©1982 AMERICAN DIABETES ASSOCIATION

How to overcome the remaining hurdles in cell survival, supply, and immune rejection BY A.M. JAMES SHAPIRO Diabetes prevalence has increased from a world estimate of 30 million in 1985 to 180 million currently, and is predicted to rise to 366 million by the year 2025. In developing countries such as China, the number of diagnosed cases is increasing at a frightening rate of 3,000 per day. Upwards of one in 400 children becomes reliant on injected insulin due to type 1 diabetes (T1DM), and even this rate is on the rise. For these individuals, injected insulin is life-sustaining and prevents acute demise from ketoacidosis. Nevertheless, it fails to prevent inexorable secondary microvascular complications: Blindness, renal failure, stroke, neuropathy, amputation, myocardial ischemia, and infarction may occur and contribute to shortened lifespan. Clearly, injected insulin falls far short of the mark. Alternative strategies to restore endogenous insulin include glucose-responsive closed-loop insulin pumps, whole-pancreas transplantation, and islet transplantation. The concept of replenishing destroyed insulin-secreting b cells through transplantation is certainly appealing, but it's not new. Efforts to transplant working islet cells from deceased donors or animals have dotted the history of diabetes research going back to the 1890s, but only within the past decade did we see real strides in relief of symptoms. Islet transplantation as offered today has proven to be incredibly effective at protecting a small subgroup of patients with T1DM from severe and incapacitating hypoglycemic reactions, but in terms of the greater need, the treatment comes nowhere close to the ultimate goal of curing diabetes for these individuals.

Three major challenges remain. First, we need to solve long-term issues with islet survival; second an unlimited cell supply source must be found that does not rely on scarce brain-dead organ donors; and third, we must find ways to overcome the immune system's natural barrier to a transplant, either through safer immunosuppression or immunological tolerance. Exciting progress is occurring on each of these fronts, but it needs to happen faster if we are to ever stem the encroaching tide of diabetes.

The Edmonton Protocol, which our group at the University of Alberta first implemented six years ago, appeared a tremendous advance, but it should be emphasized that this effort was the product of a series of incremental steps stretching back decades (see The Path to Clinical Protocols). The good news is that further incremental steps could take us the rest of the way. If solutions to the major limiting factors can be found within the next five to 10 years, then it's fair to say that a cure for T1DM is indeed within reach. Moreover, with adequate cell supply, there's no reason that we can't change the course for all patients with diabetes.

COURTESY JAMES SHAPIRO, UNIVERSITY OF ALBERTA

By five years after transplant, insulin independence can drop dramatically. But endogenous partial insulin secretion remains in more than 80% of recipients as documented by secretion of C-peptide, a protein released in the processing of proinsulin.

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Despite remarkable progress, particularly since 2000, the Edmonton group and others have detected sharp falloff in insulin-independence rates at three and five years.¹ This observation has been difficult to understand, as most of the grafts (70%) continue to make insulin and C-peptide in reasonable amounts, but at rates that are insufficient to allow permanent freedom from insulin injections. Even low-level endogenous insulin secretion provides potent protection from hypoglycemic reactions, and the degree of glycemic control is far superior to any form of injected insulin therapy. In recognition of this, Phase III trials moving forward in the United States regard the sustained improvement in glycemic control and protection from hypoglycemia as a far more important factor than complete insulin-independence.

Nevertheless, we must work to understand this falloff in insulin production. In some cases, immunological graft injury from rejection or return of autoimmunity could clearly be responsible, but nonimmune islet injury is probably an even more important factor. Islet exhaustion from chronic overstimulation of a marginal islet mass is probably the dominant reason for late partial islet dysfunction. However, at present the pathological lesions associated with nonimmune injury remain obscure.

The requirement for chronic, potent antirejection drugs remains an important limiting factor, as these treatments are often associated with risks and side-effects, and can be justified at present only in those with the most recalcitrant and unstable glycemic control despite optimal insulin therapy. Both tacrolimus and sirolimus have been associated with renal injury, and this can occasionally compound preexisting injury from diabetes. Fatigue, peripheral edema, mild tremor, mouth ulceration, anemia, menstrual irregularities, and diarrhea can be drug and treatment limiting in occasional patients.

Such therapies may also be responsible for chronic islet injury in the long term, too. Sirolimus, for instance, is an antiproliferative drug that inhibits vascular endothelial growth factor (VEGF) and islet revascularization after transplantation, and may further interfere with the islet graft's capacity for repair and regeneration over time. Many transplant scientists are therefore intensively investigating newer and more "islet-friendly" antirejection regimens that will lead to fewer clinical side effects while at the same time providing superior islet function after transplant. The recent development of CD28 costimulation blockade with fusion-protein technologies (LEA29Y [belatacept]) is one such approach presently undergoing clinical evaluation in islet transplantation.^2,3

Rich vasculature surrounds a large rat islet of Langerhans (~300 mm diameter) as seen in this scanning electron micrograph. (Reprinted with permission from Diabetes, 31: 883-9, 1982.)

