S.S. Kong

Improved functional survival of human islets of langerhans in three-dimensional matrix culture

The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.

Induction of Donor-Specific Tolerance to Rat Islet Allografts by Intrathymic Inoculation of Solubilized Spleen Cell Membrane Antigens

The development of strategies that will allow permanent survival of islet allografts without continuous host immunosuppression continues to be the most important goal in the field of pancreatic islet cell transplantation. In our study, we demonstrated that intrathymic inoculation of allogeneic spleen cell membrane antigens with a single dose of anti-lymphocyte serum induces an unresponsive state that permits survival of a subsequent pancreatic islet allograft to an extrathymic site (renal subcapsular space). This effect is donor specific and cannot be reproduced by the intravenous injection of spleen cell membrane antigens. Our results offer a potential approach for establishing donor-specific allograft acceptance in adult recipients.

Transplantation of islets of Langerhans in patients with insulin-requiring diabetes mellitus undergoing orthotopic liver transplantation - the Miami experience

Most patients with cirrhosis of the liver have detectable insulin resistance. In 60–80% of patients with cirrhosis, impaired glucose tolerance can be uncovered; approximately 20% of these patients eventually develop overt diabetes. Theoretically, insulin resistance and glucose intolerance could be improved or reversed by orthotopic liver transplantation alone or in association with a simultaneous transplant of pancreatic islet cells from the same donor. To investigate these possibilities we initiated a pilot study of simultaneous liver and pancreatic islet cell transplantation in seven patients with diabetes and liver cirrhosis. Donor bone marrow cells were also infused to enhance the acceptance of the grafts. Seven patients who received only orthotopic liver transplantation and donor bone marrow cells were used as historical controls. The preliminary results of this pilot trial suggest that islet cell transplantation in conjunction with orthotopic liver transplantation improves glucose metabolism in patients with liver cirrhosis in association with reduced insulin requirements and HbA1c levels. These results were evident in spite of pre- and post-transplant basal C-peptide levels that were unchanged. Further evaluation of the effects of orthotopic liver transplantation with or without islet cell transplantation will require a randomized prospective trial including accurate metabolic evaluation with the euglycemic insulin clamp technique.

Expression of a Specific Subset of CD44 Variant Transcripts in NOD Pancreatic Islets

Adhesion of lymphocytes to the endothelial venules inside the islets of Langerhans seems to initiate the infiltration of islets in NOD mice. An overexpression of the lymphocyte surface molecule CD44 in infiltrated NOD islets compared with peripheral blood lymphocytes was recently reported. The CD44 protein family includes a variety of molecules generated by alternative RNA splicing from 10 variant exons (v1–v10). By using reverse transcriptase–polymerase chain reaction followed by Southern blotting and hybridization to exon-specific cDNA probes, we investigated the expression of CD44 isoforms in highly purified islets of Langerhans from 4- and 10-week-old NOD mice. At least six CD44 isoforms were strongly overexpressed in NOD islets at 4 and 10 weeks when compared with age-matched BALB/c islets. Controls in different tissues indicate that these variants are specifically increased in the islets from the NOD strain. Islets from the NOD-scid/scid strain also expressed these variant exons. Splenocytes from BALB/c did not express CD44 isoforms, whereas splenocytes from 4-week-old NOD mice did express CD44 variants. Treatment with inflammatory mediators induced new isoforms; however, these transcripts have a different variant exon composition from that found in NOD mice islets. These results suggest that some isoforms are expressed very early in the development of insulitis by a component of the NOD islet itself and underscore a possible role of CD44 in islet infiltration.