Islet Transplant Recipients Research Articles

High Concentrations of Etanercept Reduce Human Islet Function and Integrity.

BackgroundMost islet transplant groups worldwide routinely use the TNFα inhibitor Etanercept in their peri-transplant protocols. Surprisingly, there have been no published dose-response studies on the effects of Etanercept on human islets. Our study aimed to address this by treating cultured human islets with increasing concentrations of Etanercept.Materials and MethodsIsolated human islets were cultured for 3–4 days in normoxic (21% oxygen) or in hypoxic (2% oxygen) atmosphere using Etanercept dissolved in a range of 2.5–40 µg/mL prior to islet characterisation.ResultsIn normoxic atmosphere, it was found that 5 µg/mL is the most efficient dose to preserve islet morphological and functional integrity during culture. Increasing the dose to 10 µg/mL or more resulted in detrimental effects with respect to viability and glucose-stimulated insulin release. When human islets were cultured for 3 to 4 days in clinically relevant hypoxia and treated with 5 µg/mL Etanercept, post-culture islet survival (P < 0.001) and in vitro function (P < 0.01) were significantly improved. This correlated with a substantially reduced cytokine production (P < 0.05), improved mitochondrial function (P < 0.01), and reduced production of reactive oxygen species (P < 0.001) in hypoxia-exposed islets.ConclusionThese findings suggest that the therapeutic window of Etanercept is very narrow and that this should be considered when optimising the dosage and route of Etanercept administration in islet-transplant recipients or when designing novel drug-delivering islet scaffolds.

1093. Infectious Complications after Pancreatic Islet Transplantation

BackgroundDespite the significant advancement in islet transplantation over the past three decades, our understanding of infectious complications post islet transplant remains limited.MethodsThis is a single center retrospective review of Islet transplant recipients at the University of Alberta between February 2006 and December 2015. All infectious episodes events occurring after transplant were categorized as opportunistic and non-opportunistic.ResultsWe analyzed 142 patients receiving a median of 2 islet transplants per patient, with 18 patients receiving 1 transplant (13%), 77 (54%) 2, 33 (23%) 3, 13 (9%) 4 and 1(1%) 5 transplants. Median age at first transplant was 50 years and 85 (47%) were male. Lymphocyte depleting agent with thymoglobulin or alemtuzumab was used for induction in 94% in first and 53% in second transplant. CMV serostatus was CMV D+/R- 61 (43%), CMVD+/R+ 52 (37%), CMVD-/R+ 16 (11%) and CMVD-/R- 13 (9%). CMV infection occurred in 21 patients (15%) [CMVD+/R- 6 (9.8%) and CMVR+ 15 (22.1%), p=0.06]. Other opportunistic infections included VZV 7 (4.9%), Nocardia 3(2.1%), and Pneumocystis jirovecii pneumonia 1. Non-opportunistic infections included skin and soft tissue infection 14 (9.9%), urinary tract infection 11 (7.7%), pneumonia 7 (4.9%) clostridium difficile infection (CDI) 4 (2.8%), and non-CDI gastroenteritis 5 (3.5%) (Table 1).Table 1: Infectious Complication post islet transplant ConclusionAlthough the rate of infections after islet cell transplant is less frequent than other types of transplants, opportunistic infections, especially CMV, are not uncommon and should be considered in this setting.Disclosures Carols Cervera, MD, PHD, Merk (Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member, Other Financial or Material Support, Lecture fees) James Shapiro, MD, PHD FRCS(Eng) FRCSC MSM FRSC, ViaCyte (Consultant) Dima Kabbani, MD, Merck (Research Grant or Support)

Chemokines as Drivers of the Autoimmune Destruction in Type 1 Diabetes: Opportunity for Therapeutic Intervention in Consideration of an Optimal Treatment Schedule.

