Development of a Quantitative Assay for the Characterization of Human Collectin-11 (CL-11, CL-K1)

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29–18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2–459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan

Erratum: Corrigendum: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

International Chicken Genome Sequencing Consortium. The Original Article was published on 09 December 2004. Nature432, 695–716 (2004). In Table 5 of this Article, the last four values listed in the ‘Copy number’ column were incorrect. These should be: LTR elements, 30,000; DNA transposons, 20,000; simple repeats, 140,000; and satellites, 4,000. These errors do not affect any of the conclusions in our paper. Additional information. The online version of the original article can be found at 10.1038/nature0315

Sodium Polyanethole Sulfonate as an Inhibitor of Activation of Complement Function in Blood Culture Systems ▿

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway

Molecular cloning, characterisation, and tissue distribution of oestrogen receptor alpha in eelpout (Zoarces viviparus).

International audienceA cDNA encoding the eelpout (Zoarces viviparus) oestrogen receptor alpha (eERalpha) has been isolated from eelpout liver, cloned and sequenced. The cDNA contains a complete open reading frame encoding 570 amino acid residues (mw: 63.0 kDa). The amino acid sequence of eERalpha showed a high degree of identity to ERalpha of other teleost species. The tissue distribution of eERalpha mRNA was examined using Northern blotting, RT-PCR and in situ hybridisation (ISH). All three methods identified a pronounced expression of eERalpha in liver, pituitary, testis and ovary. In the brain ISH experiments showed that ERalpha mRNA was highly expressed in distinct regions of the preoptic area and the mediobasal hypothalamus. We have provided evidence that the receptor is auto-regulated by 17beta-oestradiol (E(2)) not only in liver but also in the testis, indicating an important role for E(2) during spermatogenesis in male eelpout. RT-PCR analysis showed a broader expression pattern including significant expression in the brain, kidney, heart, and gut of adult eelpout. In eelpout embryos eERalpha expression has also been identified, indicating a possible role for the receptor in early development. This study contributes to the accumulating evidence that in fish E(2) is not only involved in the regulation of liver specific proteins, but has a much broader range of targets

Cell-autonomous Hedgehog signaling controls Th17 polarization and pathogenicity

Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.This work was supported by Cancer Research UK (MdlR (A22257), FB, LMOB, CK, H-CC, VC); Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (MdlR (WT107609); LMOB); Gates Cambridge Trust (AK); JH is undertaking a PhD funded by the Cambridge School of Clinical Medicine, Frank Edward Elmore Fund and the Medical Research Council’s Doctoral Training Partnership (award reference: 1954837). TA is grateful for the support from the Austrian Science Fund (FWF P33070) and the European Research Council (ERC – STG: 101039320)