The prognostic significance of glutamic acid decarboxylase antibodies in patients with chronic pancreatitis undergoing total pancreatectomy with islet autotransplantation.

AIM: Islet autotransplantation (IAT) is considered a 'non-immune' model of islet transplant, with no risk for autoimmune-mediated beta cell loss, but we have previously observed de novo type 1 diabetes in one total pancreatectomy with islet autotransplantation (TPIAT) recipient. We aimed to investigate the clinical significance of glutamic acid decarboxylase antibodies (GADA), as a sensitive marker for autoimmune diabetes mellitus (DM), in patients with chronic pancreatitis undergoing TPIAT. METHODS: We identified 9 patients undergoing TPIAT with elevated GADA pre-TPIAT (8 non-diabetic and 1 with C-peptide positive DM), otherwise demographically similar to GADA negative TPIAT recipients (n=341). Metabolic and clinical measures related to islet cell function were recorded both before and after TPIAT. RESULTS: None of the 9 TPIAT patients achieved insulin independence after surgery, vs. 33% of GADA negative patients (n=318 with 1-yr follow-up). The two patients with the highest titters of GADA (>250 IU/mL) both experienced islet graft failure, despite normoglycaemia pre-TPIAT and high islet mass transplanted (5276 and 9378 IEQ per kg), with elevated HbA1c levels post-TPIAT (8.3%, 9.6%). The remaining 7 seven were insulin dependent with partial graft function and HbA1c levels <7%. CONCLUSION: Insulin dependence was more frequent in 9 patients with elevated GADA prior to TPIAT than in GADA negative TPIAT recipients, with graft failure in 2 cases. We speculate that beta-cell autoimmunity may occur in a small subset of TPIAT recipients and that beta cell antibody testing prior to TPIAT may be warranted to identify individuals at higher risk for insulin dependence.

Unmethylated Insulin DNA Is Elevated After Total Pancreatectomy With Islet Autotransplantation: Assessment of a Novel Beta Cell Marker.

Beta cell death may occur both after islet isolation and during infusion back into recipients undergoing total pancreatectomy with islet autotransplantation (TPIAT) for chronic pancreatitis. We measured the novel beta cell death marker unmethylated insulin (INS) DNA in TPIAT recipients before and immediately after islet infusion (n = 21) and again 90 days after TPIAT, concurrent with metabolic functional assessments (n = 25). As expected, INS DNA decreased after pancreatectomy (p = 0.0002). All TPIAT recipients had an elevated unmethylated INS DNA ratio in the first hours following islet infusion. In four samples (three patients), INS DNA was also assessed immediately after islet isolation and again before islet infusion to assess the impact of the isolation process: Unmethylated and methylated INS DNA fractions both increased over this interval, suggesting death of beta cells and exocrine tissue before islet infusion. Higher glucose excursion with mixed-meal tolerance testing was associated with persistently elevated INS DNA at day 90. In conclusion, we observed universal early elevations in the beta cell death marker INS DNA after TPIAT, with pronounced elevations in the islet supernatant before infusion, likely reflecting beta cell death induced by islet isolation. Persistent posttransplant elevation of INS DNA predicted greater hyperglycemia at 90 days.

Sitagliptin Treatment After Total Pancreatectomy With Islet Autotransplantation: A Randomized, Placebo-Controlled Study.

Insulin independence after total pancreatectomy and islet autotransplant (TPIAT) for chronic pancreatitis is limited by a high rate of postprocedure beta cell apoptosis. Endogenous glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are increased by dipeptidyl peptidase 4 inhibitor therapy (sitagliptin) may protect against beta cell apoptosis. To determine the effect of sitagliptin after TPIAT, 83 adult TPIAT recipients were randomized to receive sitagliptin (n = 54) or placebo (n = 29) for 12 months after TPIAT. At 12 and 18 months after TPIAT, participants were assessed for insulin independence; metabolic testing was performed with mixed meal tolerance testing and frequent sample intravenous glucose tolerance testing. Insulin independence did not differ between the sitagliptin and placebo groups at 12 months (42% vs. 45%, p = 0.82) or 18 months (36% vs. 44%, p = 0.48). At 12 months, insulin dose was 9.0 (standard error 1.7) units/day and 7.9 (2.2) units/day in the sitagliptin and placebo groups, respectively (p = 0.67) and at 18 months 10.3 (1.9) and 7.1 (2.6) units/day, respectively (p = 0.32). Hemoglobin A1c levels and insulin secretory measures were similar in the two groups, as were adverse events. In conclusion, sitagliptin could be safely administered but did not improve metabolic outcomes after TPIAT.

Successful Application of Closed-Loop Artificial Pancreas Therapy After Islet Autotransplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) may relieve the pain of chronic pancreatitis while avoiding postsurgical diabetes. Minimizing hyperglycemia after TPIAT limits beta cell apoptosis during islet engraftment. Closed-loop (CL) therapy combining an insulin pump with a continuous glucose monitor (CGM) has not been investigated previously in islet transplant recipients. Our objective was to determine the feasibility and efficacy of CL therapy to maintain glucose profiles close to normoglycemia following TPIAT. Fourteen adult subjects (36% male; aged 35.9 ± 11.4 years) were randomized to subcutaneous insulin via CL pump (n = 7) or multiple daily injections with blinded CGM (n = 7) for 72 h at transition from intravenous to subcutaneous insulin. Mean serum glucose values were significantly lower in the CL pump group than in the control group (111 ± 4 vs. 130 ± 13 mg/dL; p = 0.003) without increased risk of hypoglycemia (percentage of time <70 mg/dL: CL pump 1.9%, control 4.8%; p = 0.46). Results from this pilot study suggest that CL therapy is superior to conventional therapy in maintaining euglycemia without increased hypoglycemia. This technology shows significant promise to safely maintain euglycemic targets during the period of islet engraftment following islet transplantation.

Development of Autoimmune-Mediated ß Cell Failure After Total Pancreatectomy With Autologous Islet Transplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) is performed for definitive treatment of chronic pancreatitis; patients are not diabetic before surgery, or have C-peptide positive pancreatogenous diabetes. Thus, TPIAT recipients are not traditionally considered at risk for autoimmune loss of the islet graft. We describe a 43-year-old female who underwent TPIAT with high mass islet graft of 6031 IEQ/kg, with no evidence of presurgical ß cell autoimmunity who developed type 1 diabetes within the first year after TPIAT, resulting in complete loss of beta cell function. The patient had positive GAD and insulin autoantibodies at 1 year and 18 months after TPIAT, not present prior, and undetectable C-peptide after mixed meal and intravenous glucose tolerance testing at 18 months. Glucagon secretion was preserved, suggesting the transplanted alpha cell mass was intact. HLA typing revealed a DR3/DR4 class II haplotype. This case highlights the need to consider de novo type 1 diabetes in patients with unexpected islet graft failure after TPIAT.

Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.

BACKGROUND: Replacement of ß-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. METHODS: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). RESULTS: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. CONCLUSIONS: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.

Defective glucagon secretion during hypoglycemia after intrahepatic but not nonhepatic islet autotransplantation.

Defective glucagon secretion during hypoglycemia after islet transplantation has been reported in animals and humans with type 1 diabetes. To ascertain whether this is true of islets from nondiabetic humans, subjects with autoislet transplantation in the intrahepatic site only (TP/IAT-H) or in intrahepatic plus nonhepatic (TP/IAT-H+NH) sites were studied. Glucagon responses were examined during stepped hypoglycemic clamps. Glucagon and symptom responses during hypoglycemia were virtually absent in subjects who received islets in the hepatic site only (glucagon increment over baseline = 1 ± 6, pg/mL, mean ± SE, n = 9, p = ns; symptom score = 1 ± 1, p = ns). When islets were transplanted in both intrahepatic + nonhepatic sites, glucagon and symptom responses were not significantly different than Control Subjects (TP/IAT-H + NH: glucagon increment = 54 ± 14, n = 5; symptom score = 7 ± 3; control glucagon increment = 67 ± 15, n = 5; symptom score = 8 ± 1). In contrast, glucagon responses to intravenous arginine were present in TP/IAT-H recipients (TP/IAT: glucagon response = 37 ± 8, n = 7). Transplantation of a portion of the islets into a nonhepatic site should be seriously considered in TP/IAT to avoid posttransplant abnormalities in glucagon and symptom responses to hypoglycemia.

Proposed thresholds for pancreatic tissue volume for safe intraportal islet autotransplantation after total pancreatectomy.

The simple question of how much tissue volume (TV) is really safe to infuse in total pancreatectomy-islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) precipitated this analysis. We examined a large cohort of CP patients (n = 233) to determine major risk factors for elevated portal pressure (PP) during islet infusion, using bivariate and multivariate regression modeling. Rates of bleeding requiring operative intervention and portal venous thrombosis (PVT) were evaluated. The total TV per kilogram body weight infused intraportally was the best independent predictor of change in PP (ΔPP) (p < 0.0001; R(2) = 0.566). Rates of bleeding and PVT were 7.73% and 3.43%, respectively. Both TV/kg and ΔPP are associated with increased complication rates, although ΔPP appears to be more directly relevant. Receiver operating characteristic analysis identified an increased risk of PVT above a suggested cut-point of 26 cmH2O (area under the curve = 0.759), which was also dependent on age. This ΔPP threshold was more likely to be exceeded in cases where the total TV was >0.25 cm(3)/kg. Based on this analysis, we have recommended targeting a TV of <0.25 cm(3)/kg during islet manufacturing and to halt intraportal infusion, at least temporarily, if the ΔPP exceeds 25 cmH2O. These models can be used to guide islet manufacturing and clinical decision making to minimize risks in TP-IAT recipients.

