TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.     

Novel polymorphism in the coding region of the IL-13 receptor alpha' gene: association study with atopic asthma in the Japanese population

Interleukin (IL)-4 and IL-13 play key roles in the development of atopic asthma. The IL-13 receptor (R) alpha' chain is a component of both IL-4R and IL-13R complexes. By screening the whole coding region of the IL-13Ralpha' gene for polymorphisms, we identified a new polymorphism at nucleotide position 1050 from the ATG start codon. The allelic frequency of the C/T polymorphism in the Japanese population was found to be 0.97:0.03. Because of the low frequency of the T allele, the association study failed to indicate any significant association between this polymorphism and atopic asthma in the Japanese population. Further studies are required in other racial groups with higher frequencies of this polymorphism to elucidate the association.     

Phosphate glasses for tissue engineering: Part 2. Processing and characterisation of a ternary-based P2O5-CaO-Na2O glass fibre system

This paper presents the results of a study of the thermal properties, solubility and dimensions of a range of phosphate-based glass fibres (PB-GFs). The glass compositions were limited by fixing the P2O5 content to 45, 50 and 55 mol%, and varying the CaO mol% at 30, 35 and 40. PB-GFs were obtained from the 50 and 55 mol% P2O5 compositions; however, we were unable to obtain fibres from the 45 mol% compositions. This was linked to the cross-linked density, network connectivity and average chain length of the compositions studied. With regards to thermal parameters investigated, initial data showed an increase of the Tg and crystallisation temperatures with increasing CaO mol% at each fixed phosphate content. A decrease in Tg temperatures was also observed with increasing P2O5 content to 55 mol%. The crystallisation temperatures obtained for compositions with fixed phosphate at 55 mol%, showed a reverse pattern, with a decrease in values as compared to the fixed 50 mol% phosphate compositions. The diameters of the fibres all decreased with increasing RPMs as expected, and the solubility also increased with increasing RPMs. This was related to the increased surface area of the higher RPM fibres. There was also a decrease seen in solubility with increasing CaO mol%.     

Suppression of eosinophilic airway inflammation by treatment with alpha-galactosylceramide

To clarify the essential role of NKT cells in allergy, we investigated the contribution of NKT cells to the pathogenesis of eosinophilic airway inflammation using alpha-galactosylceramide (alpha-GalCer), a selective ligand for NKT cells. Although continuous administration of alpha-GalCer during ovalbumin (OVA) sensitization increased OVA-specific IgE levels and worsened eosinophil inflammation, a single administration of alpha-GalCer at the time of OVA challenge completely prevented eosinophilic infiltration in wild-type mice. This inhibitory effect of alpha-GalCer was associated with a decrease in airway hyperresponsiveness, an increase in IFN-gamma, and decreases in IL-4, IL-5 and IL-13 levels in the bronchoalveolar lavage fluids. Analysis of lung lymphocytes revealed that production of IFN-gamma increased in NK cells, but not in T or NKT cells, following alpha-GalCer administration. Induction of vascular cell adhesion molecule-1 in the lungs of wild-type mice was also significantly attenuated by treatment with alpha-GalCer. These effects of alpha-GalCer were abrogated in J alpha281-/- mice, which lack NKT cells, and in wild-type mice treated with anti-IFN-gamma Ab. Hence, our data indicate that alpha-GalCer suppresses allergen-induced eosinophilic airway inflammation, possibly by inducing a Th1 bias that results in inhibition of eosinophil adhesion to the lung vessels.     

Purification and characterization of RHP (factor H) and study of its interactions with the first component of complement.

RHP has been purified from the plasma of both normal individuals and patients with rheumatoid arthritis (RA). RHP from both these sources was shown to be identical with Factor H by reaction with antisera and N-terminal amino acid sequence analysis. Factor H, from both normal and RA sera, inhibited the solubilization of immune precipitates but did not affect prevention of immune precipitation. Factor H was shown to inhibit the haemolytic activity of fluid-phase C1, but unlike C1-inhibitor, it had little effect on C1 bound to EA (EAC1). Factor H was shown to complex with intact C1, to isolated C1q and to the C1r:C1s tetramer. However, binding of factor H to C1 did not dissociate the C1 macromolecule. A C1-Factor H complex was detected in the serum and plasma from normal individuals and patients with systemic lupus erythematosus and RA. Serum levels of this complex were reduced, by EDTA-treatment of serum and by activation of complement by the classical pathway.     

Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning.

Bacillus cereus is responsible for an increasing number of food poisoning cases. By using 12 bacteriophages isolated from sewage, a typing scheme for B. cereus isolates from outbreaks or sporadic cases of food poisoning was developed. The phages belonged to three morphotypes. Ten phages with contractile tails and icosahedral heads were members of the Myoviridae family, and two phages with noncontractile tails belonged to the Siphoviridae family. Phage 11 represented a new species. It had an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known. The vast majority of 166 B. cereus strains (161, or 97%) isolated from food poisoning cases were typeable. Of 146 strains isolated from 18 outbreaks, 142 (97%) could be divided into 17 phage types. A good correlation, on the order of 80 to 100%, between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed. Most Bacillus thuringiensis strains were also typeable, providing further evidence of the close relatedness of B. cereus and B. thuringiensis. This phage typing scheme can be a valuable epidemiological tool in tracing the origins of food poisoning caused by B. cereus.     

Effect of iron on the surface, degradation and ion release properties of phosphate-based glass fibres

Phosphate-based glass fibres (PGF) have the unique characteristic of being completely soluble in an aqueous environment, releasing bioactive and biocompatible ions. They have been proposed as tissue engineering scaffolds for craniofacial skeletal muscle regeneration, where myoblasts are seeded directly onto the fibres. Studies have shown that these cells have a preference in their initial attachment to fibres of certain composition and size, which in turn control the rate of degradation. This study investigated the relationship between the surface properties, degradation properties and ion release (cationic and anionic species) by altering the chemical composition of the PGF. Iron oxide (Fe2O3) was incorporated into glasses containing P2O5 (50 mol%), CaO (30 mol%) and Na2O (20 mol%). Six glass compositions with Fe2O3 ranging from 0 to 5 mol% by replacing the equivalent Na2O mol% were investigated. Contact angle measurements showed that polar interactions occurring on the glass surfaces diminished with increasing Fe2O3 content. This behaviour was reflected in the estimated surface energies of the glasses, where the overall surface energy decreased with increasing Fe2O3 content due to the decrease in polar or acid/base component. The incorporation of up to 5 mol% Fe2O3 into PGF resulted in a significant reduction in the degradation rate (by two orders of magnitude), which can be related to the formation of more hydration resistant P-O-Fe bonds. However, the degradation rate increased with decreasing fibre diameter (comparing average diameters of 31.6 +/- 6.5 microm versus 13.1 +/- 1.3 microm) for a given mass of fibre, and this is related to the surface area to volume ratio. Taken together the results suggest that fibres with the larger diameters and containing 3-5 mol% Fe2O3 could initially be a more durable scaffold than ones with 1 or 2 mol% Fe2O3 for initial cell attachment.     

Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses

Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.     

Interleukin-4 receptor alpha chain and STAT6 signaling inhibit gamma interferon but not Th2 cytokine expression within schistosome granulomas

Compared to wild-type (WT) mice, schistosome granulomas in Stat6 knockout (KO) mice lacked eosinophils and had Th1 features. Interleukin-4 (IL-4) acts through Stat6 in assisting Th2 cell development. The importance of Stat6 for Th2-cell development within schistosome granulomas had not been explored. Therefore we studied gamma interferon (IFN-gamma), IL-4, and IL-5 production in granulomas from Stat6 KO and WT mice. Dispersed granuloma cells from Stat6 KO and WT mice made similar amounts of IL-4 and IL-5. Only Stat6 KO granuloma cells released IFN-gamma. Granuloma T cells contained most of the IL-4, IL-5, and IFN-gamma mRNA and secreted these cytokines. In Stat6 KO mice, 16.6% of the granuloma cells were CD4(+). Of these, 10.7% stained for IFN-gamma and/or IL-4 by intracytoplasmic flow analysis. Few CD4(-) T cells stained positively. The IL-4-producing T cells did not stain for DX5 or with labeled alpha-GalCer CD1d tetramer, suggesting an absence of NK T cells. Thus, conventional Th cells in Stat6 KO granulomas produce IFN-gamma and Th2 cytokines. Stat6 limits IFN-gamma production but is unnecessary for Th2-cell development or localization within the granuloma.     

Mechanism of action of an inhibitor of complement-mediated prevention of immune precipitation

Glycoprotein 60 (gp60) is a normal plasma protein (mean concentration in normal serum 34 micrograms/ml) that is present in increased levels (mean concentration 97 micrograms/ml) in the sera of patients with rheumatoid arthritis (RA). Purified gp60 binds to IgG but not to IgM, and competitively inhibits the binding of C1q. In fluid-phase studies, purified gp60 was shown to reduce immune complex-mediated complement activation in a dose-dependent manner. The addition of Fab anti-gp60 to normal serum was associated with (i) increased levels of complement-mediated prevention of immune precipitation (PIP); (ii) increased total haemolytic complement activity when EAIgG, but not when EAIgM, were used as targets; and (iii) increased immune complex-mediated complement activation. Thus gp60 appears to regulate immune complex-mediated classical pathway activation. The findings that Fab anti-gp60 (i) only partly restored PIP in RA sera showing reduced PIP levels and (ii) only partly reduced inhibition of PIP by RA sera, show that gp60 is not entirely responsible for these abnormalities.