Insulin independence and normalization of oral glucose tolerance test after islet cell allotransplantation.

To achieve permanent normoglycemia in patients with type I diabetes, it is necessary to renew the insulin-producing beta-cells by transplantation of either a vascularized pancreatic graft or isolated islets of Langerhans. Presently, about 10% of patients with type I diabetes undergoing islet allotransplantation achieve insulin independence; however, glucose intolerance remains in the majority of cases. We report a case of long-term insulin independence after islet allotransplantation in a type I diabetic patient. Three years after islet transplantation, the patient remains insulin-independent with a normal oral glucose tolerance test (OGTT). The patient therefore no longer meets the World Health Organization criteria for the diagnosis of diabetes mellitus and demonstrates that islet transplantation can cure diabetes in type I diabetic patients.

Xenogeneic islet re-transplantation in mice triggers an accelerated, species-specific rejection.

Xenogeneic islets could provide an unlimited source of tissue for the treatment of diabetes, and could in theory be transplanted repeatedly in a recipient. However, little is known on the consequences of islet re-transplantation in a recipient who has rejected a first graft. In this study, we investigated the functional consequence of xeno islet re-transplantation in mice sensitized with islets from different species. Sprague-Dawley (SD)-rat islets transplanted in sensitized C57/Bl6 mice that rejected either SD- or Lewis-rat islets underwent accelerated rejection. However, accelerated rejection was not found in mice sensitized with pig or human islets, suggesting that accelerated rejection was species specific. Immunohistochemistry showed increased binding of antibodies and accelerated leucocyte infiltration on re-grafted islets in sensitized mice. In situ apoptosis detection indicated that islet cell apoptosis was correlated with the time of leucocyte infiltration, but not with the time of antibody binding. In vitro experiments with cultured islet cells showed that although antibody binding was increased after incubation with sensitized mouse serum, islet cell cytotoxicity was not increased, suggesting that humoral immunity did not play a direct role in islet destruction. These results indicate that there is a cell-mediated, species-specific accelerated rejection after re-transplantation of xenogeneic islets.

Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations.

BACKGROUND: A series of 13 islet autotransplantations and 13 islet allotransplantations performed between 1992 and 1999 at the University Hospital of Geneva are presented. Factors affecting the outcome are analyzed. METHODS: Islet autotransplantation has been performed in seven patients with chronic pancreatitis and in six patients with benign tumors undergoing extensive pancreatectomy. Islet allografts were performed in C-peptide-negative patients simultaneously or after a kidney or lung transplantation. Each recipient received islets from one to four donors. Panel-reactive antibodies were monitored by microlymphocytotoxicity test. RESULTS: Eleven of 13 patients who underwent autotransplantation maintained insulin independence for 6 months to 5 years. Two years after autologous islet transplantation, five of nine patients were insulin independent with an glycosylated hemoglobin of 5.9%. Three late islet failures occurred in patients with chronic pancreatitis. Islet yield was significantly lower in patients with chronic pancreatitis than in patients with benign tumors (2044 equivalent islet number/gram resected pancreas versus 5184 equivalent islet number/gram; P=0.037). In islet allotransplantation, no early graft loss was found. All 13 patients who underwent allotransplantation had basal C-peptide levels above 0.3 nmol/L for 3 months to 5 years. Mean glycosylated hemoglobin decreased from 9.1% before transplantation to 5.5% at month 3. Insulin independence was achieved in two type I diabetic patients. In four of six patients with graft failure, the graft had induced panel-reactive antibodies. CONCLUSIONS: In islet autotransplantation, the reduced number of islets that can be isolated from fibrotic pancreata may be the major limiting factor. In islet allotransplantation, early graft function can now be consistently achieved. Islet allografts seem to be highly immunogenic, and chronic islet failure cannot be prevented consistently by conventional immunosuppression.

Expression of alpha-1 proteinase inhibitor in human islet microvascular endothelial cells.

