Human Metapneumoviruses (HMPV) are recently identified
Paramyxoviridae
that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful ...defense against virus infection relies on early detection by germline encoded pattern recognition receptors and activation of cytokine and type I interferon genes. Recently, the RNA helicase Retinoic acid inducible gene (RIG-I) has been shown to sense HMPV. In this study, we investigated the ability of two prototype strains of HMPV (A1 NL\1\00 and B1 NL\1\99) to activate RIG-I and induce type I interferons (IFN). Despite the ability of both HMPV-A1 and B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5’ triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells (PDC) displayed a unique ability to sense both the A1 and B1 strains and in this case sensing was
via
Toll-like receptor (TLR)-7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell-type specific manners.
Although the role of human IRF-5 in antiviral and inflammatory responses
in vitro
has been well characterized, much remains to be elucidated about murine IRF-5. Murine IRF-5, unlike the heavily ...spliced human gene, is primarily expressed as a full-length transcript, with only a single splice variant that was detected in very low levels in the bone marrow of C57BL/6J mice. This bone marrow variant contains a 288-nucleotide deletion from exons 4–6 and exhibits impaired transcriptional activity. The murine IRF-5 can be activated by both TBK1 and MyD88 to form homodimers and bind to and activate transcription of type I interferon and inflammatory cytokine genes. The importance of IRF-5 in the antiviral and inflammatory response
in vivo
is highlighted by marked reductions in serum levels of type I interferon and tumor necrosis factor α (TNFα) in Newcastle disease virus-infected
Irf5
–
/
–
mice. IRF-5 is critical for TLR3-, TLR4-, and TLR9-dependent induction of TNFα in CD11c
+
dendritic cells. In contrast, TLR9, but not TLR3/4-mediated induction of type I
IFN
transcription, is dependent on IRF-5 in these cells. In addition, IRF-5 regulates TNFα but not type I interferon gene transcription in Newcastle disease virus-infected peritoneal macrophages. Altogether, these data reveal the cell type-specific importance of IRF-5 in MyD88-mediated antiviral pathways and the widespread role of IRF-5 in the regulation of inflammatory cytokines.
While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell ...membrane. An important outcome of cytosolic recognition is the induction of IFNα and IFNβ, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Electrophoretic mobility shift assay (EMSA) is used to detect the complex of protein and nonradioisotope-labeled probe qualitatively. In this paper, we describe a modified quantitative EMSA, which ...uses biotin-labeled RNA in the complex formation. The RNA–protein complex is separated by agarose gel electrophoresis and capillary transferred to a positively charged nylon membrane. It is then detected through a secondary detection system using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT) as the colorimetric precipitating substrate. After scanning and quantification by an image analysis program, ImageQuant, it was observed that the optical density of the bands was proportional to the decadic logarithm value of standard RNA quantities in the tested range. By using the standard curve of the optical densities plotted against the logarithm values of RNA quantities in the linear range, we can calculate RNA quantities according to the optical density of the band and make a quantitative analysis of EMSA. This method was applied successfully in the study of the interactions between the AU-rich element (ARE) and HuR, which is one of the human members of the (embryonic lethal abnormal vision) ELAV family. The results reveal that the biotin-labeled AUFL transcripts in the RNA–protein complex can be detected quantitatively, while maintaining the biological features of its binding ability to the HuR protein. By this method, it is possible to conduct qualitative and quantitative analyses of the EMSA in the study of RNA–protein interactions.
The Innate immune system is the first line of defense against invading microbial pathogens. It is a fast-acting and non-antigen-specific defense system, which employs germline encoded surveillance ...systems capable of responding to a broad-spectrum of pathogens. The innate immune system involves a variety of immune cells, which express different profiles of surveillance or detection receptors. Upon sensing pathogens, these receptors trigger cell signalling to turn on transcription of inflammatory cytokines, chemokines, anti-microbial peptides and type I Interferons. These effectors have direct effects on the control of pathogen load and also activate the adaptive immune system, which is ultimately required to clear infections. The type I interferons (IFNs) are the principal cytokines strongly induced during infection with viruses and are required for direct control of viral replication and modulation of cells of the adaptive immune response. The signalling pathways induced in order to activate type I IFNs are dependent on the interferon regulatory factors (IRFs). Striving for survival, microbes have evolved various strategies to subvert/impair these critical defense molecules.
