Serial Peripheral Blood Perforin and Granzyme B Gene Expression Measurements for Prediction of Acute Rejection in Kidney Graft Recipients

Abstract

In the present study we investigated whether peripheral blood gene expression measurements may serve as an early and non-invasive tool to predict renal allograft rejection. Peripheral blood was collected twice weekly after transplantation and gene expression was measured using real-time polymerase chain reaction (PCR). Recipients with acute rejection (n = 17) had higher levels of perforin and granzyme B transcript on days 5-7, 8-10, 11-13, 17-19, 20-22, and 26-29, as compared to patients without rejection (n = 50, p < 0.05 in all cases). Rejection diagnosis using gene expression criteria, determined with receiver operating characteristic (ROC) curves, was possible 2-30 days before traditional diagnosis (median 11 days). The best diagnostic result was obtained from samples taken on days 8-10, with a specificity of 90% and a sensitivity of 82% for perforin, and a specificity of 87% and sensitivity of 72% for granzyme B. Decreases in perforin (p < 0.01) and granzyme B expression (p < 0.05) were observed after initiation of anti-rejection therapy. Our data indicate that gene expression measurement is a useful tool for the recognition of graft rejection in its earliest stages. Serial measurements could be implemented as a monitoring system to highlight patients at higher risk of rejection, making them candidates for biopsy or pre-emptive anti-rejection therapy.

Regulatory, Effector, and Cytotoxic T Cell Profiles in Long-Term Kidney Transplant Patients

“…For this molecule, measuring the mRNA appeared more sensitive than measuring the protein. We additionally show for the first time that, in contrast to acute rejection where GrzB is increased in the PBMC, 16,17,26 during CAMR GrzB is significantly decreased. Furthermore, ROC analysis revealed that measuring GrzB mRNA may have potential as a decision-making tool in clinical practice to diagnose CAMR in a minimally invasive manner.…”

“…Indeed, several groups have also clearly established the cytotoxic molecule granzyme B (GrzB) as a reliable intragraft, urine, and peripheral blood marker of acute kidney graft rejection. [12][13][14][15][16][17][18] Thus, although the implication of regulatory and effector responses has been addressed for AR, so far there have been no reports on the subject relative to later times posttransplant in cases of stable graft function or late renal allograft injury, such as calcineurin inhibitor (CNI) toxicity or chronic antibodymediated rejection (CAMR). Therefore, the potential involvement of FOXP3ϩ cells and any relationship to clinical and pathological correlates at later times posttransplant remain unknown.…”

The Role of CTLs in Persistent Viral Infection: Cytolytic Gene Expression in CD8+ Lymphocytes Distinguishes between Individuals with a High or Low Proviral Load of Human T Cell Lymphotropic Virus Type 1

“…Granzyme mRNA expression, usually assayed by qPCR, is increasingly used as a correlate of acute rejection of allogeneic transplants, in which CTLs play a critical role. Increased granzyme mRNA levels can be detected during rejection both in the graft itself (58,59), in the blood (60,61), and even in the urine (62). Measurement of granzyme mRNA is preferable to immunostaining of granzyme proteins as a correlate of CTL activity, because the granzyme proteins do not accumulate in the cell but are rapidly and continually discharged in the lytic granules.…”

Human Keratinocytes Acquire Cellular Cytotoxicity under UV-B Irradiation

“…The cDNA was synthesized using random hexamers and oligo(dT) from 4 g of mRNA and performed with the SuperScript TM First Strand Synthesis System for RT-PCR (Invitrogen). Forward and reverse primers were designed following Guilloton et al (25) for GrB and following Simon et al (26) for PFN (Table 1). Real-time PCR was performed using an iCycler thermal cycler (Applied Biosystems 7000 Real-Time PCR System, Courtaboeuf, France) according to the manufacturer's instructions.…”