Type 1 diabetes (T1D) is mainly precipitated by the destruction of insulin-producing β-cells in the pancreatic islets of Langerhans by autoaggressive T cells. The etiology of the disease is still not clear, but besides genetic predisposition the exposure to environmental triggers seems to play a major role. Virus infection of islets has been demonstrated in biopsies of T1D patients, but there is still no firm proof that such an infection indeed results in islet-specific autoimmunity. However, virus infection results in a local inflammation with expression of inflammatory factors, such as cytokines and chemokines that attract and activate immune cells, including potential autoreactive T cells. Many chemokines have been found to be elevated in the serum and expressed by islet cells of T1D patients. In mouse models, it has been demonstrated that β-cells express chemokines involved in the initial recruitment of immune cells to the islets. The bulk load of chemokines is however released by the infiltrating immune cells that also express multiple chemokine receptors. The result is a mutual attraction of antigen-presenting cells and effector immune cells in the local islet microenvironment. Although there is a considerable redundancy within the chemokine ligand-receptor network, a few chemokines, such as CXCL10, seem to play a key role in the T1D pathogenesis. Studies with neutralizing antibodies and investigations in chemokine-deficient mice demonstrated that interfering with certain chemokine ligand-receptor axes might also ameliorate human T1D. However, one important aspect of such a treatment is the time of administration. Blockade of the recruitment of immune cells to the site of autoimmune destruction might not be effective when the disease process is already ongoing. By that time, autoaggressive cells have already arrived in the islet microenvironment and a blockade of migration might even hold them in place leading to accelerated destruction. Thus, an anti-chemokine therapy makes most sense in situations where the cells have not yet migrated to the islets. Such situations include treatment of patients at risk already carrying islet-antigen autoantibodies but are not yet diabetic, islet transplantation recipients, and patients that have undergone a T cell reset as occurring after anti-CD3 antibody treatment.

Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic β cells.

It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from β cells, reflect their recent demise, and can be used to assess β cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a β cell-specific DNA methylation signature, by simultaneous assessment of 6 DNA methylation markers, that identifies β cell DNA in mixtures containing as little as 0.03% β cell DNA (less than 1 β cell genome equivalent). Based on this assay, plasma from nondiabetic individuals (N = 218, aged 4-78 years) contained on average only 1 β cell genome equivalent/mL. As expected, cell-free DNA (cfDNA) from β cells was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β cell cfDNA in a patient with KATP congenital hyperinsulinism, in which substantial β cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of β cell-derived cfDNA in autoantibody-positive subjects at risk for type 1 diabetes (N = 32), individuals with recent-onset type 1 diabetes (<4 months, N = 92), or those with long-standing disease (>4 months, N = 38). We discuss the utility of sensitive β cell cfDNA analysis and potential explanations for the lack of a β cell cfDNA signal in type 1 diabetes.

Fasting parameters for estimation of stimulated β cell function in islet transplant recipients with or without basal insulin treatment.

In order to assess β cell secretory capacity after islet transplantation, standardized mixed meal stimulation tests are often used. But these tests are cumbersome and the effect of exogenous insulin on the test results is unclear. The aim of our study was to determine to what extent fasting glycemic indices can estimate stimulated β cell function in islet transplant recipients with and without basal insulin. In total 100 mixed meal stimulation tests, including 31 with concurrent basal insulin treatment, were performed in 36 islet transplant recipients. In a multivariate model, fasting C‐peptide and fasting glucose together estimated peak C‐peptide with R 2 = .87 and area under the curve (AUC) C‐peptide with a R 2 = .93. There was a larger increase of glucose during tests in which exogenous insulin was used (+7.9 vs +5.3 mmol/L, P < .001) and exogenous insulin use was associated with a slightly lower estimated peak C‐peptide (relative change: −15%, P = .02). In islet transplant recipients the combination of fasting C‐peptide and glucose can be used to accurately estimate stimulated β cell function after a mixed meal stimulation test, whether exogenous basal insulin is present or not. These data indicate that graft function can be reliably determined during exogenous insulin treatment and that regular islet graft stimulation tests can be minimized.

Donor insulin use predicts beta-cell function after islet transplantation.

