Islet transplantation: a field on the move

Gérard Reach

INSERM U341, Diabetes Department, Hôtel-Dieu Hospital, Paris, France

Keywords: Immunosuppression; islets of Langerhans; islet transplantation; pancreas transplantation; pancreatectomized patients; type 1 diabetes

Introduction

Since 1966, more than ten thousand pancreas transplantations have been performed throughout the world, with a satisfactory rate of success indicated by low mortality and with insulin independence obtained in about 80% of the patients [1]. However, this therapeutic option is limited by the risks of immunosuppression. Pancreas transplantation is therefore only proposed when an immunosuppressive treatment is justified by the necessity of another graft, mostly of the kidney as treatment for end-stage renal failure, which itself is a complication of diabetes. At least in type 1 diabetes, the incidence of this complication is currently decreasing [2]. This may contribute to the decrease, as observed in France, in the number of patients registered on the lists of pancreas–kidney transplantation. The number of pancreas transplantations has also fallen by half during the last 5 years [3]. This can be explained in part by the general crisis of organ donation, but also by a decrease in the number of potential recipients.

The development by Paul Lacy in 1966 of a method for isolating islets of Langerhans from the rat pancreas led quickly to the demonstration of the possibility of correcting experimental diabetes in the rat by the transplantation of isolated islets. This would represent a logical alternative to the transplantation of the complete organ that is obviously more simple and therefore less risky. In 1988, a method for isolating human islets of Langerhans was developed, allowing the initiation of clinical trials of islet transplantation. However, this was achieved actually in less than 400 patients throughout the world, with the number of attempts remaining quite small (about 30 per year). Until recently, results were distressingly poor, since in contrast to transplantation of the whole organ, prolonged insulin independence was obtained only in 5–10% of patients [4]. This pessimistic view needs to be balanced by the fact that even though insulin independence was rarely achieved, the transplantation of islets brought at times a source of endogenous insulin which was sufficient to obtain an excellent glycaemic control, associated with a remarkable decrease in the risk of severe hypoglycaemia [5]. As with transplantation of the whole pancreas, transplantation of isolated islets of Langerhans also requires indefinite immunosuppression, which limits its indications.

How can one explain these poor results from human islet allografts in type 1 diabetic patients? It is remarkable that allotransplantation of islets performed in pancreatectomized patients gives much better results since interruption of insulinotherapy can be obtained in 50% of cases; autotransplantation of islets in this type of patient yields a success rate of 80% [6]. The difference between the second and the third group of patients may be due to the absence of immunosuppression in the case of the autotransplantation performed after total pancreatectomy; the difference between the first and the second group of patients may be due to the effect, in the case of allotransplantation performed in type 1 diabetic patients, of a prolonged preceding state of diabetes and its complications. These data suggest that a major element responsible for the current failure of islet allotransplantation in type 1 diabetes is the requirement of immunosuppression: as shown above, it limits the indications of transplantation of pancreatic tissue and it may also specifically jeopardize the results of the transplantation of islets of Langerhans. Actually, this is not surprising because it is known that cyclosporin is toxic for isolated islets of Langerhans [7], that cyclosporin inhibits the neovascularization of transplanted islets [8], and that cyclosporin causes insulin resistance [9]. Furthermore, it is impossible to ignore the diabetogenic effect of steroids, which by generating insulin resistance should alter the efficacy of the limited number of islets of Langerhans that can be isolated from a human pancreas. Experience with autotransplantation of islets in the dog suggested that it is necessary to transplant at least 6000 islets per kg body weight to correct diabetes [10]. When extrapolating to a 60-kg man, these data suggest that it would be necessary to transplant 360 000 islets, which represents the approximate number of islets currently isolated from a pancreas, containing normally about a million islets. But what about the number required in the presence of steroids?

Clinical progress: is islet allograft in type 1 diabetic patients finally efficient in 100% of cases?

A possible breakthrough, which became available on the internet in the spring of the year 2000 and as a full paper published in the July 2000 issue of the New England Journal of Medicine [11] was achieved by a Canadian team, which showed for the first time that it was possible to transplant in an effective way allogeneic islets of Langerhans into patients with type 1 diabetes. Briefly, A. M. Shapiro and colleagues in Edmonton, Alberta reported the success of islet allografting in seven consecutive type 1 diabetic patients. They had a mean age of 44 years, and had been diabetic for an average of 35 years. The transplanted islets were freshly isolated, not having been put into culture or cryopreserved. Transplantation was performed in premedicated patients by percutaneous transhepatic injection of the islets into the portal vein under radiological control. The preparation of islets was contained in a volume of 120 ml and was injected within 5 min under control of the portal pressure.

