Schulze Diabetes Institute (SDI), Department of Surgery
University of Minnesota
Plasma microRNA-375 – A single test to quantitate injury to porcine islet grafts and to diagnose accelerated porcine islet graft loss in nonhuman primates?
Raza Naqvi1, Wilma Suarez-Pinzon1, Amar Singh1, Chris Burlak1, Lihua Li1, Subbaya Subramanian1, Melanie L Graham1, Bernhard J Hering1.
1Department of Surgery,, University of Minnesota,, Minneapolis, MN, United States
Non Human Primates (NHP).
Background: MicroRNA-375 (miR-375) has been identified as a novel marker of mouse and human islet beta cell death. The immediate innate immune response to intraportal adult porcine islet (API) transplants (tx) varies substantially between nonhuman primates (NHP) and can cause accelerated and complete graft loss. We aimed to determine the utility of plasma miR-375 as a single test to diagnose accelerated API graft loss in NHP.
Methods: To study whether miR-375 is a marker also of API death, we cultured API aliquots from 2 preps in the presence of 0.1 mM H2O2 for 48 hrs. We also cultured API aliquots from 3 preps for 7 days in the presence of human PBMC from 3 donors. Finally, we analyzed plasma samples obtained from 6 NHP at 0, 5, 15, 30, and 60 min and at 7 and 14 days after intraportal tx of API (25 KIE/kg). All NHP received the same T- and B-cell directed immunosuppression but neither complement- nor coagulation-inhibiting agents (except for heparin). The expression of miR-375 in culture supernatants and plasma samples was determined by qRT-PCR and fold changes in expression were calculated by the ∆∆Ct method using miR-16 as an endogenous control. All fold changes were normalized to changes in islet alone cultures or in in vivo studies to pre-tx samples in each recipient.
Results: In-vitro incubation of API in the presence of H2O2 caused a 77±2.7% drop in islet viability as determined by an AO/PI assay; the corresponding miR-375 levels in supernatants increased 27 ± 4.2 fold. Co-culture of API and human PBMC for 7 days reduced islet viability by 55.5±1.1% compared with high viability in API alone cultures (90.7 ± 3.5%); miR-375 levels in supernatants of API and PBMC increased 15.03 ± 3.2 fold compared to those in supernatants of API alone (p<0.001). In-vivo, in 4 NHP that achieved prolonged insulin independence post-tx, miR-375 increased within the first 60 min post-tx 119-, 136-, 324-, and 4,329-fold, respectively. By day 7, the fold increases compared to pre-tx in miR-375 plasma levels in 3 of these 4 NHP were below 2-fold, suggesting rapid clearance of miR-375 in the absence of ongoing injury. In the 2 NHP that failed to achieve insulin independence, the miR-375 plasma levels increased 6,700 and 9,280-fold; in 1 of these 2 the plasma levels remained elevated. The fold increases in miR-375 within 60 min post-tx, calculated as AUC, were significantly lower in the 4 NHP that achieve insulin independence (21.9 ± 30.7 vs 156.5 ± 71.9; p=0.0254).
Conclusion: The in-vitro data indicates that miR-375 is a marker of porcine islet death. Our preliminary in-vivo results suggest that a massive increase in plasma miR-375 within 60 min of intraportal tx signals substantial injury to API and is diagnostic of accelerated and irreversible graft loss. Additional data is needed to determine the precise diagnostic threshold for instant post-tx islet graft loss. Equally important will be whether lower fold changes in miR-375 denote early post-tx graft injury and correlate with engrafted and functional islet mass. The utility of this assay in monitoring adaptive immunity to porcine islet tx may be limited by the rapid clearance of miR-375 from the plasma and the current sensitivity of our detection methods.
11:00 - 12:30
|Islet Xenotransplantation||Plasma microRNA-375 – A single test to quantitate injury to porcine islet grafts and to diagnose accelerated porcine islet graft loss in nonhuman primates?||Room 109|