Insulin is routinely used to manage hyperglycaemia in organ donors and during the peri-transplant period in islet transplant recipients. However, it is unknown whether donor insulin use (DIU) predicts beta-cell dysfunction after islet transplantation. We reviewed data from the UK Transplant Registry and the UK Islet Transplant Consortium; all first-time transplants during 2008-2016 were included. Linear regression models determined associations between DIU, median and coefficient of variation (CV) peri-transplant glucose levels and 3-month islet graft function. In 91 islet cell transplant recipients, DIU was associated with lower islet function assessed by BETA-2 scores (β [SE] -3.5 [1.5], P = .02), higher 3-month post-transplant HbA1c levels (5.4 [2.6] mmol/mol, P = .04) and lower fasting C-peptide levels (-107.9 [46.1] pmol/l, P = .02). Glucose at 10 512 time points was recorded during the first 5 days peri-transplant: the median (IQR) daily glucose level was 7.9 (7.0-8.9) mmol/L and glucose CV was 28% (21%-35%). Neither median glucose levels nor glucose CV predicted outcomes post-transplantation. Data on DIU predicts beta-cell dysfunction 3 months after islet transplantation and could help improve donor selection and transplant outcomes.

362-OR: The Biological Variability of the BETA-2 Score in Islet Transplant Recipients

Background: The BETA-2 score is a validated composite measure of beta cell function in islet transplantation. It is calculated from a fasting sample and lends itself to regular monitoring of islet graft function. To determine whether changes in the BETA-2 score are significant, it is important to understand inherent variability of the score. The aim of this study was to characterize the biological variability (coefficient of variation (CV)) of the BETA-2 score and to define the critical difference (CD) for the score which is the percentage change in score that reflects a significant change in islet function. Methods: Islet transplant recipients with stable graft function were included in this retrospective study. BETA-2 score was calculated weekly over 8 consecutive weeks and the CV was calculated based on the root mean square method. The CD at the 95% confidence limit was calculated as follows: CD = 2.8 x CV. Results: 21 transplant recipients were included of whom 6 were insulin dependent and 15 were insulin independent. These patients also varied in terms of kidney function: 10 subjects with eGFR ≥ 60 (76 ± 14 ml/min/1.73 m2) and 11 subjects with eGFR &amp;lt; 60 (44 ± 10 ml/min/1.73 m2). The CV for all patients was 9.3%, 95% CI [8.7, 10.0]. The CV was similar between insulin independent and insulin dependent subjects (9.0%, 95% CI [8.1, 9.9] vs. 10.2, 95% CI [9.3, 11.1], p=0.5), as well as between subjects with eGFR &amp;gt; 60 vs. eGFR &amp;lt; 60 (8.9%, 95% [8.1,9.8] vs. 9.7%, 95% CI [8.7,10.7], p=0.6). The critical difference for the BETA-2 score was 26.2% for all subjects, 25.1% for insulin independent subjects, 28.5% for insulin dependent subjects, 24.9% for those with eGFR&amp;gt;60, and 27.9% for those with GFR &amp;lt;60. Conclusions: The biological variability of the BETA-2 score appears stable across various clinical scenarios and a minimum difference of 26% between scores appears to constitute a significant change in BETA-2 score. Further analysis including more subjects and a greater range of kidney function is warranted. Disclosure R. Millott: None. R.A. Oram: None. S. Forbes: None. A. Shapiro: Consultant; Self; Viacyte, Inc. P.A. Senior: None. A. Lam: None.

359-OR: a- or ß-Adrenergic Blockade Does Not Affect Transplanted Islet Responses to Hypoglycemia in Type 1 Diabetes (T1D)

