Monitoring Signal Transduction after Kidney Transplantation

The introduction of calcineurin inhibitors (CNIs) and mycophenolic acid (MPA) to the transplantation clinic improved the outcomes of kidney transplantation. Therapeutic drug monitoring of these immunosuppressive drugs is currently based on a pharmacokinetic approach that measures whole-blood (pre-dose) drug concentrations. Unfortunately, these measurements cannot accurately predict clinical outcomes after kidney transplantation, such as rejection, and have a poor correlation with long-term outcomes after transplantation. Pharmacodynamic monitoring is an alternative and possibly complimentary way for therapeutic drug monitoring that measures the biological effects of the drug at its target site. The research described in this thesis assessed novel techniques for pharmacodynamic monitoring of immunosuppressive therapy after kidney transplantation. Two promising methods are the phospho-specific flow cytometry assay and the intracellular monitoring of NFATc1 amplification. Phospho-specific flow cytometry measures the phosphorylation of signaling molecules involved in T cell activation, such as p-p38MAPK and p-ERK and p-Akt. The assay can be applied to monitor therapeutic drug effects on both T cells and monocytes. The phospho-specific flow cytometry assay also revealed that the currently prescribed immunosuppressive drugs have only a limited effect on intracellular monocyte activation pathways, demonstrating the need for new drugs that target monocytes after kidney transplantation. Another promising method for PD monitoring of tacrolimus in T cells is the analysis of NFATc1 amplification. Measuring NFATc1 amplification is a specific whole-blood test to monitor the biological effects of tacrolimus in T cells of kidney transplant recipients. Both the phospho-specific flow cytometry assay and the NFATc1 amplification assay can measure the activation of signaling molecules in different T cell subsets. In the future, when both assays are ready for routine use we recommend that these assays should be used in combination with pharmacokinetic measurements

Pharmacodynamic Monitoring of Tacrolimus-based Immunosuppression in CD14+ Monocytes after Kidney Transplantation

Background: Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type–specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. Methods: Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14+ monocytes was quantified by phospho-specific flow cytometry. Results: Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range −28% to 77% for p-p38MAPK and 20%; range −22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range −52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = −0.65; P = 0.01 and rs = −0.58; P = 0.03, respectively). Conclusions: Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type–specific manner

Métodos para la investigación con niños : lecciones aprendidas, desafíos y propuestas desde la experiencia de Niños del Milenio en Perú

"El texto al que este capítulo sirve de introducción busca poner en manos de investigadores, docentes, estudiantes y profesionales que trabajan con niños y niñas, un conjunto de herramientas cualitativas para la investigación con niños, las cuales han sido desarrolladas y aplicadas en el Perú a lo largo de dos años, en el marco del estudio internacional Niños del Milenio (conocido internacionalmente como Young Lives). Este ensayo busca así introducir y poner en contexto los métodos y técnicas de recojo de información que se presentan en la segunda parte de este documento, así como el proceso que supuso adaptarlas al lenguaje y convenciones culturales de nuestro medio y las orientaciones éticas que guiaron el trabajo realizado."–página 11.GRADE, Grupo de Análisis para el Desarrollo; Niños del Milenio