Novel clearance of muscle proteins by muscle cells

Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells

Measurements of the DNA double-strand break response

Radiotherapy and some chemotherapeutic drugs kill cancer cells by induction of the extremely toxic DNA double-strand breaks (DSBs). Measurements of the DSB response in patients during therapy could allow personalized dosing to improve tumor response and minimize side effects. DSBs induce a strong cellular response via phosphorylation of H2AX, P-H2AX and the formation of foci. P-H2AX can be measured by flow cytometry or counted in separate cell nuclei by immunofluorescence microscopy. The overall aim of my research has been to develop and validate methods to measure P-H2AX in mononuclear cells from cancer patients undergoing radiotherapy. Initially, we wanted to characterize DNA damage induced by the chemotherapeutic drug etoposide that induces TopoII-linked DSBs and to test its ability to activate the P-H2AX response using a flow cytometry-based P-H2AX assay. We found that only 0.3% of etoposide-induced DSBs activated the H2AX response and toxicity. We concluded that the P-H2AX response was a good measure of the toxic effects of etoposide. Next, we wanted to optimize the flow cytometry assay for mononuclear cells from cancer patients undergoing radiation therapy. The P-H2AX response was measured before and after 5Gy pelvic irradiation and in in vitro-irradiated controls. We found a fraction of cells with high P-H2AX signals that corresponded to the 5Gy in vitro-irradiated blood controls. This study indicated that flow cytometry may be well suited for measurements of the P-H2AX response in mononuclear cells following local radiotherapy. To be able to implement the P-H2AX assay in clinical practice and use it in relation to clinical outcome and side effects we have also developed stable and reliable calibrators based on phosphopeptide-coated beads and fixed cells. Using these calibrators it could be possible to use the P-H2AX flow cytometry assay in the clinic in a controlled manner. Finally, using immunostaining in solution before cells are mounted on microscopic slides for quantification of single P-H2AX foci by immunofluorescence, we have the possibility to analyze 16 patient samples within few hours, which makes this method suitable for clinical use

The Liver and Kidneys mediate clearance of cardiac troponin in the rat

Cardiac-specific troponins (cTn), troponin T (cTnT) and troponin I (cTnI) are diagnostic biomarkers when myocardial infarction is suspected. Despite its clinical importance it is still not known how cTn is cleared once it is released from damaged cardiac cells. The aim of this study was to examine the clearance of cTn in the rat. A cTn preparation from pig heart was labeled with fluorescent dye or fluorine 18 (F-18). The accumulation of the fluorescence signal using organ extracts, or the 18F signal using positron emission tomography (PET) was examined after a tail vein injection. The endocytosis of fluorescently labeled cTn was studied using a mouse hepatoma cell line. Close to 99% of the cTnT and cTnI measured with clinical immunoassays were cleared from the circulation two hours after a tail vein injection. The fluorescence signal from the fluorescently labeled cTn preparation and the radioactivity from the 18F-labeled cTn preparation mainly accumulated in the liver and kidneys. The fluorescently labeled cTn preparation was efficiently endocytosed by mouse hepatoma cells. In conclusion, we find that the liver and the kidneys are responsible for the clearance of cTn from plasma in the rat