Recipients of intrahepatic islet transplantation for T1D exhibit appropriate suppression of endogenous insulin and activation of glucagon secretion in response to insulin-induced hypoglycemia with restored glucose counterregulation and protection against clinically significant hypoglycemia. Whether sympathetic activation of adrenergic receptors on transplanted islets is required for these responses is not known. To evaluate the adrenergic contribution to post-transplant glucose counterregulation, we performed a randomized, double-blind crossover study of responses during a hyperinsulinemic (1 mU·kg-1·min-1) euglycemic (EU, ∼90 mg/dL)-hypoglycemic (HYPO, ∼50 mg/dL) clamp conducted under phentolamine (0.95 µg·kg-1·min-1; α-adrenergic blockage), propranolol (0.48 µg·kg-1·min-1; β-adrenergic blockage), or placebo infusion. Subjects were 5F/4M with median (range) age 53 (34 - 63) years, T1D duration 29 (18 - 56) years, post-transplant 7 (2 - 8) years, HbA1c 5.8 (4.5 - 6.8) %, insulin in-/dependent 5/4, on tacrolimus-based immunosuppression, and spent 97 (76 - 99) % time in range 70 - 180 mg/dL and 1 (0 - 3) % time with hypoglycemia &amp;lt; 70 mg/dL by CGM. During the EU-HYPO clamp, blood pressure was lower with phentolamine and heart rate lower with propranolol vs. placebo (P &amp;lt; 0.05). There was no difference in suppression of C-peptide or activation of glucagon with phentolamine or propranolol vs. placebo. Pancreatic polypeptide was greater with phentolamine vs. placebo during EU (P &amp;lt; 0.05), and free fatty acids were lower and the glucose infusion rate higher with propranolol vs. placebo during HYPO (P &amp;lt; 0.05) with no differences seen for endogenous glucose production. These results indicate physiologic α- and β-adrenergic blockage had no effect on transplanted islet responses to hypoglycemia. Sympathetic re-innervation, as occurs with re-vascularization of intrahepatic islets, may not be necessary for post-transplant recovery of glucose counterregulation in T1D. Disclosure M.R. Rickels: Consultant; Self; Semma Therapeutics, Inc. Research Support; Self; Xeris Pharmaceuticals, Inc. M. Bellin: Research Support; Self; Dexcom, Inc., Viacyte, Inc. Other Relationship; Self; Insulet Corporation. D. Stefanovski: None. A.J. Peleckis: None. C.V. Dalton-Bakes: None. E. Markmann: None. H.T. Nguyen: None. A. Naji: None. Funding National Institutes of Health (R01DK091331, UL1TR001878, P30DK19525)

203-LB: Redosing of Suboptimally Controlled Diabetic Mice with Human Neo-islets, 3D Aggregates of Mesenchymal Stromal and Pancreatic Islet Cells, Durably Restores Euglycemia: Relevance to the Treatment of Diabetic Humans

We reported that the i.p. administration of allogeneic Neo-Islets (NIs; 3D aggregates of allogeneic, culture-expanded Islet cells (ICs) with immune- and cyto-protective Mesenchymal Stromal Cells (MSCs)), durably restores normoglycemia in autoimmune type-1 diabetic NOD mice through omental engraftment. Similarly, our ongoing, FDA-guided pilot study in insulin dependent pet dogs (INAD 012-0776) demonstrates that allogeneic canine NI therapy significantly improves both glycemic control and insulin needs (&amp;gt; 1.5 years), and this without eliciting an allo-immune response. The current study was undertaken in preparation of a Phase I Clinical Trial in order to determine whether NIs engineered from human cells (hNIs) could also reduce insulin need, and whether redosing of suboptimally controlled diabetic mice could fully restore euglycemia, as is frequently required in human recipients of islet transplants. Hyperglycemia (blood glucose &amp;gt; 350 mg/dL x 3 days) was induced with streptozotocin in adult NOD/SCID mice and blood glucose levels were controlled with insulin pellets (Linbits) until i.p. hNI (2x10e5 hNIs/kg bw) or vehicle administration (n=6/group). Blood glucose levels, weights, ip glucose tolerance tests [GTT) were closely monitored. While no mice in the hNI treated group died (vs. 4 in the control group), hNI therapy partially reduced glucose levels . Therefore, on day 64 post initial treatment, mice were re-dosed with hNIs (2x10e5 hNIs/kg bw) and followed together with controls as above. This second dose of hNI completely and durably restored euglycemia and normalized ip GTTs. We conclude that this minimally invasive, safe and highly effective therapy supports our current pursuit of an IND for the conduct of a pioneering Clinical Trial in subjects with T1DM. Disclosure S.S. Chowdhury: Employee; Self; SymbioCellTech. A. Gooch: Employee; Self; SymbioCellTech. P. Zhang: Employee; Self; SymbioCellTech. Z. Hu: Employee; Self; SymbioCellTech. C. Westenfelder: Consultant; Self; SymbioCellTech.

