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ABSTRACT: Enterohaemorrhagic Escherichia coli O157 : H7 is a major foodborne and environmental pathogen responsible for both sporadic cases and outbreaks of food poisoning, which can lead to serious sequelae, such as haemolytic uraemic syndrome. The structural subunit of E. coli O157 : H7 flagella is flagellin, which is both the antigenic determinant of the H7 serotype, an important factor in colonization, and an immunomodulatory protein that has been determined to be a major pro-inflammatory component through the instigation of host cell signalling pathways. Flagellin has highly conserved N- and C-terminal regions that are recognized by the host cell pattern recognition receptor Toll-like receptor (TLR) 5. Activation of this receptor triggers cell signalling cascades, which are known to activate host cell kinases and transcription factors that respond with the production of inflammatory mediators such as the chemokine interleukin-8 (IL-8), although the exact components of this pathway are not yet fully characterized. We demonstrate that E. coli O157 : H7-derived flagellin induces rapid phosphorylation of the epidermal growth factor receptor (EGFR), as an early event in intestinal epithelial cell signalling, and that this is required for the release of the pro-inflammatory cytokine IL-8.
Microbiology 05/2011; 157(Pt 8):2339-47. · 3.06 Impact Factor
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ABSTRACT: The Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (Norfolk QOL-DN) is a validated comprehensive questionnaire designed to capture the entire spectrum of DN related to large fiber, small fiber, and autonomic neuropathy not captured in existing instruments. We aimed to determine if the Norfolk QOL-DN could be used to capture changes in QOL that correlate with nerve fiber-specific objective measures in a placebo-controlled trial of two agents that affect different nerve fibers.
Sixty patients with DN were allocated to treatment on ruboxistaurin (RBX) (n = 18), topiramate (TPX) (n = 18), or placebo (n = 18). QOL-DN was administered and objective measures of nerve function were performed at entry and end of the study period.
Total QOL scores improved significantly in the active treatment groups (RBX -9.56 ± 4.13; TPX -12.22 ± 2.76) but not in placebo (-5.56 ± 3.49). There were differences in nerve function improvement between treatments. Neurological symptom scores (NSS) improved with TPX from 5.5 (2.3) to 4.3 (0.65) (p = .007), sensory scores improved with TPX from 15.5 (1.79) to 8.3 (1.19) (p < .001), motor scores did not change, and sensory and motor impairment scores improved with TPX from 18.8 (2.15) to 12.1 (1.71) (p = .003). Total neuropathy scores (TNS) improved with TPX from 24.35 (2.61) to 16.35 (2.02) (p = .001). Neuropathy total symptom score-6 (NTSS-6) changes were significant for both treatments: RBX 4.38 (0.75) to 1.49 (0.38) (p < .001) and TPX 7.57 (1.3) to 4.26 (0.95) (p = .036). Changes in QOL-DN large fiber subscores correlated (Spearman's rank) significantly with changes in NTSS-6 (r = 0.55; p < .0001), NSS (r = 0.31; p < .04), neuropathy impairment score (NIS) (r = 0.35; p < .02), and TNS (r = 0.48; p < .0006). Changes in QOL-DN small fiber subscores correlated significantly with changes in NTSS-6 total scores (r = 0.40; p < .005) and intraepidermal nerve fiber density (IENFD) (r = -0.29; p < .05).
Ruboxistaurin produced significant improvement in large fiber measures while TPX produced significant changes in small fiber measures. The Norfolk QOL-DN tool differentiated between these changes captured in the fiber-specific domains. Correlations were found between objective measures of neuropathy and total QOL, but those with nerve fiber domain scores were modest and reinforce the need to quantify QOL as an endpoint in neuropathy independent of other measures.
Journal of diabetes science and technology 01/2011; 5(3):714-22.
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ABSTRACT: Semliki Forest virus (SFV) infection of the laboratory mouse provides a well-characterized tractable system to study the pathogenesis of virus encephalitis and virus induced demyelination. In microMT mice, which have no antibodies, infectious virus persisted in both the serum and the brain for several weeks, indicating that antibodies are required to eliminate infectious virus. In immunocompetent mice, virus infectivity in the brain was undetectable after the first week of infection, but virus RNA levels declined slowly. Following SFV infection, lesions of demyelination were present in the brains of both immunocompetent and microMT mice, indicating that antibodies are not required to generate lesions of demyelination.
Journal of General Virology 11/2008; 89(Pt 10):2565-8. · 3.36 Impact Factor
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ABSTRACT: Semliki Forest virus (SFV) infection of the mouse provides a powerful model to study the pathogenesis of virus encephalitis. SFV and other alphavirus-based vector systems are increasingly used in biotechnology and medicine. This study analysed the strong susceptibility of this virus to type I interferon (IFN) responses. Following intraperitoneal infection of adult mice, SFV strain A7(74) was efficiently (100 %) neuroinvasive. In contrast, SFV4 was poorly (21 %) neuroinvasive. Upon entry into the brain, both viruses activated type I IFN responses. As determined by quantitative RT-PCR, activation of the IFN-alpha gene was proportional to virus RNA load. An intact type I IFN system was required for protection against both strains of SFV. IFN strongly curtailed virus spread in many cell types and in many tissues. In mice with an intact type I IFN system, infected cells were rarely observed and tissue tropism was difficult to determine. In the absence of a functional type I IFN system, the tropism and the potential for rapid and widespread infection of this virus was revealed. Virus infection was readily observed in the myocardium, endocardium, exocrine pancreas, adipose tissue, smooth muscle cells and in the brain in meningeal cells, ependymal cells and oligodendrocytes. In the brains of mice with and without type I IFN responses, virus infection of neurons remained rare and focal, indicating that the previously described restricted replication of SFV A7(74) in neurons is not mediated by type I IFN responses.
Journal of General Virology 01/2008; 88(Pt 12):3373-84. · 3.36 Impact Factor
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ABSTRACT: In 129 mice, infection with the nairovirus Dugbe virus (DUGV) was lethal following intracerebral but not intraperitoneal inoculation. Following both routes of inoculation, immunostaining of tissue sections demonstrated virus-positive cells in the brain, indicating that DUGV is neuroinvasive in mice. Many brain areas were affected and neurones were the main cell type infected. Infected cells showed punctate accumulations of viral nucleoprotein in the cytoplasm, indicative of virus replication sites. Immunostaining for activated caspase 3 demonstrated no evidence of apoptosis. The type I interferon (IFN) system plays a significant role in defence against DUGV, as 129 IFN-alpha/beta R(-/-) mice died rapidly following both intraperitoneal and intracerebral inoculations. Studies were undertaken to determine whether the IFN-inducible proteins, protein kinase R (PKR) and MxA, were important for protection; neither PKR nor constitutively expressed human MxA played significant roles.
Journal of General Virology 08/2006; 87(Pt 7):2005-9. · 3.36 Impact Factor
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ABSTRACT: Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.
Journal of Virology 07/2005; 79(11):6940-6. · 5.40 Impact Factor
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ABSTRACT: Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
Journal of Virology 06/2003; 77(9):5507-11. · 5.40 Impact Factor
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ABSTRACT: The replicase protein nsP2 of Semliki Forest virus (SFV) has a 648RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.
Journal of Virology 02/2002; 76(1):392-6. · 5.40 Impact Factor
