Sharleen Imes

High risk of sensitization after failed islet transplantation.

Human Leukocyte Antigen (HLA) antibodies posttransplant have been associated with an increased risk of early graft failure in kidney transplants. Whether this also applies to islet transplantation is not clear. To achieve insulin independence after islet transplants multiple donor infusions may be required. Hence, islet transplant recipients are at risk of sensitization after transplantation. Islet transplant recipients were screened for HLA antibodies posttransplant by flow‐based methods. A total of 98 patients were studied. Twenty‐nine patients (31%) developed de novo donor specific antibodies (DSA) posttransplant. Twenty‐three patients developed DSA while on immunosuppression (IS). Among recipients who have discontinued IS, 10/14 (71%) are broadly sensitized with panel reactive antibody (PRA) ≥50%. The risk of becoming broadly sensitized after transplant was 11/69 (16%) if the recipient was unsensitized prior to transplant. The majority of these antibodies have persisted over time. Appearance of HLA antibodies posttransplant is concerning, and the incidence rises abruptly in subjects weaned completely from IS. This may negatively impact the ability of these individuals to undergo further islet, pancreas or kidney transplantation and should be discussed upfront during evaluation of candidates for islet transplantation.

Defects in Insulin Secretion and Action in Women With a History of Gestational Diabetes

Gestational diabetes mellitus (GDM) is associated with defects in insulin secretion and insulin action, and women with a history of GDM carry a high risk for the development of non-insulin-dependent diabetes mellitus (NIDDM). Assessment of subjects with a history of GDM who are currently normoglycemic should help elucidate some of the underlying defects in insulin secretion or action in the evolution of NIDDM. We have studied 14 women with normal oral glucose tolerance who had a history of GDM. They were compared with a group of control subjects who were matched for both body mass index (BMI) and waist-to-hip ratio (WHR). All subjects underwent tests for the determination of oral glucose tolerance, ultradian oscillations in insulin secretion during a 28-h glucose infusion, insulin secretion in response to intravenous glucose, glucose disappearance after intravenous glucose (Kg), and insulin sensitivity (SI) as measured by the Bergman minimal model method. The BMI in the post-GDM women was similar to that in the control subjects (24.9 ± 1.2 vs. 25.4 ± 1.4 kg/m2, respectively), as was the WHR ratio (0.80 ± 0.01 vs. 0.76 ± 0.01, respectively). The post-GDM women were slightly older (35.2 ± 0.9 vs. 32.1 ± 1.4 years, P = 0.04). The fasting plasma glucose levels were significantly higher in the post-GDM group than in the control group (4.9 ± 0.1 vs. 4.4 ± 0.1 mmol/l, respectively, P < 0.001) and remained higher at each of the subsequent determinations during the oral glucose tolerance test, although none had a result indicative of either diabetes or impaired glucose tolerance. All measures of ultradian insulin secretory oscillations in post-GDM subjects were indistinguishable from those in the control subjects. The first-phase insulin release to intravenous glucose was lower in the post-GDM group. SI was also impaired in the post-GDM group compared with the control subjects (4.6 ± 0.5 vs. 6.8 ± 1.0·10−4·min−1· ³U−1·ml, respectively, P < 0.05). Kg was reduced in the post-GDM women compared with the control subjects (1.3 ± 0.1 vs. 2.7 ± 0.4%, P < 0.01). When the subjects were divided according to their BMI, lean post-GDM subjects (<24.2, n = 8) were more insulin resistant than the lean control subjects: SI 5.3 ± 0.6 vs. 8.8 ± 1 · 1·10−4 min −1· ³U−1·ml, P = 0.02, whereas obese post-GDM (>24.2 kg/m2, n = 6) and control subjects had a lower SI than the lean subjects, but they were not different from each other (3.6 ± 0.7 vs. 4.2 ± 1.2· 10−4· min−1 · ³U−1 · ml, respectively, P = 0.67). The acute insulin responses to glucose (0–10 min) within these groups showed that the lean post-GDM group had a significantly lower insulin response compared with control subjects (1,205 ± 179 vs. 2,404 ± 416 pmol· 1−1 min, respectively, P = 0.007), whereas the obese groups had similar responses (2,777 ± 1,112 vs. 3,114 ± 847 pmol ·1−·min, post-GDM vs. control subjects, P = 0.8). We have found defects in insulin secretion and action in post-GDM subjects who are at high risk for the development of NIDDM at a time that oral glucose tolerance is normal. These defects are present in the absence of obesity. Ultradian insulin secretory oscillations during constant glucose infusion are normal in these post-GDM subjects predisposed to NIDDM. We conclude that defects in both insulin secretion and insulin action are present before the development of hyperglycemia in women with a history of GDM.

