Production of IL-27 in multiple sclerosis lesions by astrocytes and myeloid cells: Modulation of local immune responses

The mechanisms whereby human glial cells modulate local immune responses are not fully understood. Interleukin-27 (IL-27), a pleiotropic cytokine, has been shown to dampen the severity of experimental autoimmune encephalomyelitis, but it is still unresolved whether IL-27 plays a role in the human disease multiple sclerosis (MS). IL-27 contribution to local modulation of immune responses in the brain of MS patients was investigated. The expression of IL-27 subunits (EBI3 and p28) and its cognate receptor IL-27R (the gp130 and TCCR chains) was elevated within post-mortem MS brain lesions compared with normal control brains. Moreover, astrocytes (GFAP(+) cells) as well as microglia and macrophages (Iba1(+) cells) were important sources of IL-27. Brain-infiltrating CD4 and CD8 T lymphocytes expressed the IL-27R specific chain (TCCR) implying that these cells could respond to local IL-27 sources. In primary cultures of human astrocytes inflammatory cytokines increased IL-27 production, whereas myeloid cell inflammatory M1 polarization and inflammatory cytokines enhanced IL-27 expression in microglia and macrophages. Astrocytes in postmortem tissues and in vitro expressed IL-27R. Moreover, IL-27 triggered the phosphorylation of the transcription regulator STAT1, but not STAT3 in human astrocytes; indeed IL-27 up-regulated MHC class I expression on astrocytes in a STAT1-dependent manner. These findings demonstrated that IL-27 and its receptor were elevated in MS lesions and that local IL-27 can modulate immune properties of astrocytes and infiltrating immune cells. Thus, therapeutic strategies targeting IL-27 may influence not only peripheral but also local inflammatory responses within the brain of MS patients

The Wnt pathway regulator DKK1 is preferentially expressed in hormone-resistant breast tumours and in some common cancer types

In addition to new tumour antigens, new prognostic and diagnostic markers are needed for common cancers. In this study, we report the expression of Dickkopf-1 (DKK1) in multiple common cancers. This constitutes a comprehensive analysis of the DKK1 expression profile. Dickkopf-1 expression was evaluated by classical and quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbant assay for protein determination, in cancer lines and clinical specimens of several cancer origins. For breast cancer, expression was correlated with clinicopathological parameters. Dickkopf-1 expression was confirmed in several cancer cell lines derived from breast and other common cancers. Dickkopf-1 protein secretion was documented in breast, prostate and lung cancer lines, but was negligible in melanoma. Analysis of DKK1 expression in human cancer specimens revealed DKK1 expression in breast (21 out of 73), lung (11 out of 23) and kidney cancers (six out of 20). Interestingly, DKK1 was preferentially expressed in oestrogen and progesterone receptor-negative tumours (ER−/PR−; P=0.005) and in tumours from women with a family history of breast cancer (P=0.024). Importantly, DKK1 protein production was confirmed in multiple breast cancer specimens that were positive by RT–PCR. This work establishes DKK1 as a potential prognostic and diagnostic marker for cohorts of breast cancer patients with poor prognosis. Dickkopf-1 may also become a relevant candidate target for immunotherapy of different cancers

Priming of the human neutrophil respiratory burst by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha involves regulation at a post-cell surface receptor level. Enhancement of the effect of agents which directly activate G proteins.

Abstract Over the last few years, several studies showing that production of superoxide by neutrophils in response to chemotactic factors such as FMLP is enhanced after preincubation of the cells with granulocyte-macrophage (GM)-CSF or TNF-alpha have been published. Subsequent reports have indicated that this effect of the cytokines may be mediated by modulation of the number and/or affinity of surface receptors for FMLP. In the present study we have investigated the effect of preincubation with GM-CSF and TNF-alpha on the oxidative burst induced by sodium fluoride and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-agents which directly activate guanine-nucleotide binding proteins in neutrophils. Pretreatment of neutrophils with either GM-CSF or TNF-alpha dose-dependently enhanced the production of superoxide induced by NaF, as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c. Furthermore, preincubation of neutrophils with these cytokines enhanced the production of hydrogen peroxide induced by GTP gamma S in electroporated neutrophils. Because both NaF and GTP gamma S directly activate G proteins independently of external receptor-G protein interaction, these results imply that both GM-CSF and TNF-alpha alter the neutrophil signal transduction pathway in response to subsequent agonists independently of a modulation in the expression of the cell surface receptors for such agonists.</jats:p

Fine-scale genetic diversity and relatedness in fungi associated with the mountain pine beetle

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins, 1902) forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles carry not only multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with nonsignificant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium (Rumbold) Arx possessed a larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera (Rob.-Jeffr. &amp; R.W. Davidson) Zipfel, Z.W. de Beer &amp; M.J. Wingf. and Leptographium longiclavatum S.W. Lee, J.J. Kim &amp; C. Breuil. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats.</jats:p

Fine-scale genetic structure and relatedness in fungi associated with the mountain pine beetle

The mountain pine beetle forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles not only carry multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with non-significant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium possessed larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera and Leptographium longiclavatum. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically-related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Fine-scale genetic diversity and relatedness in fungi associated with the mountain pine beetle

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins, 1902) forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles carry not only multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with nonsignificant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium (Rumbold) Arx possessed a larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. and Leptographium longiclavatum S.W. Lee, J.J. Kim & C. Breuil. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Abstract Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real‐time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non‐target species

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens

Abstract Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed