Quantitative Polymerase Chain Reaction Profiling of Immunomarkers in Rejecting Kidney Allografts for Predicting Response to Steroid Treatment

Abstract

Differences in intragraft expression profiles reflect variability in the response to antirejection treatment. In acute rejection, molecular markers, particularly those reflecting T-cell activation, offer superior prognostic value compared with conventional parameters.

Increased Metallothionein Expression Reflects Steroid Resistance in Renal Allograft Recipients

“…To investigate how the multivariate MT‐1 model (MT‐1, CYP4A11, TIMP1 and F2R) relates to the multivariate T cell mediated model from our previous study (CD25:CD3e ratio and LAG‐3) , we combined the discovery and validation cohorts. The predictive value of the MT‐1 model was slightly higher (AUC = 0.81, p = 0.000002; Figure ) than the predictive value of the T cell activation model (AUC of 0.79, p = 0.000007; Figure ).…”

“…Only patients receiving pulse therapy with methylprednisolone (3 days with 1 g bolus) as anti‐rejection treatment were included. The primary clinical endpoint was response to anti‐rejection treatment with methylprednisolone, as described previously . Steroid resistant acute rejection was defined as lack of clinical response to methylprednisolone, and a requirement for ATG treatment within 14 days after the start of the steroid therapy.…”

“…Synthesis of cDNA, primer design, and quantitative PCR (qPCR) analysis were performed according to previously described protocols . An average of 0.92 ± 0.15 µg of RNA from each frozen biopsy core was transcribed into cDNA.…”

“…). Recently, we showed that the combination of T cell activation markers CD25:CD3 ratio and LAG‐3 offers a superior prognostic value for assessing steroid response, compared with conventional parameters . We observed considerable molecular heterogeneity among the biopsy samples with acute rejection, underlining the complexity of the mechanisms involved in response to steroid therapy.…”

Simvastatin reduces CCL2 expression in monocyte-derived cells by induction of a repressive CCL2 chromatin state

“…From 1 lg of total RNA, cDNA was synthesized using SuperScript III (Invitrogen), with 250 ng random hexamers (Promega) according to manufacturer's instructions. Quantitative PCR for CCL2 was performed in duplicates on a My IQ Cycler (BioRad) with SYBR Green mix (BioRad 170-8887) using the following primer pair: forward: 5 0 -TGTGCCTGCTGCTCA-TAG-3 0 and reverse: 5 0 -CTTGCTGCTGGTGATTCTTC-3 0 , as described by Rekers and colleagues [22]. Relative transcript levels of CCL2 RNAs were calculated with the comparative Ct method and related to three reference genes by using the geometric mean of their expression.…”