A noninvasive assay for monitoring renal allograft status

Transplant rejection is a serious complication, sometimes threatening life of the patient. Although recent development of the new generation of immunosuppressive drugs reduced the incidence of acute rejection in kidney transplantation, the absence of noninvasive biomarkers of the rejection does not allow often the optimization of a prompt antirejection therapy. Serum creatinine is the most widely used marker for allograft function, however, it is not sensitive and specific enough to detect acute rejection. Other biomarkers are even less valuable for this purpose. Histological examination of renal allograft biopsy still remains the golden standard for diagnosing acute renal allograft rejection. Therefore, there is a high demand for reliable biomarkers for noninvasive monitoring of renal allograft status. Examination of urine in renal transplant recipients provides a logical and readily accessible approach for this monitoring. The high potency biomarkers for kidney allograft monitoring are fragments of DNA in recipient urine that originated from renal allograft cells. Because of the difference in the genetic origin these DNA can be distinguished from recipient DNA. Quantitative analysis of donor’s DNA, derived from cells of renal allograft, in recipient’s urine might be a reliable predictive tool for the kidney transplant rejection. We developed an assay to quantitate donor DNA content in recipient urine. Application of the technique—coamplification at lower denaturation temperature-PCR (COLD-PCR) increased the abundance of donor DNA that usually presents in recipient urine in quantities that are out of the detection range. This assay has a potential for routine application in clinical practice after statistical validation and additional modifications.

A stepwise procedure to test contractility and susceptibility to injury for the rodent quadriceps muscle

In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies.  Biological markers can also be used in measuring muscle injury, such as increased creatine kinase levels in the blood, but these are not always correlated with loss in muscle function (i.e. loss of force production).  This is even true of histological findings from animals, which provide a “direct measure” of damage, but do not account for loss of function.  The most comprehensive measure of the overall health of the muscle is contractile force.  To date, animal models testing contractile force have been limited to the muscle groups moving the ankle.  Here we describe an in vivo animal model for the quadriceps, with abilities to measure torque, produce a reliable muscle injury, and follow muscle recovery within the same animal over time.  We also describe a second model used for direct measurement of force from an isolated quadriceps muscle in situ.

Method for the quantitative measurement of collecting lymphatic vessel contraction in mice

Collecting lymphatic vessels are critical for the transport of lymph and its cellular contents to lymph nodes for both immune surveillance and the maintenance of tissue-fluid balance. Collecting lymphatic vessels drive lymph flow by autonomous contraction of smooth muscle cells that cover these vessels. Here we describe methods using intravital microscopy to image and quantify collecting lymphatic vessel contraction in mice. Our methods allow for the measurement of the strength of lymphatic contraction of an individual lymphangion in a mouse, which has not yet been demonstrated using other published methods. The ability to study murine collecting lymphatic vessel contraction—using the methods described here or other recently published techniques—allows the field to dissect the molecular mechanisms controlling lymphatic pumping under normal and pathological conditions using the wide variety of molecular tools and genetic models available in the mouse. We have used our methods to study lymphatic contraction in physiological and inflammatory conditions. The methods described here will facilitate the further study of lymphatic function in other pathological conditions that feature lymphatic complications