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For clinicians and scientists involved with islet transplantation, these are particularly exciting times. The remaining challenges are all within reach, and all it will take is a series of carefully thought-out, testable hypotheses and interventional strategies to make further leaps in improved clinical outcomes for this field. Just how many leaps will be needed to transition from the level of effective treatment to durable cure of diabetes may be much harder to define.

Olle Korsgren and the Nordic Network islet transplant team have focused their efforts on enhancing islet survival after transplant through inhibition of what they termed the instant blood-mediated inflammatory reaction (IBMIR). They have identified a panel of potent IBMIR inhibitors that they anticipate will not only optimize early islet survival but may also reduce susceptibility of islets to injury-mediated immune attack.⁴ Growth-factors such as epidermal growth factor, gastrin, or other cocktails may prove fruitful both in expanding islet numbers in the culture dish and after transplantation in patients. Furthermore, these drugs might also stimulate islet recovery within the native pancreas if persistent autoimmune injury can also be held in check.⁵ Another extremely promising avenue is the use of a synthetic extract originally identified in the saliva of the Gila monster. GLP-1 analogues (e.g., exenatide) is currently being tested for its ability to stimulate islet growth and improve function after transplantation in mice and in patients.⁶

Immunological tolerance to islet and other solid-organ transplants has tantalized the transplant community since Peter Medawar and colleagues described this phenomenon in mice more than 50 years ago. The Immune Tolerance Network was set up by the National Institutes of Health and the Juvenile Diabetes Research Foundation with the goal of translating tolerance induction strategies from mice to the clinic. Three major avenues of study have emerged for testing in patients: 1) creation of mixed chimerism by combining bone marrow together with solid-organ transplants; 2) treatment with costimulation blockade antibodies to prevent T cell activation and favor anergy; or 3) infusion of regulatory T cells. Results are eagerly awaited and will eventually have major impact on all forms of clinical transplantation.

Finding an unlimited source of insulin-secreting tissue will be an essential component if cellular replacement therapy is to meet demand. Beyond the ethical and political issues that accompany embryonic stem cell and therapeutic cloning techniques, substantial challenges pervade this entire field. Control of cell growth, differentiation, transdifferentiation, and prevention of tumor development remain major issues beyond the hurdles of physiologically regulated insulin secretion. The use of adult stem cells may avoid emotional and religious issues, but it is also cloaked in controversy relating to the true reserve of human islets in terms of replication.^7,8

THOM GRAVES MEDIA

Improving Islet Transplantation
Click to view enlarged diagram

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The use of pig islet tissue as a potential xenogeneic source of cells for human transplantation just got closer with two recent reports of more than six months of insulin independence in pig-to-monkey transplants. In the first report, Bernhard Hering at the University of Minnesota used adult pig islets and gave potent combination immunosuppression including use of an anti-CD40L antibody. Unfortunately this antibody was associated with alarming rates of clinical and subclinical pulmonary emboli in the treated monkeys.⁹

In a subsequent paper, Christian Larsen and colleagues at Emory University and the University of Alberta used neonatal pig islets for transplantation in primates, and used combination immunosuppressant based on a different anti-CD40L antibody together with an anti-IL2R antibody, costimulation blockade with belatacept, and sirolimus.¹⁰ While concerning rates of opportunistic infections were observed, clinically evident pulmonary emboli were not observed. Both of these studies have generated considerable excitement and demonstrate that the pig-to-primate xenograft response is indeed controllable and can provide full and sustained islet function. Clearly, further refinements are needed to optimize the safety profile of the immunosuppressant regimens if clinical trials are to be considered in the near future.

Cellular replacement therapy is here to stay. A plethora of exciting and key scientific questions remain unanswered but solvable with intense and coordinated efforts between collaborating groups. Just as incremental steps brought about the giant leaps leading up to our present situation, further leaps could take us to the stage of durable cure.

A.M. James Shapiro is clinical research chair in transplantation, and the director of the Clinical Islet Transplant Program at the University of Alberta in Edmonton. The author receives funding from a Clinical Center Grant from the Juvenile?Diabetes Research Foundation, from the National Institutes of Health (NIDDK), from the Immune Tolerance Network, and through a clinical research Chair in Transplantation Research funded jointly by the Canadian Institutes for Health Research and from Wyeth Canada.amjshapiro@the-scientist.com