INCREASED GRANZYME B AND RANTES mRNA IN REJECTING LUNG ALLOGRAFTS

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TITLE: INCREASED GRANZYME B AND RANTES mRNA IN REJECTING LUNG ALLOGRAFTS– QUANTITATIVE ANALYSIS BY TAQMAN

Kathy Spichty,¹ Alin Girnita,¹ Ken McCurry,² Aldo Iacono,³ James Dauber,³ Gill Burckart,⁴ Adriana Zeevi.¹

¹Pathology, University of Pittsburgh; ²Surgery; ³Medicine; ⁴Pharmacy, University of Pittsburgh, Pittsburgh, PA

The gold standard for diagnosis of lung rejection is the histological evaluation of transbronchial biopsies. The detection of pro–inflammatory cytokines with competitive PCR has also been correlated with ACR. In this study, we evaluated the TaqmanTM based real time PCR for the determination of granzyme B (GB a cytolytic effector molecule), RANTES (a chemokine for activated T cells and monocytes) and IL–15 (T cell growth factor). Real time PCR was carried out on 35 bronchoalveolar lavage (BAL) samples from 12 lung transplant recipients. We used DNA–free RNA, gene–specific primers and SYBR Green PCR reagents and ABI PRISM 7700 sequence detection system (Applied Biosystems Inc.). Each assay was done in triplicate and human 18s rRNA was simultaneously amplified as an internal control for data normalization. The 7700 Sequence Detection Software was used for instrument control, automated data collection and data analysis. The 2–ddCt methods were used for data analysis. In 10 patients who experienced AC, we determined the cytokine gene expression sequentially before rejection (quiescence), during and after treatment of rejection. The gene expression of GB and RANTES increased significantly (p = 0.001) during rejection in comparison to BAL samples from non–rejecting transplant recipients. The mean Ct for GB during rejection was 14.84+1.96 (10 BAL samples) while during quiescence (20 BAL samples) was significantly higher 18.7+1.6, indicating about 4 cycles less during rejection. A similar pattern was observed for RANTES (the mean Ct for rejection 13.23+ 1.95 vs. no rejection 16.38+1.69). In contrast, the levels of IL–15 did not change significantly throughout the post–transplant follow–up (mean Ct for rejection 13.79+1.81 vs. no rejection 15.34+2.05). We propose to use this new TaqmanTM based real PCR analysis to monitor the expression of pro–inflammatory cytokines in BAL samples during rejection and for the determination of effectiveness of immunosuppressive treatment.