Pretransplant HLA antibodies are associated with reduced graft survival after clinical islet transplantation.

Despite significant improvements in islet transplantation, long‐term graft function is still not optimal. It is likely that both immune and nonimmune factors are involved in the deterioration of islet function over time. Historically, the pretransplant T‐cell crossmatch and antibody screening were done by anti‐human globulin—complement‐dependent cytotoxicity (AHG‐CDC). Class II antibodies were not evaluated. In 2003, we introduced solid‐phase antibody screening using flow‐based beads and flow crossmatching. We were interested to know whether pretransplant human leukocyte antigen (HLA) antibodies or a positive flow crossmatch impacted islet function post‐transplant. A total of 152 islet transplants was performed in 81 patients. Islet function was determined by a positive C‐peptide. Results were analyzed by procedure. Class I and class II panel reactive antibody (PRA) > 15% and donor‐specific antibodies (DSA) were associated with a reduced C‐peptide survival (p < 0.0001 and p < 0.0001, respectively). A positive T‐ and or B‐cell crossmatch alone was not. Pretransplant HLA antibodies detectable by flow beads are associated with reduced graft survival. This suggests that the sirolimus and low‐dose tacrolimus‐based immunosuppression may not control the alloimmune response in this presensitized population and individuals with a PRA > 15% may require more aggressive inductive and maintenance immunosuppression, or represent a group that may not benefit from islet transplantation.

Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon

Background. Previous small clinical trials indicate that the two-layer method (TLM) for pancreas preservation improves islet isolation outcome. However, the effect of TLM has not been evaluated in large-scale study. In addition, a direct benefit of TLM on islet transplantation outcome has not been addressed in the setting of any randomized controlled trials. Methods. Between April 2003 and October 2005, human pancreata from brain-dead donors were preserved by TLM using preoxygenated perfluorocarbon (n=75) or in University of Wisconsin (UW) solution (n=91) prior to islet isolation. Islet isolation and transplantation outcomes were compared between the two groups. Results. We did not find any significant differences in adenosine triphosphate content in pancreatic tissue after preservation, pre and postpurification islet yields, in vitro insulin secretory function, or utilization ratio of transplantation between the two groups. Transplanted mass and functional viability of islet isolated from TLM-preserved pancreas were similar to those from UW-preserved pancreas. Patients receiving the TLM-islet or the UW-islet showed a marked decrease in insulin requirement after transplantation. However, no significant difference was observed in a decrease in insulin requirement between patients receiving the TLM-islet and the UW-islet. Conclusions. No beneficial effect of TLM on islet isolation and transplantation outcomes was observed. Our findings bring into question the true merit of routine use of TLM prior to islet isolation.

Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success

Background. Successful islet transplantation can result in insulin independence in many patients with type 1 diabetes mellitus, but it often requires more than one islet infusion. The ability to achieve insulin independence with a single donor is an important goal in clinical islet transplantation due to the limited organ supply. Methods. We examined factors that may be associated with insulin independence after islet transplantation with islets from a single donor, using univariate and multivariate analysis. Results. Thirteen of 85 (15.3%) achieved insulin independence after single-donor islet transplantation. Using multivariate analysis, only the use of insulin and heparin infusions peritransplant was a significant factor associated with insulin independence, with an adjusted odds ratio of 8.6 (95% confidence interval 2.0-37.0). Patients who had received insulin and heparin infusions peritransplant had greater indices of islet engraftment and a greater reduction in insulin use (80.1%±4.3% vs. 54.2%±2.8%, P<0.001) even if insulin independence was not achieved. Conclusions. Peritransplant intensive insulin and heparin enhances islet transplantation outcomes likely related in part to mitigation of the effects of the instant blood-mediated inflammatory reaction, combined with islet rest and avoidance of inflammation. It would be important to further investigate the effects of peritransplant insulin and heparin infusions on islet engraftment.

Histologic graft assessment after clinical islet transplantation.

Background. An accurate monitoring would help understanding the fate of islet grafts after transplantation. Methods. This work assessed the feasibility of needle biopsy monitoring after intraportal islet transplantation (n=16), and islet graft morphology was studied with the addition of autopsy samples (n=2). Pancreas autopsy samples from two nondiabetic individuals were used as control. Results. Islet tissue was found in five needle samples (31%). Sampling success was related to size (100% sampling for the four biopsies of 1.8 cm in length or higher, P≤0.01). Mild liver abnormalities included localized steatosis (n=8), mild nodular regenerative hyperplasia and mild portal venopathy (n=3), and hepatocyte swelling (n=2). Endocrine cell composition and distribution were similar between islet grafts and normal islets within the native pancreas. There was no or minimal immune cell infiltrate in patients on and off exogenous insulin, including two patients with ongoing negative metabolic events (increasing HbA1c or insulin requirement). The infiltrate was mainly composed of CD4- and CD8-positive cells. Conclusion. This study demonstrates that needle biopsy is feasible after clinical islet transplantation but with a limited practical value because of its low islet sampling rate using current sampling and analysis methods. Both biopsy and autopsy samples demonstrated the well-preserved islet endocrine composition after transplantation and the presence of focal areas of steatosis. Islet grafts showed no or minimal immune cell infiltration, even in the case of ongoing islet loss. On the basis of the findings, possible reasons for allograft islet loss are discussed.

Changes in liver enzymes after clinical islet transplantation

Background. Clinical islet transplantation (ITx) shows insulin independence with adequate metabolic control in patients with type 1 diabetes. The aim of this study was to characterize the pattern of elevation in liver enzymes observed after ITx and to investigate any correlation between these elevations and graft characteristics or graft functional outcome. Methods. Eighty-four consecutive ITx procedures were performed in 42 recipients. Liver function tests (LFT) were assessed during the first 40 days posttransplant. LFT elevated greater than or equal to 2.5 times above the upper limit of normal (ULN) were considered relevant. Results. In 54% of the transplants, the aspartate aminotransferase (AST) increased by more than 2.5 times above ULN. A 5-fold increase in AST was observed in 27% of the procedures. The highest AST levels were observed after the first ITx. AST for all transplants peaked at 7±0.5 days at a value of 162±23 U/L (P <0.001, compared with the pretransplant values). Changes in alanine aminotransferase were similar to AST. Alkaline phosphatase increased more than 2-fold above ULN in 12% of the procedures. LFT normalized in 90% of the recipients within 4 weeks posttransplant. The remaining 10% normalized within 2 months after ITx. Graft characteristics and graft function were not significantly different when comparing LFT with greater than 5-fold versus less than 2.5-fold increase above ULN. The mean bilirubin remained within the normal range. Conclusions. After intraportal ITx, a significant increase in LFT levels was noticed in more than 50% of the procedures. These levels normalized spontaneously in 90% of the recipients within 4 weeks. No correlation between the increase in LFT and graft characteristics or graft function was found.

