Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation.

The differentiation of human CD4(+) T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel-based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC-MS/MS is required to provide a reference dataset for proteome-based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 (http://proteomecentral.proteomexchange.org/dataset/PXD001066).

Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.

Monoclonal antibody-polyethyleneimine conjugates targeting Her-2/neu or CD90 allow cell type-specific nonviral gene delivery.

We have developed an efficient and cell-specific nonviral gene delivery system using monoclonal antibodies (mAb) coupled with branched 25-kDa polyethyleneimine (PEI). The system was evaluated for two model antibodies with different well-characterized antigen specificities: the mouse anti-human IgG1 mAb AS02 recognizing human CD90 (hThy-1) which is expressed on human fibroblasts, and the humanized anti-Her-2/neu mAb Trastuzumab recently introduced for the treatment of Her-2/neu-positive breast cancer. Efficacy and selectivity of gene delivery were evaluated for covalent mAb-PEI conjugates coupled with N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP) or N-succinimidyl-4-(maleimidomethyl)-cyclohexancarboxylate (SMCC), or, as a newly introduced coupling reagent, noncovalent complexes of mAb with 3-(2-(2-(vinylsulfonyl)ethylthio)ethyl)quinazoline-2,4(1H,3H)-dione (IBFB 110001) coupled to PEI. An enhanced green fluorescent protein (EGFP)-expressing reporter plasmid was used to monitor transfection efficiencies of cell lines expressing different levels of hCD90 or Her-2/neu. While mAb-PEI conjugates coupled with SPDP resulted in antigen-nonspecific EGFP expression, conjugates coupled with SMCC or IBFB 110001 enabled antigen-specific gene delivery. Thus, Her-2/neu-PEI conjugates prepared with IBFB 110001 allowed to transfect 23+/-2% of Her-2/neu-positive SKOV-3 versus 0.5+/-2% of Her-2/neu-negative MB-468 cells. Proof of principle for specific antibody-mediated gene transfer was demonstrated by saturating the Her-2/neu receptor with free anti-Her-2/neu, thereby blocking subsequent transfection with anti-Her-2/neu-PEI/DNA complexes.

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3.

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.