Human Islet Isolation for Autologous Transplantation: Comparison of Yield and Function Using SERVA/Nordmark Versus Roche Enzymes

Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean ± standard deviation; 216 417 ± 79 278 islet equivalent [IEQ] in the LH group; 227 958 ± 58 544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.     

Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children—digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio < 10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.     

The effect of epitope‐based ligation of ICAM‐1 on survival and retransplantation of pig islets in nonhuman primates

Background

Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long‐term survival of the graft. MD‐3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM‐1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long‐term survival of adult wild‐type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD‐3 mAb and maintenance with anti‐CD154 mAb and sirolimus.

Methods

Eleven rhesus monkeys were assigned to three groups. Group 1 (n = 4) involved treatment with MD‐3 induction, short‐term (<4 months) administration of anti‐CD154 mAb, and maintenance therapy with sirolimus. Group 2 (n = 4) involved treatment with MD‐3 induction and long‐term maintenance therapy with anti‐CD154 mAb and sirolimus. Group 3 (n = 3) involved only maintenance therapy with anti‐CD154 mAb and sirolimus. Diabetes was induced in monkeys by streptozotocin, and pig islets (61 000‐112 000 IEQ/kg for each transplant; up to 280 000 IEQ/kg per recipient) were infused through the portal vein. The in vivo functional potency of the isolated islets was tested by minimal model transplant in streptozotocin‐induced diabetic NOD/SCID mice, and the mean AUC of blood glucose level divided by the number of follow‐up days was calculated.

Results

The islet grafts survived more than 6 months (between 225 and 727 days) in nine of 12 transplants of MD‐3‐treated groups 1 and 2, whereas in the absence of MD‐3 mAb, survival was <40 days. In three transplants of the MD‐3‐treated Group 2, functional graft survival was only for 104, 125, and 154 days. In these cases, a retrospective analysis suggested that the relatively short survival duration was associated with the relatively high AUC value in the NOD/SCID bioassay. Notably, when retransplantation was performed in Group 3, blood glucose control was extended up to 956 days, which was supported by MD‐3 mAb‐based suppression of adaptive immunity. No replication of cytomegalovirus genes was observed.

Conclusions

Long‐term survival of pig islet xenografts and successful retransplantation were achieved with MD‐3 mAb‐based immunosuppression regimen in this pig‐to‐monkey transplantation model. It should be emphasized that these encouraging results were achieved following the transplantation of islets from pigs that had not been genetically modified. Considering that it is possible to further substantially reduce the destruction of grafted islet using genetically modified pig islet, the islet requirement could be reduced and much longer graft survival can be achieved.     

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.

     

Effect of hypoxia‐inducible VEGF gene expression on revascularization and graft function in mouse islet transplantation

For gene transfer strategies to improve islet engraftment, vascular endothelial growth factor (VEGF) expression should be regulated in a way that matches the transient nature of revascularization with simultaneously avoiding undesirable effects of overexpression. The aim of this study was to investigate the effects of hypoxia‐inducible VEGF gene transfer using the RTP801 promoter on islet grafts. We implanted pSV‐hVEGF transfected, pRTP801‐hVEGF transfected or nontransfected mouse islets under the kidney capsule of streptozotocin‐induced diabetic syngeneic mice. Human VEGF immunostaining of day 3 grafts revealed that the pRTP801‐hVEGF transfected group had higher hVEGF expression compared with the pSV‐hVEGF transfected group. BS‐1 staining of day 3 grafts from the pRTP801‐hVEGF transfected group showed the highest vascular density, which was comparable with day 6 grafts from the nontransfected group. In 360 islet equivalent (IEQ)‐transplantation which reverted hyperglycemia in all mice, the area under the curve of glucose levels during intraperitoneal glucose tolerance test 7 weeks post‐transplant was lower in mice transplanted with pRTP801‐hVEGF transfected grafts compared with mice transplanted with nontransfected grafts. In 220 IEQ‐transplantations, diabetic mice transplanted with pRTP801‐hVEGF islets became normoglycemic more rapidly compared with mice transplanted with pSV‐hVEGF or nontransfected islets, and diabetes reversal rate after 50 days was 90%, 68%, and 50%, respectively. In conclusion, our results indicate that regulated overexpression of hVEGF in a hypoxia‐inducible manner enhances islet vascular engraftment and preserves islet function overtime in transplants.     

Development of Autoimmune‐Mediated β 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 β 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.     

Distant processing of pancreas islets for autotransplantation following total pancreatectomy.

