Ray V. Rajotte

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.

Variables in organ donors that affect the recovery of human islets of Langerhans.

In an attempt to reduce the variability in the yields of human islets isolations and to identify donor factors that were potentially deleterious, we retrospectively reviewed 153 human islets isolations in our center over a 3-year period. Isolations were performed using controlled collagenase perfusion via the duct, automated dissociation, and Ficoll purification. Factors leading to successful isolations (recovery of >100,000 islet equivalents at a purity >50%) were analyzed retrospectively using univariate and multivariate analysis. Critical factors in the multiorgan cadaveric donors that were identified using univariate analysis included donor age (P<0.01), body mass index (BMI)(P<0.01), cause of death (P<0.01), and prolonged hypotensive episodes (systolic blood pressure <90 mmHg or mean arterial pressure <60 mmHg for > 15 min) requiring high vasopressors (>15 microgram/kg/min dopamine or >5 microgram/kg/min Levophed) (P>0.01). Independent analysis of 19 donor variables using multivariate logistic stepwise regression showed six factors were statistically significant. Odds ratio (OR) showed that donor age (OR 1.1, P<0.01), local procurement team (OR 10.9, P<0.01), and high BMI (OR 1.4, P<0.01) had a positive correlation with islet recovery. In contrast, hyperglycemia (all blood glucose >10 mmol/L) (OR 0.63, P<0.01), frequency and duration of cardiac arrest (OR 0.7, P<0.01), and increased duration of cold storage before islet isolation (OR 0.83, P<0.01) had negative correlation. Using these combinations of factors, the prediction of success was 85% accurate. By donor age, success was 13% for 2.5- to 18-year-old donors (n=23), 37% for 19- to 28-year-old donors (n=30), 65% for 29- to 50-year-old donors (n=70), and 83% for 51- to 65-year-old (n=29) donors. However, when vitro function was assessed by perifusion, the insulin secretory capabilities of islets isolated from the >50-year-old donor group was significantly reduced as compared with the 2.5- to 18-year-old group (P<0.02). Multiple regression analysis using postdigestion and postpurification islet recovery as outcome variables identified BMI, procurement team, pancreas weight, and collagenase digestion time factors tht can affect the recovery of human islets. Locally procured pancreases and donors with elevated minimum blood glucose levels were identified as factors that affect the insulin secretory capabilities of the isolated islets. This review of parameters suggests an improved approach to the prediction of successful islet isolation from human pancreases. Selection of suitable pancreases for processing may improve consistency in human islet isolation and thereby decrease costs.

Cotransplantation of Allogeneic Islets With Allogeneic Testicular Cell Aggregates Allows Long-Term Graft Survival Without Systemic Immunosuppression

We prepared single-cell suspensions of Lewis rat (RT11/1) testicular cells and cultured these in vitro for 48 h under conditions that promoted the formation of cellular aggregates. In the absence of systemic immunosuppression, the transplantation of a sufficient quantity of these aggregates (containing 11 × 106 cells, (75% Sertoli cells), together with 2,000 purified Lewis rat islets, reversed the diabetic state for >95 days in 100% (5/5) of the chemically diabetic Wistar-Furth (RT1u/u) recipients. Similar grafts consisting of islets alone or islets plus 50% fewer testicular cell aggregates survived for only 10 days. Functioning composite allografts harvested from normoglycemic animals at ü100 days showed healthy β-cells in close association with Fas ligand–expressing Sertoli cells. Because no gene therapy protocol is required, the transplantation of composite grafts consisting of purified human allogeneic islets plus human allogeneic testicular cell aggregates can be applied in clinical islet transplantation as soon as it has been proven in a large animal model.

Granzyme B: a natural born killer

Summary:  A main pathway used by cytotoxic T lymphocytes (CTLs) and natural killer cells to eliminate pathogenic cells is via exocytosis of granule components in the direction of the target cell, delivering a lethal hit of cytolytic molecules. Amongst these, granzyme B and perforin have been shown to induce CTL‐mediated target cell DNA fragmentation and apoptosis. Once released from the CTL, granzyme B binds its receptor, the mannose‐6‐phosphate/insulin‐like growth factor II receptor, and is endocytosed but remains arrested in endocytic vesicles until released by perforin. Once in the cytosol, granzyme B targets caspase‐3 directly or indirectly through the mitochondria, initiating the caspase cascade to DNA fragmentation and apoptosis. Caspase activity is required for apoptosis to occur; however, in the absence of caspase activity, granzyme B can still initiate mitochondrial events via the cleavage of Bid. Recent work shows that granzyme B‐mediated release of apoptotic factors from the mitochondria is essential for the full activation of caspase‐3. Thus, granzyme B acts at multiple points to initiate the death of the offending cell. Studies of the granzyme B death receptor and internal signaling pathways may lead to critical advances in cell transplantation and cancer therapy.

