time consuming; it can be completed within 2-3 days. The new method may be regarded as a modification of the standard Northern blot analysis.

Blotting, Northern↗
PubMed2001

[Cloning, sequence analysis and high-level expression in Escherichia coli and activity assay of pac-1 gene from Schizosaccharmyces pombe].

The Schizosaccharmyces pombe pac-1 gene product is a kind of dsRNA dependent ribonuclease, which has potential to degrade the dsRNA viral genome, the replication form of ssRNA viral genome and viroid genome. Therefore, to introduce the pac-1 gene into plants conferring them resistance to viruses is a new method of establishing the anti-virus transgenic plant. The pac-1 gene from the S. pmobe genome DNA isolated from China was cloned by means of PCR amplification. The pac-1 gene was inserted into the cloning vector pGEM-7Zf(+) by using restriction endonuclease Kpn I/BamHI. Sequencing analysis shows that it is a complete gene with 1095 necleotides. Compared to the reported pac-1 gene, its homology is significant, but with 5 nucleotides differences, leading to only one amino acid difference. Pac-1 gene was inserted into the prodaryotic expression vector pET-21(a) by using the restriction endonuclase Nde I/BamHI. It was induced by the IPTG in E. coli BL21 harbouring the recombinant vector pET-pac-1. The pac-1 gene product is analyzed by the SDS-PAGE. The result shows the product of pac-1 gene exists in the supernatant part as soluble form and in the precipitant part as inclusion bodies after the cells were lysed by ultrasonic wave. The supernatant was applied to detect the enzyme activity of pac-1 gene product. We concluded that pac-1 gene has the biological activity of degrading the CMV-dsRNA.

Cloning, Molecular↗
PubMed2001

[Inhibition of gene expression by administration of homologous double-stranded RNA in Drosophila melanogaster cell culture].

Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitor effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs from a special plasmid did not inhibit expression of the reporter construct.

Animals↗