BACKGROUND: Small duct chronic pancreatitis is associated with intractable pain and failure to thrive, usually unresponsive to conventional management approaches. Total pancreatectomy is considered after failure of medical intervention. The major morbidity following total pancreatectomy is diabetes mellitus with its associated complications. This adverse outcome can be mitigated through autotransplantation of islets recovered from the pancreatectomy specimen. This approach has been limited historically owing to the absence of an on-site islet processing facility. We present the results from 5 pancreatectomized patients whose islets were prepared 1,500 miles away. METHODS: Five patients (4 women, 1 man, average age 42 years) who failed medical therapy and were not candidates for longitudinal pancreaticojejunostomy underwent total/completion pancreatectomy (4 total, 1 completion) for intractable symptoms from idiopathic small duct chronic pancreatitis. The resected pancreata were preserved in ViaSpan solution and were transferred to an islet processing laboratory by commercial airliner and returned. The dispersed pancreatic islet tissue was infused into a portal vein tributary through an operatively placed catheter after systemic heparinization. RESULTS: All 5 patients experienced complete relief from pancreatic pain; 2 had significant residual discomfort from underlying Crohn's disease. Three of the 5 patients had minimal or no insulin requirement after autotransplantation (median follow-up of 23 months); 1 patient continued with glycemic control difficulties related to Crohn's disease. One patient died 17 months following autotransplantation from an unrelated pneumonia. CONCLUSION: Total pancreatectomy with autologous islet transplantation can offer patients with idiopathic small duct chronic pancreatitis pain relief without the sequelae of diabetes mellitus and can be performed without an on-site islet processing facility. All patients undergoing total/ completion pancreatectomy should be considered candidates for this procedure.     

Total pancreaticoduodenectomy with autologous islet transplantation 14 years after liver‐contained composite visceral transplantation

Chronic pancreatitis (CP) is a severely disabling disorder with potential detrimental effects on quality of life, gut function, and glucose homeostasis. Disease progression often results in irreversible morphological and functional abnormalities with development of chronic pain, mechanical obstruction, and pancreatic insufficiency. Along with comprehensive medical management, the concept of total pancreatectomy and islet autotransplantation (TP‐AIT) was introduced 40 years ago for patients with intractable pain and preserved beta‐cell function. With anticipated technical difficulties, total excision of the inflamed‐disfigured gland is expected to alleviate the incapacitating visceral pain and correct other associated abdominal pathology. With retrieval of sufficient islet‐cell mass, the autologous transplant procedure has the potential to maintain an euglycemic state without exogenous insulin requirement. The reported herein case of CP‐induced recalcitrant pain and foregut obstruction is exceptional because of the technical challenges in performing native pancreaticoduodenectomy in close proximity to the composite visceral allograft with complex vascular and gut reconstructions. Equally novel is transplanting the auto‐islets in the liver‐contained visceral allograft. Despite intravenous nutrition shortly after birth, liver transplantation at age 13, retransplantation with liver‐contained visceral allograft at age 17 and TP‐AIT at age 31, the 38‐year‐old recipient is currently pain free with full nutritional autonomy and normal glucose homeostasis.     

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.     

Liraglutide,a long‐acting human glucagon‐like peptide 1 analogue,improves human islet survival in culture

The culture of human islets is associated with approximately 10–20% islet loss, occasionally preventing transplantation. Preconditioning of the islets to improve postculture yields would be of immediate benefit, with the potential to increase both the number of transplanted patients and their metabolic reserve. In this study, the effect of liraglutide, a long‐acting human glucagon‐like peptide 1 analogue, on cultured human islets was examined. Culture with liraglutide (1 μmol/l) was associated with a preservation of islet mass (significantly more islets at 24 and 48 h, compared to control; P ≤ 0.05 at 24 and 48 h) and with the presence of larger islets (P ≤ 0.05 at 48 h). These observations were supported by reduced apoptosis rates after 24 h of treatment. We also demonstrated that human islet engraftment is improved in C57Bl/6‐RAG^?/? mice treated with liraglutide 200 μg/kg sc twice daily (P ≤ 0.05), suggesting that liraglutide should be continued after transplantation. Overall, these data demonstrate the beneficial effect of liraglutide on cultured human islets, preserving islet mass. They support the design of clinical studies looking at the effect of liraglutide in clinical islet transplantation.     