Characterizing Donor‐Specific T Cells in Islet Transplant Recipients Using scRNA‐seq

Organ transplants remain an effective treatment of various end‐stage diseases. Induction of tolerance has remained the elusive goal of transplant community to overcome the mortality and morbidity associated with chronic immunosuppression for long term function of the transplanted organs. We have developed a tolerogenic protocol using apoptotic donor leukocytes (ADLs) that circumvents the need for immunosuppressants and have established the efficacy of ADLs for islet transplants in macaques with diabetes. However, the mechanism by which ADLs induce tolerance has not been fully elucidated. Single‐cell RNA‐sequencing (scRNA‐seq) has proven to be a powerful tool for precisely tracking changes to specific cell types and cell developmental trajectories. Flow sorting using fluorochrome‐labelled MHC class II tetramers was used to isolate donor‐specific T cells from macaques that did and did not receive ADLs prior to an islet transplant, and scRNA‐seq on the 10X Genomics platform was used to characterize these cells. Transcription profiling, TCR fingerprinting and functional clustering of the scRNA‐seq data was used to describe how treatment with ADLs altered specific T cell subtypes, as well as how they develop and interconvert between subtypes. Additionally, combining scRNA‐seq data with the macaques’ clinical outcomes will enable identification of biomarkers that can alert physicians to the fate of the transplanted islets (tolerance or rejection). The results of this project enable a better understanding of the fundamental molecular mechanisms of tolerance induced by ADLs, and further validation studies of the identified biomarkers could greatly aid the physicians’ ability to non‐invasively monitor transplant recipients.Support or Funding InformationFunding from Diabetes Research Wellness Foundation

Characterization of pre-transplant psychosocial burden in an integrated national islet transplant program

ABSTRACT The psychological burden experienced by people with diabetes prior to islet transplantation is recognized but has not been studied comprehensively, especially in relation to glycemia. Therefore, we conducted a rigorous pre-operative psychosocial profile of UK islet transplant recipients, and compared groups with higher/lower HbA1 c to test the null hypothesis that pre-transplant hypoglycemia awareness and psychosocial burden would not be related to baseline HbA1 c in this high-risk cohort. Pre-transplant, recipients (n = 44) completed validated hypoglycemia awareness questionnaires and generic/diabetes-specific measures of psychological traits and states. Scores were compared in groups, dichotomized by HbA1 c (≤8% versus >8%). Participants were aged (mean±SD) 53 ± 10 years; 64% were women; with HbA1 c 8.3 ± 1.7%. Median rate of severe hypoglycemia over the preceding 12 months was 13 events/person-year and 90% had impaired awareness of hypoglycemia (Gold/Clarke score ≥4). Participants had elevated fear of hypoglycemia (HFS-II Worry), impaired diabetes-specific quality of life (DQoL) and low generic health status (SF-36; EQ-5D). One quarter reported scores indicating likely anxiety/depression (HAD). Dispositional optimism (LOT-R) and generalized self-efficacy (GSE) were within published ‘norms.’ Despite negative perceptions of diabetes (including low personal control), participants were confident that islet transplantation would help (BIPQ). Hypoglycemia awareness and psychosocial profile were comparable in lower (n = 24) and higher (n = 20) HbA1 c groups. Islet transplant candidates report sub-optimal generic psychological states (anxiety/depressive symptoms), health status and diabetes-specific psychological states (fear of hypoglycemia, diabetes-specific quality of life). While their generic psychological traits (optimism, self-efficacy) are comparable with the general population, they are highly optimistic about forthcoming transplant. HbA1 c is not a proxy measure of psychosocial burden, which requires the use of validated questionnaires to systematically identify those who may benefit most from psychological assessment and support.

The Role of Vitamin D and Omega-3 PUFAs in Islet Transplantation.

Recurrence of autoimmunity and allograft rejection represent major challenges that impact the success of islet transplantation. Despite the remarkable improvements achieved in immunosuppression strategies after the publication of the Edmonton protocol, long-term data of intra-hepatic islet transplantation show a gradual decline in beta-cell function. Therefore, there is a growing interest in the investigation of novel, safe and effective anti-inflammatory and immunomodulatory strategies able to promote long-term islet graft survival and notable improvements in clinical outcomes of islet transplant recipients. Vitamin D has been shown to exert anti-inflammatory and immunomodulatory effects. Pre-clinical studies investigating the use of vitamin D and its analogs (alone or in combination with immunosuppressive agents and/or other anti-inflammatory agents, such as omega-3 polyunsaturated fatty acids) showed beneficial results in terms of islet graft survival and prevention of recurrence of autoimmunity/allograft rejection in animal models of syngeneic and allogeneic islet transplantation. Moreover, epidemiologic studies demonstrated that vitamin D deficiency is highly prevalent after solid organ transplantation (e.g., heart, liver or kidney transplantation). However, studies that critically assess the prevalence of vitamin D deficiency among islet transplant recipients have yet to be conducted. In addition, prospective studies aimed to address the safety and efficacy of vitamin D supplementation as an adjuvant immunomodulatory strategy in islet transplant recipients are lacking and are therefore awaited in the future.