There is a microcirculation system within the islets of Langerhans. However, little is known about the phenotypic and functional characterization of islet microvascular endothelial cells (MVEC). In this study, we purified MVEC from human pancreatic islets by using Ulex europaeus (Sigma, St. Louis, MO) agglutinin-1 (UEA-1)-coated dynabeads (Dynal A.S., Oslo, Norway). These purified human islet MVEC (HI-MVEC) express von Willebrand factor, take up high levels of acetylated LDL, and upregulate endothelial cell leukocyte adhesion molecule 1 in response to tumor necrosis factor-alpha. Ultrastructure examination shows the presence of microvilli and fenestrations on the cell surface, Weibel-Palade bodies in the cytoplasm, and tight junctions between cells. Furthermore, we show that vascular endothelial cell growth factor contributes to the formation of surface fenestrations on cultured HI-MVEC. After purification, HI-MVEC exhibit a very low proliferation capacity and are strongly resistant to trypsin, compared with other original MVEC. We also demonstrate that alpha-1 proteinase inhibitor (Api) is expressed on HI-MVEC and specifically located at the area of cell-cell junctions. By reverse transcription-polymerase chain reaction, a significant messenger RNA band of Api was found only in HI-MVEC, but not in other organ-derived MVEC, indicating that expression of Api is islet MVEC specific. Antibodies to Api significantly reversed the resistance to trypsin and promoted proliferation of HI-MVEC, suggesting that these specific functional characteristics of HI-MVEC are related to the expression of Api. These results indicate that HI-MVEC exhibit some specific morphological and functional characteristics that differ from MVEC derived from other organs.

Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model.

Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno-islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF)-decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels. Deposition of IgM and C3 on grafted islets in vivo or on isolated islets in vitro (after incubation with normal and decomplementized mouse serum), as well as CD4- and CD8-positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement-mediated cytotoxicity on rat islet cells was evaluated by a 3-(4, 5-dimethythiazolyl)-2.5-diphenyl-2H-tetrazolium-bromide (MTT) assay. A significant C3 deposition was found on grafted islets from the first day after transplantation in vivo, as well as on isolated islets after incubation with mouse serum in vitro. By MTT assay, complement-mediated cytotoxicity for islet cells was found. Decomplementation by CVF decreased C3 deposition on either isolated or grafted islets, delayed CD4- and CD8-positive leucocyte infiltration, led to significant inhibition of complement-mediated cytotoxicity for islet cells, and prolonged graft survival (mean survival time 21.3 versus 8.5 days; P<0.01). Our results indicate that decomplementation can prolong the survival time of devascularized xenografts across concordant species. The deposition of complement on transplanted islets may contribute to xenograft rejection by direct cytotoxicity and by promoting leucocyte infiltration.

Autologous bone marrow transplantation for recurrent malignant lymphoma after liver transplantation.

BACKGROUND: Cancer chemotherapy in chronic carriers of hepatitis B virus is known to promote viral replication, and, when immunosuppressive treatment is stopped, the return of immune competence can be followed by a fulminant hepatitis. Liver transplantation may be required and has been successfully performed for this condition. However, malignancy recurrence after transplantation has not been reported yet. METHODS AND RESULTS: We here report the case of an asymptomatic hepatitis B surface antigen carrier who developed a malignant lymphoma, which was treated by chemotherapy. After cessation of chemotherapy, he developed a fulminant hepatitis, requiring liver transplantation. Three years later, he developed a recurrent malignant lymphoma, which was treated successfully by autologous bone marrow transplantation. In order to prevent viral replication, lamivudine and intermittent administration of fresh-frozen plasma highly concentrated in anti-HBs immunoglobulin was initiated before the bone marrow transplantation. The patient remains well 12 and 56 months after autologous bone marrow and liver transplantation, respectively. CONCLUSIONS: This experience suggests that all hepatitis B surface antigen-positive patients for whom chemotherapy is indicated would benefit from prophylactic antiviral hepatitis B virus therapy. Furthermore, successful autologous bone marrow transplantation is possible after liver transplantation.

Xenoreactive natural antibodies.