Regulatory challenges in manufacturing of pancreatic islets

At the present time, transplantation of pancreatic islet cells is considered an experimental therapy for a selected cohort of patients with type 1 diabetes, and is conducted under an Investigational New Drug (IND) application. Encouraging results of the Edmonton Protocol published in the year 2000 sparked a renewed interest in clinical transplantation of allogeneic islets, triggering a large number of IND applications for phase I clinical trials. Promising results reported by a number of centers since then prompted the Food and Drug Administration (FDA) to consider the possibility of licensing allogeneic islets as a therapeutic treatment for patients with type 1 diabetes. However, prior to licensure, issues such as safety, purity, efficacy, and potency of the islet product must be addressed. This is complicated by the intricate nature of pancreatic islets and limited characterization prior to transplantation. In this context, control of the manufacturing process plays a critical role in the definition of the final product. Despite significant progress made in standardization of the donor organ preservation methods, reagents used, and characterization assays performed to qualify an islet cell product, control of the isolation process remains a challenge. Within the scope of the FDA regulations, islet cells meet the definition of a biologic product, somatic cell therapy, and a drug. In addition, AABB standards that address cellular therapy products apply to manufacturing facilities accredited by this organization. Control of the source material, isolation process, and final product are critical issues that must be addressed in the context of FDA and other relevant regulations applicable to islet cell products.     

Clinical use of donation after circulatory death pancreas for islet transplantation

Due to a shortage of donation after brain death (DBD) organs, donation after circulatory death (DCD) is increasingly performed. In the field of islet transplantation, there is uncertainty regarding the suitability of DCD pancreas in terms of islet yield and function after islet isolation. The aim of this study was to investigate the potential use of DCD pancreas for islet transplantation. Islet isolation procedures from 126 category 3 DCD and 258 DBD pancreas were performed in a 9-year period. Islet yield after isolation was significantly lower for DCD compared to DBD pancreas (395 515 islet equivalents [IEQ] and 480 017 IEQ, respectively; p = .003). The decrease in IEQ during 2 days of culture was not different between the two groups. Warm ischemia time was not related to DCD islet yield. In vitro insulin secretion after a glucose challenge was similar between DCD and DBD islets. After islet transplantation, DCD islet graft recipients had similar graft function (AUC C-peptide) during mixed meal tolerance tests and Igls score compared to DBD graft recipients. In conclusion, DCD islets can be considered for clinical islet transplantation.     

Beneficial Effects of Desferrioxamine on Encapsulated Human Islets—In Vitro and In Vivo Study

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 μM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose‐induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF‐1α (Hypoxia inducible factor‐1 alpha), caspase‐3 and caspase‐8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase‐3 and caspase‐8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6‐ and 2.5‐fold at 24 and 72 h, respectively, with a concomitant increase in HIF‐1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF‐1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.     

In‐hospital and 90‐day outcomes after total pancreatectomy with islet autotransplantation for pediatric chronic and acute recurrent pancreatitis

Total pancreatectomy with islet autotransplantation (TPIAT) is used to treat debilitating chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP) that has failed medical and endoscopic therapy. We performed a retrospective review of TPIAT patients at a free‐standing children's hospital to evaluate perioperative outcomes. Twenty patients (median age 13, 65% female) underwent TPIAT (2015 through 2017). Of the 20 patients, 95% had CP and 1 patient (5%) had ARP alone. Seventy‐five percent of the patients had a pancreatitis‐associated genetic mutation; 40% had pancreas divisum. The median surgical time was 757 (IQR 657 to 835) minutes. Median islet equivalents per kg of body weight (IEQ/kg) were 6404 (IQR 5018 to 7554). At 90 days postoperatively vs preoperatively, significantly fewer patients were receiving parenteral nutrition (0% vs 25%, P = .006) and opioids (45% vs 75%, P = .01). Short Form 36‐Item Health Survey (SF‐36) physical health module scores and total scores improved (34.0 preoperatively vs 54.6 at 90 days, P = .008, and 47.1 vs 65.3, P = .007, respectively); SF‐10 physical health scores also improved (13.4 vs 33.1, P = .02). Insulin requirement decreased from 0.5 unit/kg/day to 0.4 unit/kg/day between discharge and 90 days (P = .02). TPIAT is an effective option when debilitating disease persists despite maximal medical and endoscopic therapy. Opioid, parenteral nutrition, and exogenous insulin use can successfully be weaned within 90 days after TPIAT, with gains in health‐related quality of life.     

Prolonged Survival of Allogeneic Islets in Cynomolgus Monkeys After Short‐Term Anti‐CD154‐Based Therapy: Nonimmunologic Graft Failure?