260-LB: Continuous Glucose Monitoring Metrics in Islet Transplant Recipients with Long-Term Insulin Independence

Continuous glucose monitoring (CGM) following islet transplantation (ITx) has shown improved time-in-range, reduced glycemic variability (GV) and hypoglycemia prevention despite subnormal beta-cell function. However, it is unclear if improvement by CGM persists long-term in insulin independent (Ifree) ITx recipients. We evaluated 5 ITx recipients who remained Ifree for &amp;gt;5 years. Subjects completed 7-day non-blinded CGM (Dexcom G4 Platinum). HbA1c, 90min C-peptide, Clarke score, and BETA-2 score (composite measure of beta-cell function after ITx) were obtained. Before ITx, mean age was 47.2±4.0 years, diabetes duration 34.6±9.4 years, HbA1c 7.0±1.2%, insulin/Kg/day 0.44±0.08 units and Clarke score 5 (range 4-7). Subjects received 1-2 islet infusions. Induction immunosuppression consisted of ATG or daclizumab and dual maintenance with sirolimus, tacrolimus or mycophenolate mofetil. At time of CGM, subjects were Ifree and 10.7±3.5 years post-ITx (range 7.7-16.0). Two subjects were on non-insulin adjunct therapies. CGM and metabolic data, BETA-2 and Clarke scores are shown in Table 1. In Ifree ITx recipients with very good long-term graft function (BETA-2 score&amp;gt;15) and restoration of hypoglycemia awareness, CGM shows near-normalization of time-in-range and GV with minimal time-in-hypoglycemia. ITx is a successful long-term cellular therapy for T1D in a selected group of patients. Disclosure D. Baidal: None. A.M. Alvarez Gil: None. N. Padilla: None. C. Ricordi: Advisory Panel; Self; Zone Labs. R. Alejandro: None. Funding National Institute of Allergy and Infectious Diseases/National Institute of Diabetes and Digestive and Kidney Diseases (U01-DK-070460); Immune Tolerance Network (NIS01); Diabetes Research Institute Foundation; State of Florida; University of Miami Clinical and Translational Science Institute (1UL1TR000460); JDRF International (4-2004-361)

220-OR: Is There Any Prognostic Value of Diabetes-Associated Autoantibody Levels for Predicting Islet Graft Function in Patients with Long-Standing Type 1 Diabetes Mellitus?

Introduction: Correlation between increasing titers for GADA, IA-2A, and ZnT8A and graft loss due to recurrent autoimmunity has been reported, but not confirmed in other studies. Here, we assessed the prognostic value of DAA levels for predicting graft function in our cohort of islet transplant recipients. Methods: Twelve patients with T1DM with hypoglycemic unawareness underwent islet allotransplantation (ITx). Eight of those patients were randomly assigned to receive Reparixin (CXCR1/2 inhibitor) and remaining 4 to placebo in addition to thymoglobulin, tacrolimus and mycophenolate mofetil for immunosuppression. Titers of DAAs were monitored before IT and 0, 7, and 75 days and every 3 months post-operatively. Results: IA-2A and ZnT8A levels were undetectable prior to and after IT in all 12 T1DM patients at any time points, median follow-up of 56 months (24-66) so had no value in islet monitoring. No difference was observed for trends of GADA titers between 4 patients with long-term insulin independence after IT and the remaining 8 who experienced a decline in islet graft function. Prior to and after IT, GADA levels did not vary between patients receiving Reparixin and placebo with most GADA negative (65% and 50%, respectively). Most patients converted from GADA seronegative to seropositive or increased antibody titers [5/8 (62.5%) and 4/4 (100%), respectively] and converted back to seronegative by 1 year post-transplant and maintained partial or complete islet function over 4 years. Conclusion: We found no correlation between trends of anti-GAD65 autoantibody levels and IT outcome in our 12-patient cohort. Levels of DAAs targeting IA-2 and ZnT8 were not detectable in all patients prior to and after IT and did not correlate with IT outcome. Differences in immunosuppression and anti-inflammatory regimens and patient characteristics between studies may contribute to inconsistency among reported results. Disclosure P.J. Bachul: None. J.E. Golebiewska: None. F. Antic: None. M. Para: None. L. Basto: None. A.C. Lucander: None. K. Golab: None. L. Perea: None. M.L. Tibudan: None. L. Wang: None. C.C. Thomas: None. P. Witkowski: None. Funding State of Illinois; Dompé

Standardisation of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients.