In this thesis work, I have used Human Metapneumoviruses (HMPVs), a relatively newly described family of paramyxoviruses as model viruses to explore the role of pattern recognition receptors (PRRs) and the IRF family of transcription factors in the innate immune response. These studies revealed that the recognition of HMPV viral pathogen-associated molecular patterns (PAMPs) by immune cells is different in different cell types. Retinoic acid-inducible gene-I (RIG-I), a cytosolic RNA helicases senses HMPV-A1 virus for triggering type I IFN activation by detecting its 5’- triphosphate viral RNA in most human cells, including cell lines and primary monocytes. An exception to these findings was plasmacytoid dendritic cells (PDCs), where Toll-like receptor (TLR)-7 is the primary sensor involved in detecting HMPV viruses. By comparing the innate immune response to two HMPV strains, we found that these two closely related strains had very different immune stimulatory capabilities. HMPV-1A strain triggered type I IFNs in monocytes, PDCs and cells of epithelial origin. In contrast, a related strain, HMPV-B1 failed to trigger IFN responses in most cell types. Our studies suggested that the phosphoprotein (P) of HMPV-B1 could prevent the viral RNA from being detected by RIG-I, thus inhibiting the induction of type I IFN production in most cell type examined. This finding adds to our understanding of the mechanisms by which viruses are sensed by surveillance receptors and also unveils new means of viral evasion of host immune responses.
Although IRFs are extensively studied for their role in regulating type I IFN activation, especially in TLR and RIG-I like receptor (RLR) signalling pathways upon viral infection, a clear understanding of how this family of transcription factors contributes to anti-viral immunity was lacking. Studies conducted as part of this thesis revealed that in addition to IRF3 and IRF7, which play a central role in anti-viral immunity downstream of most PRRs (e.g. TLRs, RLRs, DNA sensors), the related factor IRF5 was also an important component of innate anti-viral defenses. Using IRF5-deficient mice we studied in detail the role of IRF5 in coordinating antiviral defenses by examining its involvement in signalling downstream of TLRs. These studies led us to examine the role of IRF5 in the regulation of type I IFNs as well as inflammatory cytokines in different cell types. While most TLRs that induced IFNβ showed normal responses in IRF5-deficient mice, CpG-B-induced IFNβ production in CD11c+CDCs isolated from mouse spleen but not those generated in vitro from bone marrow required IRF5. This was in contrast to responses with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (polyIC), ligands for TLR4 and 3, respectively. Moreover, we found that in contrast to IRF3 and/or IRF7, IRF5 was important in coordinating the expression of inflammatory cytokines such as TNFα downstream of some TLRs. In addition to our studies to examine the requirement for IRF5 in TLR signaling, we also showed that muramyl peptide (MDP) from Mycobacterium tuberculosis (Mtb) could activate type I IFNs via IRF5. This was the first evidence linking IRF5 to a non-TLR-driven pathway. IRF5 activation in this case was downstream of a novel nucleotide-binding oligomerization domain containing (NOD)-2/receptor-interacting serine-threonine kinase (RIP)-2 signaling pathway.
Collectively, the studies outlined in this thesis have assisted in providing a framework to understand the role of TLRs, RLRs and IRFs in the immune response to paramyxoviruses and have unveiled new mechanisms of activation of the IRFs as well as new mechanisms by which pathogens subvert or evade these important innate defense mechanisms.
•Use PSO algorithm to optimize the parameters of VMD.•Signal construction is based on energy ratio.•The clearance factor and kurtosis factor are selected as the feature vectors.•The SVM is selected ...as the pattern recognition method.•A set of five different leak apertures are adopted for leak detection.
Leak detection is critical for the safety management of pipelines since leakages may cause serious accidents. The present paper aims to develop an efficient method able to detect the presence and importance of leaks in pipelines.
This method relies on adequate signal processing of acoustic emission (AE) signals, and improves the variational mode decomposition (VMD) for signal de-noising. In order to optimize the governing parameters, i.e. the penalty term and the mode number of VMD, the particle swarm optimization (PSO) algorithm is coupled to a fitness function based on maximum entropy (ME). After the signal reconstruction, based on the energy ratio of each VMD sub-mode, the waveform feature vectors for leak detection are extracted. Finally, the support vector machine (SVM) is employed for leak pattern recognition. For calibration purposes, artificial input signal is simulated. The results show that the proposed PSO-VMD method is capable of de-noising background noise.
For validation purposes, experiments have been conducted on metal pipelines, with water flow. For sensitivity analysis, a set of five different leak apertures are adopted: aperture diameters as {10; 12; 15; 20; 27} mm, whereas the pipeline diameter is 108 mm. A database of AE signals is collected for each leak aperture, and different time sequences. The proposed method appears to be efficient since the classification accuracy of the SVM method reaches up to 100% in identifying the size of the leak on the basis of the AE signals collected in the database for the same leak size, and 89.3% on the basis of the whole database.
PDF
