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Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application.

Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

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Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival. Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13-/- mice. Chemotactic properties of A. fumigatus -activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm). Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus -activated serum of wildtype and knock-out mice. Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections. Disclosures No relevant conflicts of interest to declare.