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50–300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.

Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions.

Pancreatic islet transplantation has become an established approach to β-cell replacement therapy for the treatment of insulin-deficient diabetes. Recent progress in techniques for islet isolation, islet culture, and peritransplant management of the islet transplant recipient has resulted in substantial improvements in metabolic and safety outcomes for patients. For patients requiring total or subtotal pancreatectomy for benign disease of the pancreas, isolation of islets from the diseased pancreas with intrahepatic transplantation of autologous islets can prevent or ameliorate postsurgical diabetes, and for patients previously experiencing painful recurrent acute or chronic pancreatitis, quality of life is substantially improved. For patients with type 1 diabetes or insulin-deficient forms of pancreatogenic (type 3c) diabetes, isolation of islets from a deceased donor pancreas with intrahepatic transplantation of allogeneic islets can ameliorate problematic hypoglycemia, stabilize glycemic lability, and maintain on-target glycemic control, consequently with improved quality of life, and often without the requirement for insulin therapy. Because the metabolic benefits are dependent on the numbers of islets transplanted that survive engraftment, recipients of autoislets are limited to receive the number of islets isolated from their own pancreas, whereas recipients of alloislets may receive islets isolated from more than one donor pancreas. The development of alternative sources of islet cells for transplantation, whether from autologous, allogeneic, or xenogeneic tissues, is an active area of investigation that promises to expand access and indications for islet transplantation in the future treatment of diabetes.

Effects of Moderate Cycling Exercise on Blood Glucose Regulation Following Successful Clinical Islet Transplantation.

Islet transplantation is effective in preventing hypoglycemia in patients with type 1 diabetes (T1D). However, it is unknown whether transplanted islets regulate plasma glucose concentrations appropriately during and after exercise in human islet transplant recipient (ITxs). To determine the effect of exercise on plasma glucose, insulin, and glucagon concentrations in ITxs compared with control subjects (CONs) without diabetes. Participants completed two conditions in random order: 45 minutes of aerobic exercise (60% VO2peak) and 45 minutes of seated rest. Blood samples were drawn at baseline, immediately after exercise or rest, and every 15 minutes throughout a 60-minute recovery period. Postexercise (24 hours) interstitial glucose was monitored with continuous glucose monitoring (CGM). Twenty-four participants (12 ITxs, 12 CONs) completed the protocol. Plasma glucose decreased more over time with exercise in ITxs compared with CONs [main effects of treatment (P = 0.019), time (P = 0.001), and group (P = 0.012)]. Plasma glucose was lower during exercise vs rest in ITxs but not CONs [treatment by group interaction (P = 0.028)]. Plasma glucose decreased more during exercise than during rest [treatment by time interaction (P = 0.001)]. One ITx and one CON experienced plasma glucose concentrations <3.5 mmol/L at the end of exercise, both of whom returned above that threshold within 15 minutes. Nocturnal CGM glucose <3.5 mmol/L was detected in two CONs but no ITxs. Despite a greater plasma glucose decline during exercise in ITxs, hypoglycemia risk was similar during and after exercise in ITxs compared with CONs.

Current state and future evolution of pancreatic islet transplantation.

Pancreatic islet transplantation provides an effective treatment option for patients with type 1 diabetes (T1D) with intractable impaired awareness of hypoglycemia and severe hypoglycemic events. Currently, the primary goal of islet transplantation should be excellent glycemic control without severe hypoglycemia, rather than insulin independence. Islet transplant recipients were less likely to achieve insulin independence, whereas solid pancreas transplant recipients substantially had greater procedure‐related morbidity. Excellent therapeutic effects of islet transplantation as a result of accurate blood glucose level–reactive insulin secretion, which cannot be reproduced by current drug therapy, have been confirmed. Recent improvement of islet transplantation outcome has been achieved by refinement of the pancreatic islet isolation technique, improvement of islet engraftment method, and introduction of effective immunosuppressive therapy. A disadvantage of islet transplantation is that donors are essential, and donor shortage has become a hindrance to its development. With the development of alternative transplantation sites and new cell sources, including porcine islet cells and embryonic stem/induced pluripotent stem (ES/iPS)‐derived β cells, “On‐demand” and “Unlimited” cell therapy for T1D can be established.