Comparison of Human Islet Isolation Outcomes Using a New Mammalian Tissue-Free Enzyme Versus Collagenase NB-1

Background. After the discontinuation of the manufacturing Liberase HI because of a small potential for prion disease transmission, Roche Diagnostics (Indianapolis, IN) developed a new enzyme product (Liberase MTF [mammalian tissue free]), which is similar to Liberase HI with the exception that no mammalian tissue is used in the manufacture of the collagenase component. We report our experience using the MTF enzyme in clinical islet isolations compared with Serva NB-1 with modified enzyme delivery method. Methods. Islets were isolated from 41 pancreata using MTF enzyme (n=17) or NB-1 enzyme (n=24). NB-1 enzymes were delivered using a modified (nonsimultaneous) enzyme delivery method whereas isolations using MTF used the standard method of simultaneous collagenase and thermolysin perfusion. Islets were purified on a COBE 2991 Cell Blood Processor and subsequently cultured. Results. The average islet mass after purification was 392±36×103 islet equivalent (IE) for MTF versus 371±40×103 IE for Serva NB-1 (P=0.63). Post-IE/cm3 of tissue was 110±9×103 IE/cm3 and 91±11×103 IE/cm3 for MTF and NB-1, respectively (P=0.07). The isolation success rate (>400,000 IE) for MTF was 53% compared with 33% for Serva (P=0.33). Conclusion. We conclude that MTF may be successfully used for high-yield human islet isolation and clinical transplantation and provides similar quality islets to those derived using NB-1.

Single-Donor Islet Transplantation and Long-term Insulin Independence in Select Patients With Type 1 Diabetes Mellitus:

Background Islet transplantation is a recognized treatment option for select patients with type I diabetes mellitus. However, islet infusions from multiple donors are often required to achieve insulin independence. Ideally, insulin independence would be achieved routinely with only a single donor. Identification of factors associated with insulin independence after single-donor islet transplantation may help to select recipient-donor combinations with the highest probability of success. Methods Subjects undergoing islet transplantation at a single center (Edmonton, Canada) between March 1999 and August 2013 were included. Recipient, donor, and transplant characteristics were collected and compared between recipients who became insulin independent after one islet transplantation and those who did not. Results Thirty-one patients achieved insulin independence after a single-donor islet transplantation, and 149 did not. Long-term insulin-free survival was not different between the groups. Factors significantly associated with single-donor success included recipient age, insulin requirement at baseline, donor weight, donor body mass index, islet transplant mass, and peritransplant heparin and insulin administration. On multivariate analysis, pretransplantation daily insulin requirements, the use of peritransplantation heparin and insulin infusions, and islet transplant mass remained significant. Conclusion We have identified clinically relevant differences defining the achievement of insulin independence after single-donor transplantation. Based on these differences, a preoperative insulin requirement of less than 0.6 U/kg per day and receiving more than 5,646 islet equivalents (IEQ)/kg have a sensitivity of 84% and 71% and specificity of 50% and 50%, respectively, for insulin independence after single-donor islet transplantation. With ideal patient selection, this finding could potentially increase single-donor transplantation success and may be especially relevant for presensitized subjects or those who may subsequently require renal replacement.

Long-term graft function after allogeneic islet transplantation.

Islet transplants are emerging as a viable option for the treatment of type 1 diabetes mellitus. From 1989 to 1995 we conducted a series of simultaneous islet–kidney transplants in six uremic type 1 diabetic patients. We report two of these patients who have shown persistent islet graft function over many years. Two female patients with duration of diabetes of 27 and 37 years underwent simultaneous islet–kidney transplant under steroid- and cyclosporine-based immunosuppression. Freshly isolated islets were supplemented with cryopreserved islets from our low-temperature bank of frozen islets. A total islet mass of 9,866 and 15,061 islet equivalents/kg body weight, respectively, was transplanted into the liver through portal vein. Reasonable blood glucose control has been achieved for up to 6 years posttransplant in one patient, but there was minimum clinical benefit from the islet graft at 10 years. In contrast, sustained insulin secretion with nearly normal HbA1c at 13 years follow-up was observed in another patient, providing hope for improving long-term graft outcomes for islet transplant recipient.