Shortages of human organs for transplantation have made it necessary to examine the possibility of using nonhuman organs for xenotransplantation the transplantation of tissues between different species. Pigs are now regarded as the most likely species to serve as donors for clinical xenotransplantation. However, rejection of pig tissues and organs, mediated by the host's immune system, remains a major barrier to successful xenotransplantation. The primary immunological hurdle to overcome is rejection mediated by antibodies in the host that recognize antigens present on xenogeneic tissues. Since these antibodies are produced naturally in the host without immunization, they are termed natural antibodies. Here, we review the nature of xenoreactive natural antibodies directed toward pig tissues, and summarize recent progress in the field of xenotransplantation directed at overcoming humoral rejection of porcine xenografts.

Human islet allotransplantation: world experience and current status.

Currently type-I diabetes mellitus is treated with exogenous insulin administration, but traditional insulin therapy does not prevent long-term systemic complications and therefore alternatives should be sought. Presently the only option is to substitute the insulin-producing beta cells in order to obtain a more physiological way to deliver insulin. Simultaneous kidney and pancreas transplantation is an established procedure. Pancreas transplantation, however, is a major surgical procedure with a high rate of complications. Transplantation of the isolated insulin-secreting islets of Langerhans is an alternative approach, which is easier and safer than whole organ transplantation. Since 1990, clinical trials of islet transplantation have begun in a few specialized centers worldwide and the International Islet Registry counting 305 human islet allografts at the end of 1995. Insulin independence at 1 year was achieved in 8% of the patients, but 20% of cases showed a graft function with a normal basal C peptide and improved glycemic regulation. We review here the different aspects of human islets of Langerhans allotransplatation, namely historical aspects, indications, isolation and purification procedures and the results obtained.

[Isolation of the pig islets of Langerhans: evaluation of in vitro and in vivo function]. / Isolement d'îlots de Langerhans porcins: évaluation de la fonction in vitro et in vivo.

Pig islets are considered the best alternative to human islets in the treatment of insulin-dependent diabetes. Pigs could represent a potential islet donor for xenotransplantation in humans because of the close similarity between human and porcine insulin and the theoretically unlimited availability of porcine pancreas. From November 1991 to January 1997 we performed 221 pig islet isolations from 3 pig sources: group 1: minipigs (age 9-18 months) and white pigs (3-8 months), group 2: large white pigs (5-8 months), group 3: large white pigs (12-24 months). Islets were isolated according to a semi-automated method using enzymatic digestion and purification through discontinuous Euro-Ficoll gradients. The pancreases were surgically removed in our laboratory for group 1, while pancreases from groups 2 and 3 were removed at the slaughterhouse with an average warm ischemia time of 15 minutes. In vitro islet function was assessed by static incubations and perifusions, and in vivo islet function by transplantation under the kidney capsule of nude diabetic mice. The results were as follows: [table: see text] Insulin secretion increased twofold after in vitro glucose stimulation. We obtained restoration of euglycemia in diabetic mice which survived > 3 months after the graft and returned to diabetes after nephrectomy. This study shows that our isolated pig islets are viable and functional in vitro and in vivo after transplantation.

Islet autotransplantation in a cirrhotic liver.

Chronic pancreatitis resulting from alcohol abuse might in some rare cases require a total surgical resection of the pancreas to treat severe local complications. We have learned from the new techniques developed for islet isolation that it is now possible to obtain a sufficient number of good quality islets from one single pancreas to be transplanted into one recipient. We present a case of total surgical pancreatectomy for chronic pancreatitis in a previously non-diabetic patient with immediate islet isolation and autotransplantation. At operation, a cirrhotic liver was found, but no portal hypertension. We still decided to embolize a non purified preparation of endocrine tissue into the liver without alteration of liver function or durable modification of the portal pressure. One year after this procedure, the patient remains insulin-independent with a close to normal glycemic regulation as demonstrated by stimulation tests. Islet autotransplantation does not appear to be generally contra-indicated in the presence of a cirrhotic liver; provided the portal pressure is within normal limits. Under these circumstances, satisfactory glycemic control is achieved.

[Islands of Langerhans autotransplantation after pancreatic resection for benign pathology]. / Autotransplantation d'îlots de Langerhans après résection du pancréas pour une pathologie bénigne.