Metabolic assessment prior to total pancreatectomy and islet autotransplant: utility, limitations and potential.

Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass ≥2500 IEQ/kg. The acute C-peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values -0.33 to -0.40, p ≤ 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C-peptide on MMTT ≥4 ng/mL were associated with greater odds of receiving an islet mass ≥2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87-1.0; OR 7.9 for C-peptide, CI 1.75-35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C-peptide ≥4 ng/mL on MMTT conveyed eight times the odds of receiving ≥2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.

Severely fibrotic pancreases from young patients with chronic pancreatitis: evidence for a ductal origin of islet neogenesis.

While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28 years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize postsurgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon, and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both ß-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double immunofluorescent labeling, we found insulin-positive cells to be present within the ductal lumens, among the cytokeratin-positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.

Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes.

The seemingly inexorable decline in insulin independence after islet transplant alone (ITA) has raised concern about its clinical utility. We hypothesized that induction immunosuppression therapy determines durability of insulin independence. We analyzed the proportion of insulin-independent patients following final islet infusion in four groups of ITA recipients according to induction immunotherapy: University of Minnesota recipients given FcR nonbinding anti-CD3 antibody alone or T cell depleting antibodies (TCDAb) and TNF-α inhibition (TNF-α-i) (group 1; n = 29); recipients reported to the Collaborative Islet Transplant Registry (CITR) given TCDAb+TNF-α-i (group 2; n = 20); CITR recipients given TCDAb without TNF-α-i (group 3; n = 43); and CITR recipients given IL-2 receptor antibodies (IL-2RAb) alone (group 4; n = 177). Results were compared with outcomes in pancreas transplant alone (PTA) recipients reported to the Scientific Registry of Transplant Recipients (group 5; n = 677). The 5-year insulin independence rates in group 1 (50%) and group 2 (50%) were comparable to outcomes in PTA (group 5: 52%; p>>0.05) but significantly higher than in group 3 (0%; p = 0.001) and group 4 (20%; p = 0.02). Induction immunosuppression was significantly associated with 5-year insulin independence (p = 0.03), regardless of maintenance immunosuppression or other factors. These findings support potential for long-term insulin independence after ITA using potent induction therapy, with anti-CD3 Ab or TCDAb+TNF-α-i.

Islet transplantation in type 1 diabetics using an immunosuppressive protocol based on the anti-LFA-1 antibody efalizumab.

The applicability of islet transplantation as treatment for type 1 diabetes is limited by renal and islet toxicities of currently available immunosuppressants. We describe a novel immunosuppressive regimen using the antileukocyte functional antigen-1 antibody efalizumab which permits long-term islet allograft survival while reducing the need for corticosteroids and calcineurin inhibitors (CNI). Eight patients with type 1 diabetes and hypoglycemic unawareness received intraportal allogeneic islet transplants. Immunosuppression consisted of antithymocyte globulin induction followed by maintenance with efalizumab and sirolimus or mycophenolate. When efalizumab was withdrawn from the market in mid 2009, all patients were transitioned to regimens consisting of mycophenolate and sirolimus or mycophenolate and tacrolimus. All patients achieved insulin independence and four out of eight patients became independent after single-islet transplants. Insulin independent patients had no further hypoglycemic events, hemoglobin A1c levels decreased and renal function remained stable. Efalizumab was well tolerated and no serious adverse events were encountered. Although long-term follow-up is limited by discontinuation of efalizumab and transition to conventional imunnosuppression (including CNI in four cases), these results demonstrate that insulin independence after islet transplantation can be achieved with a CNI and steroid-free regimen. Such an approach may minimize renal and islet toxicity and thus further improve long-term islet allograft survival.

Prolonged insulin independence after islet allotransplants in recipients with type 1 diabetes.

We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received one to two islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor-alpha blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 +/- 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A(1c) levels and C-peptide production) and for adverse events associated with the study protocol. Of the six recipients, five were insulin-independent at 1 year, and four continue to be insulin-independent at a mean of 3.4 +/- 0.4 years posttransplant. None of the six recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 +/- 21.2 mL/min/1.73 m(2) pretransplant to 82.6 +/-19.1 mL/min/1.73 m(2) at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in four of six recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.