Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

BACKGROUND: The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. METHODS: In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR). RESULTS: Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. CONCLUSION: The design of new approaches to identify such markers is warranted

Central CD4+ T cell tolerance: deletion versus regulatory T cell differentiation

The diversion of MHC class II-restricted thymocytes into the regulatory T (Treg) cell lineage, similarly to clonal deletion, is driven by intrathymic encounter of agonist self-antigens. Somewhat paradoxically, it thus seems that the expression of an autoreactive T cell receptor is a shared characteristic of T cells that are subject to clonal deletion and those that are diverted into the Treg cell lineage. Here, we discuss how thymocyte-intrinsic and -extrinsic determinants may specify the choice between these two fundamentally different T cell fates

Overwintering of Ampelomyces mycoparasites on apple trees and other plants infected with powdery mildews

Apple shoots and aerial parts of 13 other plant species infected with powdery mildews during the previous season were collected in late winter and early spring between 1998 and 2003 at a total of 34 sample sites in Hungary. Samples were examined for the presence of overwintering structures of Ampelomyces, common mycoparasites of powdery mildews. Pycnidia and resting hyphae resembling those of Ampelomyces were found on six plant species, including apple. Their viability and subsequent mycoparasitic activity of the hyphae emerging from the overwintered fungal structures were studied in vitro to determine whether they can serve as sources of primary inocula of Ampelomyces in the spring. Overwintered pycnidia of Ampelomyces collected in the spring, and produced in both the ascomata and the conidiophores of powdery mildews during the previous season, initiated the life cycle of these mycoparasites when placed close to fresh powdery mildew colonies in vitro. Similarly, thick-walled resting hyphae, found in the dried powdery mildew mycelia which covered the overwintered aerial parts of the host plants, also germinated and gave rise to new intracellular pycnidia of Ampelomyces when powdery mildew colonies were inoculated with them in vitro. On apple trees, Ampelomyces mycoparasites overwintered as resting hyphae in the dried powdery mildew mycelia covering the shoots and in the parasitized ascomata of Podosphaera leucotricha on the bark and the scales of the buds. Approximately 31% of the field samples collected from apple trees in spring between 1998 and 2003 contained overwintered structures of Ampelomyces. Artificial bursting of apple buds in the laboratory showed that both P. leucotricha and Ampelomyces start their life cycle during or soon after bud burst, but Ampelomyces can only slowly follow the spread of its mycohost on infected leaves. Most probably, the mycoparasites did not overwinter in the dormant hyphae of P. leucotricha in the buds, but only on the bark and the bud scales, as their hyphae were not found in the young hyphae of apple powdery mildew that appeared on the leaf tissues during bud burst. This study demonstrated that Ampelomyces mycoparasites can survive the winter in the field as pycnidia and as resting hyphae in the dried mycelia of their mycohosts