In these seven patients, it was possible to stop insulin administration, with a follow-up of from 4 to 14 months at the time of publication. In six patients it was necessary to perform a second injection of islets 14–70 days after the first injection, and one patient (body weight 90 kg) had to receive a third islet injection. Metabolic results in these previously C-peptide-negative patients were impressive: there was a sustained increase in the rate of C-peptide secretion, a strict normalization of glycated haemoglobin, a spectacular decrease in the amplitude of glycaemic variations in the absence of any hypoglycaemic episode and oral glucose tolerance tests showed only minor abnormalities. The evaluation of insulin sensitivity by the HOMA method gave normal results.

How does one explain this success? Analysis of previous islet transplantation trials suggested that positive outcomes depended on transplanting islets in the absence of immunosuppressive therapy, or at least by using an immunosuppressive strategy less toxic for the graft and which would not cause insulin resistance in the recipient, and performing the transplantation earlier in the evolution of the disease. This was indeed the strategy adopted by the Edmonton team.

First, the immunosuppressive therapy was free of steroids. It consisted of a combination of sirolimus (Rapamune®) and tacrolimus (Prograf®). It had been shown that the combination of these two agents with steroids led to a low rate of rejection in hepatic, renal, and pancreatic transplantations. To avoid the diabetogenic effect of steroids, the immunosuppressive treatment included a monoclonal antibody against the receptor of interleukin-2, daclizumab (Zenapax®). This combination prevents an activation of the immune cascade by inhibiting T-lymphocyte activation, interleukin-2 production as well as that of other cytokines, interleukin-2 receptor binding to its ligand, and clonal lymphocyte expansion. In theory, such a treatment should protect islets not only against graft rejection but also against autoimmunity. Thus, the major point was the absence of steroid and cyclosporin use, and their replacement by an immunosuppressive treatment which should not lead to insulin resistance, as was shown by the HOMA test.

The second original result from this work concerned the recipients: these trials were performed in patients presenting either frequent severe hypoglycaemic episodes or uncontrolled diabetes. The absence of end-stage renal failure (mean serum creatinine level was 1.3 mg/dl) suggests the absence of microangiopathy in general (more information on the ophthalmological status of these patients would be interesting), which may have jeopardized the neovascularization of the transplanted islets in previous trials. One would have also liked to have known the state of insulin resistance in these patients before transplantation.

The third original result of this work concerns the transplanted islets. First, these were freshly isolated islets, having been neither cultivated nor cryopreserved. Second, during islet isolation, the use of any xenoprotein, such as fetal calf serum, was avoided. It is possible that this prevented the aggression of islets, which are made visible to the immune system by a coating of animal proteins. Last but not least, iterative transplantations were achieved, leading to the graft of 11 500 islets/kg on average. This is almost double than that previously considered necessary, and was done in the absence of insulin resistance.

This report is important for two reasons: it demonstrates that it is possible successfully to transplant isolated islets of Langerhans by using a new strategy that actually confirms hypotheses developed to explain previous failures. In addition to the hope that it can generate for diabetic patients, this procedure also presents a theoretical interest and opens new perspectives of research. But it also generates further possibilities which are to revisit the indications for transplantation of islets of Langerhans. Until now, it was thought that transplantation of pancreatic tissue, because of the necessary immunosuppressive treatment, was reserved for patients requiring another organ transplantation. Here, the authors proposed a transplantation of islets of Langerhans and a long-term immunosuppressive treatment to ‘patients with serum glucose concentration remaining uncontrolled despite exogenous insulin therapy, who had to have recurrent severe hypoglycemia with coma or metabolic instability to such an extent that the risk of the transplantation and immunosuppression was judged to be less than the risk of continued uncontrolled diabetes’ [11].

There is no doubt that by preventing the occurrence of these hypoglycaemic episodes, the quality of life of these patients was transformed dramatically. One would have appreciated having more details, first on the real severity of the disease in these patients (for example, the number of severe hypoglycaemic episodes per year, the number and the duration of hospitalizations etc.), and on the basis for evaluation of the relative risks of the immunosuppressive treatment and of the hyperglycaemia. Secondly, a discussion of the long-term potential toxicity of this novel immunosuppressive treatment, especially for the kidney, would have strengthened the paper. Finally, it would be interesting to know if such an immunosuppressive treatment can be used in the more usual indication of islet allograft associated with kidney transplantation, and whether it would protect not only the islets but also the transplanted kidney. Thus, this real progress in the history of the transplantation of islets of Langerhans could be applied to a therapeutic strategy which is already the object of a consensus in the treatment of the diabetic patient with end-stage renal failure.

New approaches to create a tissue that secretes insulin: science fiction?

