Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

IgG and IgM western blot assay for diagnosis of congenital toxoplasmosis

The aim of this study was to evaluate the utility of western blot (WB) analysis as a diagnostic tool for congenital toxoplasmosis in 215 newborn infants. The children were submitted to clinical examinations to assess macular, neurological and hearing signals. The WB results obtained were compared to the persistence of IgG antibodies at the end of 12 months, which is regarded as the "gold standard" diagnosis of congenital toxoplasmosis. Association between the WB results and the clinical signs presented by the infants was also assessed. Of the 215 children, 177 had a confirmed congenital toxoplasmosis diagnosis and 38 were uninfected. IgG-WB showed a sensitivity of 73.5% and a specificity of 97.4%. IgM-WB showed a sensitivity of 54.8% and a specificity of 94.7%. The IgG-WB and IgM-WB combination increased the sensitivity to 86.5%. The IgM-WB-positive children had a 1.4-fold greater risk of presenting active macular lesions than did those that were IgM-WB-negative. This study showed that the WB assay is a useful tool to confirm a diagnosis of congenital toxoplasmosis and that the IgM-WB-positive results can indicate active macular lesions in newborn infants.     

Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

Spondylitis in a humpback whale (Megaptera novaeangliae) from the southeast Pacific

A 7.25 m long male humpback whale (Megaptera novaeangliae) with spondylitis was found beached on August 13, 1994 at Ancon, Ecuador (2 degrees 23' S, 80 degrees 47' W). The condition involved at least 11 vertebrae, 7 lumbar (L4 to L11) and 4 caudal (Ca1 to Ca4). Partial fusion of vertebrae was observed as a result of intervertebral bony proliferation, likely impeding full motion. The relatively young age of this specimen and the severity of the deformities suggest an infectious, rather than degenerative, process. The gross findings are most consistent with some type of spondyloarthritis. Although this condition has previously been identified in a number of cetacean species, the pathogenesis, population impact and ecologic implications have not been fully assessed. This is the third case described for humpback whales and the first for a humpback whale from the SE Pacific.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Genetic structure in a tropical lek-breeding bird, the blue manakin (Chiroxiphia caudata) in the Brazilian Atlantic Forest

Determining the genetic structure of tropical bird populations is important for assessing potential genetic effects of future habitat fragmentation and for testing hypotheses about evolutionary mechanisms promoting diversification. Here we used 10 microsatellite DNA loci to describe levels of genetic differentiation for five populations of the lek-mating blue manakin (Chiroxiphia caudata), sampled along a 414-km transect within the largest remaining continuous tract of the highly endangered Atlantic Forest habitat in southeast Brazil. We found small but significant levels of differentiation between most populations. F(ST) values varied from 0.0 to 0.023 (overall F(ST)=0.012) that conformed to a strong isolation by distance relationship, suggesting that observed levels of differentiation are a result of migration-drift equilibrium. N(e)m values estimated using a coalescent-based method were small (相似文献   

Synthesis and potent antitumor activity of new arylamino derivatives of nor-beta-lapachone and nor-alpha-lapachone

Several arylamino derivatives of nor-beta-lapachone were synthesized in moderate to high yields and found to show very potent cytotoxicity against six neoplastic cancer cells: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast), HL-60 (leukaemia), PC-3 (prostate), and B-16 (murine melanoma), with IC(50) below 1 microg/mL. Their cytotoxicities were compared to doxorubicin and with their synthetic precursors, beta-lapachone and nor-beta-lapachone. The activity against a normal murine fibroblast L-929 showed that some of the compounds were selective against cancer cells. The absence of hemolytic activity (EC(50)>200 microg/mL), performed with erythrocyte suspensions, suggests that the cytotoxicity of the compounds was not related to membrane damage of mouse erythrocytes. For comparison purposes, one isomeric compound based on nor-alpha-lapachone was also synthesized and showed lower activity than the related ortho-derivative. The modified arylamino quinones appear as interesting new lead compounds in anti-cancer drug development.