Differential Expression of GAD65 and GAD67 in Human, Rat, and Mouse Pancreatic Islets

The smaller form of the autoantigen glutamic acid decarboxylase, GAD65 (formerly the 64,000 Mr autoantigen), is a major target of humoral autoimmunity in type I diabetes. Human autoantisera have been used extensively to characterize the GAD65 antigen in both rat and human islets, but the protein has escaped detection in mouse islets. We have now analyzed the expression of GAD65 and GAD67, the larger glutamic acid decarboxylase protein, in human, rat, and mouse islets of Langerhans and brain, using human monoclonal islet cell autoantibodies, human autoantisera, and experimentally raised antibodies to glutamic acid decarboxylase. Human monoclonal autoantibodies and experimentally raised antibodies reacted with mouse GAD65 produced in a baculovirus expression system by Western blotting and immunoprecipitation and with GAD65 in mouse brain by immunohistochemistry but failed to detect GAD65 in mouse islets by the latter two methods. However, analysis of mouse islets by Western blotting technique, using the most sensitive experimentally raised antibody, showed that mouse islets express both GAD65 and GAD67 but at levels that are severalfold lower than those in mouse brain or in human and rat islets. Furthermore, both human and rat islets predominantly express GAD65, whereas GAD67 is the major glutamic acid decarboxylase protein in mouse islets. Human islets are significantly distinct from mouse and rat islets and from brain because they only express GAD65, which is consistent with the predominant role of this form as a target of autoantibodies associated with β-cell destruction in humans. Human as well as rat islet GAD65 are found in both membrane-bound and soluble forms. The low level of glutamic acid decarboxylase expression in mouse islets compared with human and rat islets is likely to have implications for both the development of tolerance to glutamic acid decarboxylase as well as the homing of glutamic acid decarboxylase-specific lymphocytes to the mouse β-cell. In this context, the results suggest 1) that the mouse is ideal for studies of the consequences of an expression of high levels of glutamic acid decarboxylase in the β-cell from a transgene and 2) that the rat may be better suited than the mouse for development of nontransgenic animal models of glutamic acid decarboxylase autoimmunity by immunization.

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Combination Therapy With Sirolimus and Interleukin-2 Prevents Spontaneous and Recurrent Autoimmune Diabetes in NOD Mice

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet beta-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas beta-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (gamma-interferon-positive and IL-2(+)) were decreased the most, and Th2-type cells (IL-4(+) and IL-10(+)) and Th3-type cells (transforming growth factor-beta1(+)) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet beta-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.

Portal venous pressure changes after sequential clinical islet transplantation.

Background. Sequential pancreatic islet transplantation via the portal vein has led to insulin independence in patients with type 1 diabetes. Complications associated with the injection of islets into the portal vein have been reported; therefore, in this study we sought to further characterize changes in portal venous pressure associated with islet infusion. Methods. Pre- and posttransplant portal venous pressures were recorded in 50 consecutive transplant procedures in 26 patients receiving highly purified, heparinized allogeneic islet preparations via a radiologically placed portal venous cannula. Doppler ultrasound scans of the portal vein were completed within 24 hr of transplantation. Results. Posttransplant portal vein pressures rose significantly with sequential transplantation (12.4 mm Hg vs. 17.3 mm Hg, P <0.05). Portal pressure change correlated significantly with islet packed cell volume (r =0.66, P <0.001) and also with the number of islets transplanted (r =0.49, P <0.001). Segmental portal vein thrombosis was radiologically detected after two procedures (4%). Conclusion. Multiple sequential islet transplants can be safely performed via the portal vein, provided that care is taken with islet purification and attention is paid to portal venous monitoring.

Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits Calcify and Fail From Xenograft Rejection

Background— Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. Methods and Results— Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. Conclusions— G-F xenografts have cellular/humoral rejection and calcify secondarily.