Update on the Provisional Estimation of Developing Iatrogenic Variant Creutzfeldt-Jakob Disease From Human Islet Cell Transplantation Procedures (2014)

Dear Editor Improvements to the method of transplanting human islet cells into adult patients with type 1 diabetes have reduced the effects of the disease to recipients. The donor islet cells are implanted into the livers of patients through a relatively simple outpatient procedure called the Edmonton Protocol (1). The success of the procedure relies on a high yield of islet cells obtained from gentle tissue dissociation using Liberase, a purified enzyme blend of collagenase (isoforms I and II from Clostridium histoliticum and thermolysin from Bacillus thermoproteolyticus) (Roche Applied Science, Basel, Switzerland) (2). Previously, C. histoliticum was grown in fermentation media consisting of brain heart infusion and gelatin. The source of brain heart infusion was bovine brain and porcine heart, whereas the gelatin was of porcine origin. Concerns were raised that the preparation method for islet cells may potentially put recipients at risk for iatrogenic transmission of variant Creutzfeldt-Jakob disease (vCJD) because enzyme components used to prepare islet cells for transplantation may have been exposed to infective cattle brain materials during production (3, 4). vCJD is a rare, fatal human neurodegenerative disease characterized by progressive dementia. Only a small number of vCJD cases have been attributed to iatrogenic transmission (5, 6). Moreover, the risk of vCJD from islet transplants may be increased because patients receive multiple rounds of islet cell infusions over time. Since the research described in this communication was performed, another method of collagenase preparation was developed and implemented using plant-based growth media; this alternative collagenase product is currently in clinical use. Prion protein is highly expressed in neurons and all endocrine cells of fetal and adult pancreatic islet cell types (7). Experimental diabetes-prone rodent model systems also report ubiquitous, high expression levels of cytosolic cellular prion protein aggregates in beta cells (8). Prion diseases progress by prion misfolding, but the exact mechanism and conditions involved remains to be fully characterized (9). It is unknown whether islet cells with cytosolic cellular prion protein aggregates provide a greater mechanistic risk for misfolding and disease propagation if contaminated with prion protein (scrapie isoform) (PrPSc). Thus, endocrine pancreatic cells are of particular concern as a possible increased iatrogenic transmission risk route during islet cell transplantation procedures. Previously, the level of iatrogenic vCJD risk from islet cell transplant procedures was estimated using a theoretical, quantitative risk assessment. The potential risk of exposure to contaminated prions per islet cell procedure was estimated at one in one hundred million (1.12×10−8). The 95% confidence interval around the uncertainty of this result was also below or at the one in a million de minimis risk level (95% confidence interval, 3×10−9, 1×10−6) often used by regulators. The theoretical estimation suggested that the probability of vCJD transmission by this exposure route was negligible (4, 10). The preliminary quantitative risk assessment identified the different steps of the enzyme manufacturing process to determine the factors that augmented or reduced the overall vCJD infection risk (10). Values for some of the input variables were obtained from the manufacturers or derived from the literature. Because of the limited literature on prion protein infectivity in pancreatic islets assumptions was made about the behavior of prion protein during islet cell recovery. No evidence base for prion clearance factors during islet cell isolation or the degree to which prions may associate with islet cells was available (10). To clarify this assumption regarding whether “prion material could stick to islet cells” (10), we undertook a transgenic mouse bioassay. Liberase enzyme (Roche Applied Science) (11) was used to dissociate Tg20 mouse pancreases using an established protocol (12). Islet cells were isolated and intracranially injected (ic) into 6-week-old Tg20 mice (n=6 experimentals and n=6 vehicle controls). For the experimental group, the Liberase used for pancreatic dissociation was “spiked” at a level of 5% w/w contamination (10% RML mouse brain homogenate/Liberase enzyme) before pancreatic dissociation. This was chosen as the maximum level of contamination because collagenase production quality control shows the enzyme preparations to be at least 95% pure after production (13). All mice were killed after 175 days, and brain tissue recovered for Western blot analysis to determine the presence or absence of protease resistant prion protein. Figure 1 shows that all mouse brain tissues tested in the bioassay contained no detectable proteinase K–resistant prions. The transgenic mouse bioassay result supports the original theoretical risk assessment assumption, namely, that infectious prions likely do not copurify with islet cells during the pancreatic dissociation process. The risk to type I diabetes islet transplant recipients of acquiring vCJD, as determined by the original risk assessment (4, 10), remains negligible.FIGURE 1: Western blot results of transgenic mouse bioassay. (A) 10 µL of experimental Tg20 mice bioassay 10% brain homogenate in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% NP40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.8) with (+) or without (−) 100 µg/mL PK treatment carried out at 37 °C for 1 hr.Mousebrain tissues from the bioassaywere isolated 175 days after ic injection. The initial islet cells used for ic injections were isolated from adult Tg20 mice. For the experimental group (n=6), Liberase enzyme was “spiked” with a 5% w/w amount of 10% RML sheep scrapie-infected mouse brain homogenate before pancreatic digestion. Islet cells harvested from this digestion of three adult mice pancreases were suspended in a small volume (0.5 mL) of 10% Fetal Bovine Serum/Hank’s Balanced Salt Solution and frozen at −20°C until used for ic injections. PrPSc-positive control (10% RML sheep scrapie-infected mouse brain homogenate) with (+) and without (−) PK digestion shown onWestern blot as a positive control (RML). Protein molecular weightmarkers also shown (M). Primary antibody used for detection was 6D11, this antibody crossreacts to both prion protein (normal cellular isoform) (PrPc) and PrPSc as indicated in the RML-positive control. (B) Control mice (n=6) that received islet cell ic injection mix prepared with Liberase that was not “spiked” with 10% mouse brain RML sheep scrapie homogenate. ic, intracranially injected; PK, proteinase K.Michael G. Tyshenko 1 Lisa Bertram2,3 Li Li2,3 Susie ElSaadany4 John Samis5 Daniel Krewski1,6 Neil R. Cashman2,3 1 McLaughlin Centre for Population Health Risk Assessment, University of Ottawa Ottawa, Ontario, Canada 2 Brain Research Centre University of British Columbia Vancouver British Columbia, Canada 3 PrioNet Canada, Vancouver, British Columbia, Canada 4 Professional Guidelines and Public Health Practice Division, Centre for Infectious Disease Prevention and Control Public Health Agency of Canada Ottawa Ontario, Canada 5 Faculty of Health Sciences, University of Ontario Institute of Technology Oshawa Ontario, Canada 6 Department of Epidemiology and Community Medicine Faculty of Medicine, University of Ottawa Ottawa, Ontario, Canada ACKNOWLEDGMENTS Dr. Neil Cashman provided laboratory space, technical support and use of level 2 enhanced facilities for the islet cell isolation experiments. PrioNet Canada’s Bioassay and Pathogenesis Platform was invaluable for completion of the mouse bioassay.