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Reutlingen, Germany

Schneiderhan-Marra N.,Reutlingen University | Schneiderhan-Marra N.,University of Ulm | Sauer G.,University of Ulm | Kazmaier C.,Reutlingen University | And 5 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated. © 2010 Springer-Verlag.

Herberth M.,University of Cambridge | Rahmoune H.,University of Cambridge | Schwarz E.,University of Cambridge | Koethe D.,Central Institute of Mental Health | And 9 more authors.
Clinical Schizophrenia and Related Psychoses | Year: 2014

Objectives: Alterations in immunological parameters have been reported for schizophrenia although little is known about the effects of inflammatory status on immune-related functional changes at disease onset. Here, we have investigated such T cell-dependent molecular changes in first-onset, antipsychotic-naive schizophrenia patients using a novel ex vivo blood culture system. Methods: Blood samples from patients (n=17) and controls (n=17) were collected into stimulant-containing or null control TruCulture™ tubes, incubated 24 hours and the concentrations of 107 immune and metabolic molecules measured in the conditioned media using the HumanMAP™ immunoassay system. Results: Nine molecules showed altered release from schizophrenia blood cells compared to those from controls and this was replicated in an independent cohort. In silico pathway analysis showed that these molecules had roles in endothelial cell function, inflammation, acute phase response and fibrinolysis pathways. Importantly, five of these molecules showed altered release only after stimulation. Conclusions: This study has identified a reproducible peripheral molecular signature associated with altered immune function in first-onset schizophrenia subjects. This suggests that immune status can affect the biomarker profile which could be important for personalized medicine strategies. Furthermore, whole blood culture analysis may be useful in the identification of diagnostic tools or novel treatment strategies due to ease-of-use and clinical accessibility.

Slow-healing wounds benefit considerably from treatment with platelet-rich plasma (PrP). The drawback of using PrP is its laborious preparation, which requires expensive technical equipment (centrifuges) and well-trained personnel. Methods. The authorsĝ€™ new method overcomes these issues and provides the practitioner with an innovative tool to freshly prepare a platelet mediator mix with PrPĝ€™s known biological activities, but is much simpler to obtain. This is achieved by employing the sedimentation of a blood sample at regular gravity (no centrifuge necessary) in the presence of an anti-coagulant and a sedimentation accelerator. Thereafter, the supernatant containing the platelets is concentrated on a unique filter, which causes these platelets to release their mediators (different biologically active molecules resembling the substance mix that is released by the platelets upon degranulation). This solution is eluted from the filter, providing a sterile-filtered, enriched fraction of biologically active mediators (TGF-2, PDGF, IGF-1, etc.), most of which are active in wound healing disorders. Results. This preparation triggers in-vitro proliferation of fibroblasts and osteoblasts, the secretion of IL-6 by osteoblasts, and differentiation of fibroblasts into cells with an endothelial morphology resembling cells during angiogenesis. Conclusion. By providing the practioner a sterile concentrate of a whole range of autologous platelet mediators within 1 hour, this new method has the potency to become a substitute of PrP in wound-healing therapy. PMC (platelet mediator concentrate) eases the manufacturing of such preparations, thereby making them not only more widely applicable, but also reducing treatment costs.

Rimmele S.,University of Tubingen | Gierschik P.,University of Ulm | Joos T.O.,University of Tubingen | Joos T.O.,EDI GmbH | Schneiderhan-Marra N.,University of Tubingen
Journal of Molecular Recognition | Year: 2010

Bead-based interaction assays are excellently suited to study protein-protein interactions, as they require only minimal amounts of sample material. Miniaturized protein-protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK). Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase-Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis. Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1. © 2010 John Wiley & Sons, Ltd.

Albrecht A.,University of Cambridge | Rahmoune H.,University of Cambridge | Leedjarv K.,Storkbio Ltd. | Knorpp T.,EDI GmbH | And 5 more authors.
Proteomics | Year: 2013

We describe the development of a novel, robust assay system for determining the changes in activity of proprotein converting enzymes. An assay for prolyl oligopeptidase (POP) activity was constructed using a peptide-streptavidin substrate coupled to magnetic microspheres and cleavage was detected by loss of streptavidin on the MAGPIX reader. Test analysis of postmortem pituitary extracts from schizophrenia patients showed an increase in POP activity compared to controls. The results were validated using both fluorometric and Western blot analyses for POP activity and immunoreactivity, respectively. The assays can be multiplexed for measuring the activity of multiple proprotein cleaving enzymes simultaneously in laboratory and clinical settings and should add valuable new information for conditions such as neuropsychiatric diseases, diabetes, endocrine dysfunction, and cancer, where effects on proteolysis of biologically active peptides play a key role.© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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