Conventional drug therapy and several anti-CD154 mAb-based regimens were tested in the nonhuman primate (NHP) islet allograft model and found to be inadequate because islets were lost to rejection. Short-term therapy with an optimized donor-specific transfusion (DST) + rapamycin (RPM) + anti-CD154 mAb regimen enables immunosuppression drug-free islet allograft function for months following cessation of therapy in the NHP islet allograft model. After a substantial period of drug-free graft function, these allografts slowly and progressively lost function. Pathologic studies failed to identify islet allograft rejection as a destructive islet invasive lymphocytic infiltration of the allograft was not detected. To evaluate the mechanism, immunologic versus nonimmunologic, of the late islet allograft loss in hosts receiving the optimized therapeutic regimen, we performed experiments with islet autografts and studied islet function in NHPs with partial pancreatectomy. The results in both experiments utilizing autologous islet allografts and partially pancreatectomized hosts reinforce the view that the presence of a marginal islet mass leads to slowly progressive nonimmunological islet loss. Long-term clinically successful islet cell transplantation cannot be realized in the absence of parallel improvements in tolerizing regimens and in the preparation of adequate numbers of islets.     

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo‐reactivity

Modification of human islets prior to transplantation may improve long‐term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo‐rejection. Intragraft incorporation of stem cells secreting beta (β)‐cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three‐dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture‐assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo‐response in discrete T‐cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo‐response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T‐cell subsets to AEC‐bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre‐transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo‐reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long‐term graft survival in islet transplantation.     

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.     

BMX‐001, a novel redox‐active metalloporphyrin,improves islet function and engraftment in a murine transplant model

Islet transplantation has become a well‐established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX‐001 (MnTnBuOE‐2‐PyP⁵⁺ [Mn(III) meso‐tetrakis‐(N‐ b ‐butoxyethylpyridinium‐2‐yl)porphyrin]) and its earlier derivative, BMX‐010 (MnTE‐2‐PyP [Mn(III) meso‐tetrakis‐(N‐methylpyridinium‐2‐yl)porphyrin]) could improve islet function and engraftment outcomes. Long‐term culture of human islets with BMX‐001, but not BMX‐010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24 hours with 34 μmol/L BMX‐001 exhibited improved insulin secretion (n = 3 isolations, P < .05) in response to glucose relative to control islets. In addition, 34 μmol/L BMX‐001–supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P < .05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34 μmol/L BMX‐001–treated islet recipients became euglycemic (n = 11 of 19) compared with 19% of nontreated control islet recipients (n = 3 of 19, P < .05). Of murine recipients receiving a marginal dose of human islets cultured with 34 μmol/L BMX‐001, 92% (n = 12 of 13) achieved euglycemia compared with 57% of control recipients (n = 8 of 14, P = .11). These results demonstrate that the administration of BMX‐001 enhances in vitro viability and augments murine marginal islet mass engraftment.     

The risks of total pancreatectomy and splenic islet autotransplantation

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence. two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.     

Prevention of primary nonfunction of canine islet autografts by treatment with pravastatin.

BACKGROUND: Nonspecific inflammation is the primary cause of early islet graft loss. We have shown in mice that pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, prevents primary nonfunction of islet isografts by reducing inflammatory reactions at the graft site. This study was designed to test the effectiveness of this agent in a large animal model, dogs, by transplanting autologous islets. METHODS: After total pancreatectomy, islets were isolated by using a two-step digestion method, followed by discontinuous gradient centrifugation on EuroFicoll. A known number of freshly isolated islets were immediately transplanted back into the same dog via the portal vein. RESULTS: First, we determined the minimal islet number required to reverse diabetes by transplanting 3,000-10,000 IEQ/kg with no additional treatment. The number was found to be 4,000 IEQ/kg, and islets less than 4,000 IEQ/kg consistently failed. To test the effect of pravastatin, 3,000 IEQ/kg were transplanted into dogs that either received no further treatment or were treated daily with 20 mg/kg of pravastatin from days -2 to 14. Without pravastatin, this number of islets lowered blood glucose only transiently, and all four of these dogs became hyperglycemic within 1 week. In contrast, four of the five dogs treated with pravastatin became normoglycemic (<150 mg/dL) and maintained this level during the observation period of 12 weeks (P<0.05). Postprandial plasma glucose and insulin levels returned to normal, and K values of intravenous glucose tolerance tests were significantly higher in pravastatin-treated dogs than in controls (P<0.04 at week 2 and P<0.01 at week 4). CONCLUSION: Peritransplant pravastatin treatment reduced the number of autologous islets required to reverse diabetes in totally pancreatectomized dogs. These results suggest that pravastatin may also facilitate better islet graft survival and function in clinical transplantation.