Let us dream and imagine that a totally non-toxic method of immunosuppression is available, or that a method is developed making it possible to transplant cells in the absence of any immunosuppression, for example by induction of tolerance [12] or cellular immuno-isolation [13]. This process could be applied to the transplantation of islets of Langerhans for all the diabetic patients. The problem of a source of transplantable tissue would then arise, given the scarcity of transplantable organs and the huge number of potential recipients.

Two approaches are possible:

(i) Animal islets of Langerhans could be used, or more precisely porcine islets i.e. islet xenograft. Transplantation of porcine insulin-secreting cells into diabetic patients has already been performed in Sweden in the 90s, and showed the presence of porcine C-peptide in the urine of the transplanted patients over a number of weeks, demonstrating the feasibility of this approach [14]. Previously, and in anticipation of this perspective, the isolation of islets of Langerhans from the pancreas of specific pathogen-free (SPF) pigs had been developed. In addition, the breeding of transgenic pigs is possible, allowing the harvesting of islets of Langerhans, which could present certain advantages in term of xenotransplantation.

However, this option presents a risk that remains to be assessed. By avoiding transmission in the transplanted patient of a conventional zoonosis, this risk can be limited by the use of SPF pigs. Actually, the real risk is linked to the presence in the pig of endogenous retroviruses which could transfect the recipient cells. A modification of the virus in the host could give birth to an infectious pathogenic virus, and could lead in a devastating scenario to the generation of a new viral disease. These major concerns were the object of a debate at the beginning of 1998, leading some to ask for a moratorium on any xenotransplantation clinical trials [15]. Clearly, this question is especially important in the case of islet xenografts: it cannot be considered as a life-saving therapy, since diabetes can be treated with insulin. On the other hand, the risk that a rare event occurs would be increased by the repetition of the procedure in a large population of patients. The future of islet xenografting will therefore depend on the results from this debate.

(ii) Another option consists in the use of cell lines that secrete insulin. Such cell lines have been developed from spontaneous or experimentally induced tumours or were produced by cell molecular engineering [16]. This option presents the advantage of reproducibility of the tissue and control of the infectious risk. It presents the inconvenience of relying on the production of insulin by cells which may not exhibit the fine regulation present in primary pancreatic beta cells. This could decrease the efficiency of these cells in correcting diabetes in the patients to whom they would be transplanted. Risks inherent to this cell engineering approach remain largely unknown.

Islets of Langerhans produced from stem cells correct experimental diabetes in the mouse
A report published in the March 2000 issue of Nature Medicine [17] described the production of functional islets of Langerhans from stem cells isolated from the pancreas of young NOD mice (an auto-immune model of diabetes) sampled before the mice became diabetic. These cells formed neo-islets which by successive culture during 3 years culminated in the generation of a number of islets that was 10 000 times greater than the number of islets that can be isolated from a mouse pancreas. These neo-islets secreted insulin in response to glucose in vitro, and it was shown in a small number of experiments that they were able to correct diabetes in NOD mice in the absence of any immunosuppression. This is interesting because transplanted ‘normal’ islets should have been rejected by the auto-immune process.

One could imagine that by taking a fragment of pancreas from a patient at the onset of diabetes, to generate islets of Langerhans from stem cells present in this fragment, re-implantation would be possible when the patient develops overt insulin-dependent diabetes [18]. Although less ambitious, one could imagine the use of this stem-cell technology to produce an important source of neo-islets of human origin which would be available for allograft in type 1 diabetic patients.

It is possible to produce in vivo insulin-secreting cells from stem cells present in the liver
Transcription of the factor PDX-1 (pancreatic and duodenal homeobox gene 1), also called STF-1, IPF-1 [19], is involved in the control of the expression of numerous genes, including insulin, the glucose transporter Glut-2, and glucokinase, which plays a key role in the mechanism of insulin secretion in response to glucose. Furthermore, it plays a major role in the differentiation of pancreatic stem cells into islets of Langerhans. For example, the absence of the gene coding for PDX-1 leads to the agenesis of the pancreas [20]. Mutations of the gene are associated with a form of maturity diabetes in the young [21].

A recent report by Ferber et al. [22] describes the effect of intravenous injection into non-diabetic mice of a recombinant adenovirus bearing the sequence coding for PDX-1. The gene was expressed in the liver of some of these mice. In these animals, the expression of the gene was accompanied by that of two genes, one coding for insulin and the other for the enzyme that transforms pro-insulin into insulin. The livers of these animals contained some mature insulin which was secreted into the blood stream and their plasma insulin concentration was three times as high as that of the control mice. Secondly, the experiment was repeated in mice made diabetic by streptozotocin. The glycaemia of these animals improved gradually, reaching normal values within 10 days (unfortunately, the figure presenting these results ‘stops’ when glycaemia is normalized, and one would have liked to have known whether glycaemia continued to decrease).

