Raffaella Melzi

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Aims/hypothesisEfficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation.MethodsSince 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome.ResultsBy univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071).Conclusions/interpretationThese data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

CXCR1/2 inhibition enhances pancreatic islet survival after transplantation

Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation.

Rapamycin unbalances the polarization of human macrophages to M1

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon‐γ or interleukin‐4 (IL‐4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor‐β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL‐6, tumour necrosis factor‐α and IL‐1β release but reduced CD206 and CD209 expression and IL‐10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll‐like receptor 4‐stimulated peripheral blood mononuclear cells showed a clear shift to an M1‐like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2‐related diseases through mTOR inhibitor treatment.

Mesenchymal Cells Appearing in Pancreatic Tissue Culture Are Bone Marrow‐Derived Stem Cells With the Capacity to Improve Transplanted Islet Function

Adherent fibroblast‐like cells have been reported to appear in cultures of human endocrine or exocrine pancreatic tissue during attempts to differentiate human β cells from pancreatic precursors. A thorough characterization of these mesenchymal cells has not yet been completed, and there are no conclusive data about their origin.

Alloantibody and Autoantibody Monitoring Predicts Islet Transplantation Outcome in Human Type 1 Diabetes

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.

Autologous Pancreatic Islet Transplantation in Human Bone Marrow

The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans.

Bone marrow as an alternative site for islet transplantation

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

Effects of calcitonin gene-related peptide and amylin on human osteoblast-like cells proliferation.

Expression of mRNA for calcitonin gene-related peptide (CGRP) and CGRP receptor has been detected in osteoblasts indicating that CGRP could play a role in bone metabolism. In the present study, we evaluated the effect of CGRP on primary culture of human osteoblast-like cells proliferation. The peptide was able to stimulate [3H]thymidine incorporation in human osteoblast-like cells with a maximal effect at 10(-8) M. The proliferating activity of CGRP was not inhibited by the two antagonists, CGRP-(8-37) or amylin-(8-37), whereas amylin fragment antagonized the proliferating activity of amylin. In human osteoblast-like cells CGRP, but not amylin, was able to stimulate adenylyl cyclase activity and this effect was completely antagonized only by CGRP-(8-37) and not by amylin-(8-37). These data suggest that the CGRP induced stimulation of cAMP is not involved in the peptide proliferating effect in human osteoblast-like cells and that in this cell population there are receptor subtypes for CGRP, distinct from that of amylin.

Role of CCL2/MCP-1 in islet transplantation.

High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent proinflammatory effects that influence engraftment and graft acceptance. Both in mice and humans CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released “proinflammatory” IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islets into syngenic recipients did not improve the transplant function. Transplantation of islets into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islets CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of “inflamed” islets without having a direct role in graft failure. On the other hand, a causal effect for developing detrimental proinflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

Extending indications for islet autotransplantation in pancreatic surgery.

Objective: To assess metabolic and oncologic outcomes of islet autotransplantation (IAT) in patients undergoing pancreatic surgery for either benign or malignant disease. Background: IAT is performed to improve glycemic control after extended pancreatectomy, almost exclusively in patients with chronic pancreatitis. Limited experience is available for other indications or in patients with pancreatic malignancy. Methods: In addition to chronic pancreatitis, indications for IAT were grade C pancreatic fistula (treated with completion or left pancreatectomy, as indicated); total pancreatectomy as an alternative to high-risk anastomosis during pancreaticoduodenectomy; and distal pancreatectomy for benign/borderline neoplasm of pancreatic body-neck. Malignancy was not an exclusion criterion. Metabolic and oncologic follow-up is presented. Results: From November 2008 to June 2012, 41 patients were candidates to IAT (accounting for 7.5% of all pancreatic resections). Seven of 41 did not receive transplantation for inadequate islet mass (4 pts), patient instability (2 pts), or contamination of islet culture (1 pt). IAT-related complications occurred in 8 pts (23.5%): 4 bleeding, 3 portal thromboses (1 complete, 2 partial), and 1 sepsis. Median follow-up was 546 days. Fifteen of 34 patients (44%) reached insulin independence, 16 patients (47%) had partial graft function, 2 patients (6%) had primary graft nonfunction, and 1 patient (3%) had early graft loss. Seventeen IAT recipients had malignancy (pancreatic or periampullary adenocarcinoma in 14). Two of them had already liver metastases at surgery, 13 were disease-free at last follow-up, and none of 2 patients with tumor recurrence developed metastases in the transplantation site. Conclusions: Although larger data are needed to definitely exclude the risk of disease dissemination, the present study suggests that IAT indications can be extended to selected patients with neoplasm.