One way to prevent the occurrence of insulin-dependent diabetes after major pancreatic resection is to perform islet of Langerhans autotransplantation. Thus far, we have performed nine autotransplantations. The last three autotransplantations were performed in patients with benign tumoral pathology (one corporeal mucinous cyst, one isthmic insulinoma and one corporeal cystadenoma). In these three cases, we performed a distal 40%, 75% and 80% pancreatectomy respectively, since enucleation was not indicated or not feasible. After resection and removal of the tumoral lesion, pancreatic segments were injected intraductally with collagenase and digested according to a modified semi-automated Ricordi's technique. We obtained 105,000, 415,000 and 144,300 non-purified islets which were then embolized into the liver by intraportal injection during the same operative procedure. After surgery, all patients were insulin-independent. There was no morbidity or mortality. In a patient who presented acute pancreatitis of the residual pancreas five months after transplantation, insulin therapy was introduced. More than one year after the graft, the two other patients remain insulin-independent. In conclusion, we propose islet autotransplantation after pancreatic resection for benign focal pathology, to prevent or delay the occurrence of insulin-dependent diabetes.

[Isolation and cryopreservation of human islets of Langerhans]. / Isolement et cryopréservation d'îlots de Langerhans humains.

Islet transplantation represents an alternative to whole pancreas transplantation for the treatment of patients suffering from diabetes type I. The transplantation of a sufficient number of islets is an essential condition for successful allograft. Islet cryopreservation allows the storage of islet preparations for subsequent pooling, at the time of transplantation, of cryopreserved islets with a fresh preparation in order to increase the mass of transplanted pancreatic endocrine tissue. From May 1994 to April 1995, islets were isolated from 22 human pancreases using a modified automated method, and 19 preparations were cryopreserved. The function of cryopreserved islets was tested in vitro (static incubation and perifusion). The results of static incubation experiments confirmed that the insulin secretion of cryopreserved human islets in response to glucose stimulation was comparable to the response of islets that have not been frozen. In static incubation experiments, the mean (+/- SEM) insulin secretion of islets, prior to cryopreservation, was 239.3 (+/- 58.9) and 479.5 (+/- 59.5) pg/islet/15 min at 2.8 mM glucose and 16.7 mM glucose respectively. The mean (+/- SEM) insulin secretion of cryopreserved islets was 274 (+/- 103.2) and 468.5 (+/- 191.9) pg/islet/15 min at 2.8 mM and 16.7 mM glucose respectively. The perifusion experiments also demonstrated a significant increase of insulin secretion from cryopreserved islets perifused with a stimulating glucose concentration. Our experiments allow us to envisage the use of cryopreserved islet preparations for allotransplantation in diabetic patients.

Combined islet-lung transplantation in a cystic fibrosis patient.

The prevalence of insulin-dependent diabetes mellitus (IDDM) in cystic fibrosis patients ranges from 2 to 8% and glucose intolerance up to 15%. In recent years, lung transplantation has helped to prolong life expectancy of cystic fibrosis patients and represents 10 to 30% of all indications for lung transplantation. The postoperative need for immunosuppressive therapy using diabetogenic agents has decompensatory effects on glucose regulation and will probably increase the number of insulin-dependent cystic fibrosis patients. We report the case of an insulin-dependent cystic fibrosis patient transplanted with a combined islet-lung allograft. The pre-transplantation C-peptide level was below 0.04 nmol/l and preoperative insulin requirement was some 100 U per day. A sequential bipulmonary lung transplantation was performed and, using the pancreas of the same donor, we isolated and purified the islets of Langerhans by a modified automated method. We obtained 232,200 islets equivalent, which were injected into the liver by portal embolization. The postoperative course was uncomplicated, the insulin requirement decreased to 50% of the preoperative need and the C-peptide value increased to normal values and remained with the normal range during a follow-up period of 15 months. In conclusion, we believe that, besides type I diabetic patients, insulin-dependent cystic fibrosis patients with a negative C-peptide value could also be good candidates for combined islet-lung allotransplantation.