Correlation of Dynamic Immune Cell Profile with Individual Alloresponse and Outcome in Type 1 Diabetes Islet Transplantation Recipients

To evaluate dynamic whole blood immune profile(WBIP) of T1D islet transplant(Tx) recipient(R) pre &post-transplant(Tx), and evaluate for correlation with Tx outcomes. Materials and Methods WBIP of 9 T1D non-Tx patients & 8 volunteers was analyzed by FACS for absolute numbers of WCC, granulocyte(GR), monocyte(MO), B, NK, &T cells including NKT. WBIP of 4 Tx recipients(R1-4) was performed pre-Tx &post Tx [(at 2 weeks(w), &1, 3, 6, &12 months(m)]. R1 received 3 Tx, R2 received 2 Tx, and R3 & R4 had 1 Tx. The fold change of cell populations following Tx was compared to levels prior to 1st Tx. The induction immune suppression was ATG/Tacrolimus/MMF/Etanercept(Eta), except R1 didn't receive Eta at his 1st Tx. Results All analyzed subjects (for R1-4, prior to their 1st Tx) had levels within normal range for all assessed immune cells; except R2 who had an elevated WCC(1272cells/uL) due to high GR(9383cells/ul). The current clinical progress of the recipients is summarized in table 1. R1 had a significant reduction in insulin requirement after the 2ndTx, and was insulin free after the 3rd. In R1 during the 13m post-1st Tx, T cells were greatly reduced(3.7/43fold), and to a lesser extent MO, B & NK cells(1/3.8fold)(Fig1). Minor reductions in GR & NKT cells was observed(≤1.5fold) after 1st Tx in R1, but much greater reductions were seen (3/13.5, vs 9/11fold) within the 1st m following his 2nd and 3rd Txs. Of note, Eta was not given with the 1st Tx(Fig.1). R2 remains on insulin after 2 Txs, but with a partial reduction in insulin use. The degree of decline of T cells during the 6m post Tx(2.5/6.7fold) was less marked than in R1, R3 & R4 at the same time points. Slight reductions in GR, MO, NK, NKT & B cells(1.2/3.2fold) was also observed, while there was an increase in NK(1.5fold) and NKT(1.3fold) at 3m and 1m respectively post-Tx (Fig.1). In R3 remains on insulin after 1 Tx, but dosage has halved. Over the first 6m post Tx, there was a decline in T cells (4/8.8fold), B cells (1.5/2.4fold), NK (1.4/2.8fold) and GR(1/1.2fold) while MO increased slightly (1.1fold) at 2w post-Tx, but then declined at subsequent time points(Fig.2). In R4 after 1 Tx who has a partial reduction in insulin dosage, there were marked reductions in T cells and NKT (27/77fold, & 15.2fold) within the 6 months following Tx; while declines in B (2/3fold), NK(5/10fold), and MO (1.4/2.9fold) cells was seen(Fig 2). However, there was a rise in the GR (2.7fold) at 1m Post-Tx, which subsequently returned to pre-tx levels (Fig 2). Discussions We observed the changes of dynamic WBIP to immunosuppression differed between individual Tx recipients despite similar regimens, and the reduction degree in T cells did not correlate with successful outcomes. Conclusion Dynamic WBIP after Tx can be performed and used to evaluate an individual’s response to Tx and immunosuppression. Monitoring WBIP may be important after Tx to guide the usage of immunosuppression.

Ten Year Insulin-Independence in Select Islet Transplant Recipients Receiving CNI-Sparing Immunosuppression with Either Costimulation Blockade or Anti-LFA1

Ten Year Insulin-Independence in Select Islet Transplant Recipients Receiving CNI-Sparing Immunosuppression with Either Costimulation Blockade or Anti-LFA1