Testing lupus anticoagulants in a real-life scenario - a retrospective cohort study

Introduction: Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients. Materials and methods: In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test. Results: The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 – 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests. Conclusions: Among the parameters evaluated, aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics

Inhibition of Collagen XVI Expression Reduces Glioma Cell Invasiveness

Background/Aims: The effects of a 10% α-hydroxy acid (AHA) oil/water (O/W) emulsion on the pH of human skin surface (pHss) and stratum corneum (SC; pHsc) were evaluated in vivo. Methods: The AHA O/W emulsion was applied to an area on the volar forearm of male volunteers (n = 12), and then wiped off after 10 min. Prior to application and over the following 3 h, the pHss was measured. We used glass electrode measurements and time domain dual lifetime referencing (tdDLR) with luminescent sensor foils. In another experiment (n = 5), the impact of the AHA O/W emulsion on the pHsc gradient was measured by tape stripping of the SC of the volar forearm after application of the AHA O/W emulsion. Results: Compared with pHss values prior to treatment [5.2 ± 1.7 (tdDLR) or 4.8 ± 0.5 (electrode)], the pHss was significantly reduced 10 min after application [4.0 ± 0.3 (tdDLR) or 4.1 ± 0.1 (electrode)] and the pHss remained significantly reduced over the measurement period of 3 h [after 3 h: 4.4 ± 0.2 (tdDLR) or 4.5 ± 0.3 (electrode)]. The AHA O/W emulsion significantly reduced the pHsc even down to deep layers of the SC. Conclusion: After a 10-min application time, the 10% AHA O/W emulsion reduces the pHss (for at least 3 h) and pHsc in deep layers of the SC

Studies on the molecular interactions of collagen type XVI. Part I: The role of collagen XVI in pathological disorders Part II: Extablishment of a retroviral mediated gene silencing model

Molecular interactions of collagen XVI in Crohn’s disease In Crohn’s disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF), provided by intact tissue is disturbed due to inflammation and cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original extracellular matrix (ECM) leads ISEMF to exit this cycle, however, in fibrosis ISEMF remain. It is known that ISEMF produce and deposit collagen types I, III, IV and V; but synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and the effect of collagen XVI on cell proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall and ISEMF reveal increasing gene and protein expression of collagen XVI with concurrent increasing inflammation. ISEMF display more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This might involve recruitment of α1 integrin, since its cell surface expression on ISEMF is increased in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of α1ß1 integrin into the focal contacts. Mechanistical studies of collagen XVI by a retroviral mediated knockdown model in murine fibroblasts Collagen XVI, a member of FACIT collagens (fibril associated collagens with interrupted triple helices) is described as macro-molecule of the ECM. Very little is known about its role in cell-matrix-interactions and in cell signalling. However it has been demonstrated that cells interact with collagen XVI via integrin α1β1 and α2β1. These interactions presumably determine organization of extracellular components and their communication with cells. We have established a retroviral mediated collagen XVI knockdown in NIH3T3 fibroblasts to investigate the role of collagen XVI in cell-matrix interactions. Specific shRNAs against collagen XVI were utilized in parallel to a viral luciferase vector construct as control. After successful transduction, positive cell clones were further selected by antibiotic resistance. Knockdown efficiency was determined on mRNA and protein level and further downstream experiments were performed with respect to adhesion and proliferation. Differential protein expression in knockdown cells was compared to the control by 2D-gelelectrophoresis. The knockdown resulted in 80-90 % inhibition of collagen XVI gene and protein expression and mass spectrometry revealed several differentially expressed proteins. Collagen XVI inhibited cell lysates showed a lack of macrophage migration inhibitory factor (MIF) and prolylpeptidyl-cis-trans-isomerase A (PPIA). The gene expression of these proteins was slightly up-regulated, however, western blot analysis confirmed 2D-gelelectrophoresis results. Proliferation of knockdown cells was generally reduced and was not influenced by the presence of collagen XVI as cell culture substrate. The cytokine MIF influences migration and proliferation of fibroblasts during wound healing. MIF induces synthesis of collagens in fibroblasts (collagen I, III, IV, V and VI) whereas PPIA contributes to correct protein folding in the cytoplasm of cells. Collagen XVI acts as adapter molecule in organizing suprastructures. Therefore, we assume that the lack of collagen XVI detains fibroblasts in arranging fibrillar stuctures which results in disturbed initial adhesion. The amelioration of cellular adhesion to the available matrix indicates a compensation in integrin expression pattern. Reduced proliferation together with decreased MIF expression hints at changes in the differentiation stage and has to be further elucidated

Collagen XVI is a neural component of the developing and regenerating dorsal root ganglia extracellular matrix.

International audienceCollagen XVI is a homotrimeric molecule harbouring similarities to the FACIT (fibril-associated collagens with interrupted triple helices) family of collagens (Grassel et al., 1996). Collagen XVI is expressed in skin and cartilage where it is integrated into tissue specific aggregates (Grassel et al., 1999; Kassner et al., 2003). In the nervous system, collagen XVI has been detected at lowlevel in the brain and a strong expression was also reported in spinal root fibers during development (Lai and Chu, 1996). In dorsal root ganglia (DRG), analysis of SAGE banks performed by our group during development and after nerve injury (Mechaly et al., 2006) shows a fluctuation of collagen XVI expression between the different conditions and prompted us to study it further. DRGs contain the cell bodies of neurons, the axons of which transmit sensory information from the periphery to the central nervous system. While it is well known that during development and regeneration, neurites require extracellular matrix molecules for growth and guidance (Hari et al., 2004), the composition and the role of the matrix surrounding neurons in the ganglia itself have solicited little interest. Here, we show that collagen XVI is a component of the developingDRG extracellular matrix, that following nerve injury, its expression is increased around neuronal cell bodies and that neurons express collagen XVI in the peripheral nervous system