Rapid evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos.

Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p < 0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.

Mass spectrometric analyses of CL(39), CL(41) and H(1), H(2), H(3) confirm identity with fetidin and lysenin produced by earthworm leukocytes.

Antibacterial proteins realize effective immunological mechanisms against invading pathogens. Some of them exert hemolytic and agglutinating properties. Here, we analyzed two hemolysins isolated from cell lysate (CL(39) and CL(41)) and three hemolytic proteins isolated from coelomic fluid (H(1), H(2) and H(3)) of the annelid Eisenia fetida using mass spectrometry and bioinformatics. We demonstrated the identity of CL(39,41) with fetidin and lysenin; these have been described earlier. H(1-3) share sequence components with fetidin but they seem to be glycosylated as shown for H(1). The results help to resolve a long debate concerning nomenclature and identity of these hemolytic proteins. They support: (1). the concept that the hemolytic proteins originate from chloragocytes; (2). their origin to some extent from large coelomocytes; and (3). the view that they are secreted into CF.

Hexadecylphosphocholine selectively upregulates expression of intracellular adhesion molecule-1 and class I major histocompatibility complex antigen in human monocytes.

U 937 cells are widely used as a model system to study human monocytes, since they express typical human monocyte markers and properties. Hexadecylphosphocholine (HPC) is the main representative of a class of synthetic phospholipids, the alkylphosphocholines (APCs), and is able to form stable multilamellar vesicles (MLVs = liposomes) to deliver HPC to monocytes/macrophages. Here we report the ability of both micellar and liposomal HPC (L-HPC) to interact with human monocytes and upregulate specific adhesion molecules. Whereas CD14 could neither be induced by HPC nor by L-HPC on U 937 cells, intracellular adhesion molecule-1 and class 1 major histocompatibility complex (MHC-1) antigen were upregulated by both HPC and L-HPC in a dose-dependent manner. These data support and complete previous studies on HPC-induced activation of U 937 cells and provide additional mechanistic information on the initial steps of HPC-mediated recruitment of macrophages and their antitumor activity.

S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells.

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.