This fascinating work shows that the expression of PDX-1 in the liver leads to the transformation of still unidentified hepatic stem cells into cells having a phenotypic expression of cells capable of secreting insulin into the circulation, and which can produce a hypoglycaemic effect. If these data were applicable to man, one could imagine giving diabetic patients the possibility of producing, from their own hepatic cells, cells capable of secreting insulin. Obviously, it would be necessary to demonstrate that these cells respond to glucose in vivo in a way that achieves glycaemic homeostasis, that they are not vulnerable to the auto-immune process, and of course that it is possible to control the transforming effect in order to avoid the generation of a hepatic insulinoma.

The development of a novel or, as here, a revolutionary therapy, necessitates a careful assessment of risk. Risk, a relative notion, depends on the context: if it can be acceptable in a given situation (serious disease without therapeutic alternatives, which is not the case in diabetes), it must be also compared with its potential benefit. It will be necessary more than ever to apply the Aristotelian phronesis; i.e. prudence, the mother of the principle of precaution (the Latin word for ‘be prudent, be ware’, caute, was written on Spinoza's seal). ‘What characterizes the prudent man is the comprehensive consideration which allows him to avoid immoderation, to appreciate obstacles, to take into account particular cases, to choose the appropriate moment, and even to foresee the unpredictable’ (Ethics to Nicomaque, VI).

Notes

Correspondence and offprint requests to: Gérard Reach, INSERM U341, Diabetes Department, Hôtel-Dieu Hospital, 1 Place du parvis Notre-Dames, F-75181 Paris Cedex 04, France. Back

References

  1. Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE. Pancreas and islet transplantation for patients with diabetes. Diabetes Care2000; 23: 112–116[Free Full Text]
  2. Bojestig M, Arnqvist HJ, Hermansson G, Karlberg BE, Ludvigsson J. Declining incidence of nephropathy in insulin-dependent diabetes mellitus. N Engl J Med1994; 330: 15–18[Abstract/Free Full Text]
  3. Etablissement Français des Greffes, Annual report, 1997
  4. Bretzel RG, Hopt UT, Schatz H. Transplantations in patients with diabetes mellitus. Exp Clin Endocrinol Diabetes2000; 108: 241–242[ISI][Medline]
  5. Federlin K, Pozza G. Indications for clinical islet transplantation today and in the foreseeable future—the diabetologist's point of view. J Mol Med1999; 77: 148–152[ISI][Medline]
  6. Oberholzer J, Triponez F, Mage R et al. Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations. Transplantation2000; 69: 1115–1123[ISI][Medline]
  7. Basadonna G, Montorsi F, Kakizaki K, Merrell RC. Cyclosporin A and islet function. A J Surg1988; 156: 191–193[ISI]
  8. Vajkoczy P, Vollmar B, Wolf B, Menger MD. Effects of cyclosporine A on the process of vascularization of freely transplanted islets of Langerhans. J Mol Med1999; 77: 111–114[ISI][Medline]
  9. Menegazzo LA, Ursich MJ, Fukui RT et al. Mechanism of the diabetogenic action of cyclosporin A. Horm Metab Res1998; 30: 663–667[ISI][Medline]
  10. Warnock GL, Rajotte RV. Critical mass of purified islets that induce normoglycemia after implantation into dogs. Diabetes1988; 37: 467–470[Abstract]
  11. Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med2000; 343: 230–238[Abstract/Free Full Text]
  12. Waldmann H. Transplantation tolerance—where do we stand? Nat Med1999; 5: 1245–1248[ISI][Medline]
  13. Lee MK, Bae YH. Cell transplantation for endocrine disorders. Adv Drug Deliv Rev2000; 42: 103–120[ISI][Medline]
  14. Groth CG, Korsgren O, Tibell A et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet1994; 344: 1402–1404[ISI][Medline]
  15. Bach FH, Fineberg HV. Call for moratorium on xenotransplants. Nature1998; 391: 326
  16. Efrat S. Prospects for gene therapy of insulin-dependent diabetes mellitus. Diabetologia1998; 41: 1401–1409[ISI][Medline]
  17. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nature Medicine2000; 6: 278–282[ISI][Medline]
  18. Sachs DH, Bonner-Weir S. Editorial: New islets from old. Nature Medicine2000; 6: 250–251[ISI][Medline]
  19. Sander M, German MS. The beta cell transcription factors and development of the pancreas. J Mol Med1997; 75: 327–340[ISI][Medline]
  20. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet1997; 15: 106–110[ISI][Medline]
  21. Stoffers DA, Stanojevic V, Habener JF. Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein. J Clin Invest1998; 102: 232–241[Abstract/Free Full Text]
  22. Ferber S, Halkin A, Cohen H et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med2000; 6: 568–572[ISI][Medline]




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