A novel device for islet transplantation providing immune protection and oxygen supply.

Islet transplantation as a biological ß-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'ßAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.

Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells.

Flavonoids are polyphenolic compounds that occur ubiquitously in plants. They are discussed to represent cancer preventive food components in a human diet that is rich in fruits and vegetables. To understand the molecular basis of the putative anticancer activity of flavonoids, we investigated whether and how the core structure of the flavones, 2-phenyl-4H-1-benzopyran-4-one (flavone) affects proliferation, differentiation, and apoptosis in HT-29 human colon cancer cells. Moreover, the effects of flavone in transformed epithelial cells were compared with those obtained in nontransformed primary mouse colonocytes. Proliferation, differentiation, and apoptosis in transformed as well as nontransformed colon cells were measured by fluorescence-based techniques. Apoptosis was also determined by changes in membrane permeability, FACScan analysis, and detection of DNA fragmentation. Semiquantitative reverse transcription PCR was performed to assess the effects of flavone on transcript levels. Flavone was found to reduce cell proliferation in HT-29 cells with an EC(50) value of 54.8 +/- 1.3 microM and to potently induce differentiation as well as apoptosis. The flavonoid proved to be a stronger apoptosis inducer than the clinically established antitumor agent camptothecin. The effects of flavone in HT-29 cells were associated with changed mRNA levels of cell-cycle- and apoptosis-related genes including cyclooxygenase-2 (COX-2), nuclear transcription factor kappaB (NF-kappaB), and bcl-X(L). Moreover, flavone, but not camptothecin, displayed a high selectivity for the induction of apoptosis and of growth inhibition only in the transformed colonocytes. In conclusion, the plant polyphenol flavone induces effectively programmed cell death, differentiation, and growth inhibition in transformed colonocytes by acting at the mRNA levels of genes involved in these processes. Because these genes play a crucial role in colon carcinogenesis, flavone may prove to be a potent new cytostatic compound with improved selectivity toward transformed cells.

Adiponectin is functionally active in human islets but does not affect insulin secretory function or beta-cell lipoapoptosis.

CONTEXT: The adipokine adiponectin has insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Mouse and human adiponectin receptor-1 and -2 have been cloned, both of which are expressed in various tissues and mediate effects of globular and full-length adiponectin. Whether adiponectin affects insulin secretion and beta-cell apoptosis and whether plasma adiponectin is associated with beta-cell function in humans is under investigation. DESIGN AND METHODS: In human islets from multiorgan donors, we investigated expression of adiponectin receptor-1 and -2. Furthermore, glucose-stimulated insulin secretion was determined by RIA. In addition, we investigated fatty acid-induced beta-cell apoptosis by terminal dUTP nick end labeling and flow-cytometric cell cycle analysis (sub-G1 formation). In humans in vivo, insulin secretory function was measured during hyperglycemic clamps in 65 normal glucose-tolerant subjects. We determined first and second phase of glucose-stimulated, glucagon-like peptide-1-stimulated, and arginine-stimulated insulin secretion. RESULTS: Adiponectin receptor-1 and -2 are expressed in human islets at the mRNA and protein level. Moreover, full-length adiponectin induces phosphorylation of acetyl coenzyme A carboxylase. However, adiponectin did not affect basal or glucose-stimulated insulin secretion or basal or fatty acid-induced beta-cell apoptosis. In vivo, fasting plasma adiponectin concentrations were not associated with glucose-stimulated first- and second-phase insulin secretion or with glucagon-like peptide-1- or arginine-stimulated insulin secretion (all P > 0.42). CONCLUSIONS: These data support a regulatory role of adiponectin in human islets; however, adiponectin does not seem to affect insulin secretion or basal/fatty acid-induced beta-cell apoptosis in humans.

Persistent reversal of diabetes by transplantation of fetal pig proislets into nude mice.

Facing the limited availability of human adult and fetal pancreases, fetal pig proislets (pancreatic islet precursors) were investigated in view of several inherent advantages. Six litters of fetuses of mean +/- SE gestational age 75 +/- 3 days were obtained from commercially available farm pigs. Pancreatic tissue was gently digested with collagenase, then a 10-day culture was performed. During culture, fetal proislets showed no insulin response to glucose alone but a significant response to glucose plus theophylline. The insulin content per microgram of DNA in the cultured proislets continuously increased. Histological examination by immunoperoxidase staining showed that, apart from single insulin- and glucagon-positive cells, there were no discrete islets in the pancreatic tissue and the cultured proislets. Diabetes was induced with streptozocin (STZ) in eight nude mice 3-4 wk after proislet transplantation and in another eight nude mice without transplantation. During the initial week, blood glucose levels of mice in both groups increased rapidly. The mean +/- SE peak value of blood glucose levels in the transplanted group was 20.4 +/- 2.0 mM and was 20.1 +/- 1.3 mM in the group without transplantation. Simultaneously, body weight decreased from 29.5 +/- 0.7 to 21.5 +/- 0.9 g and from 27.9 +/- 0.7 to 19 +/- 1 g in the groups, respectively. Afterward, blood glucose levels of mice in the transplanted group gradually decreased, and normoglycemia was achieved in all mice within 50 +/- 13 days after injection of STZ, i.e., 74 +/- 13 days after transplantation. The group without transplantation persistently maintained blood glucose levels greater than 16.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Progressive islet graft failure occurs significantly earlier in autoantibody-positive than in autoantibody-negative IDDM recipients of intrahepatic islet allografts.

Alloimmunity has been uncovered to be a cause of graft loss representing a major barrier for clinical islet transplantation, and several studies are designed to evaluate new strategies for immunosuppression to prevent alloimmunity. In contrast, the significance for autoimmune destruction of transplanted beta-cells has remained somewhat controversial. Recently, two case reports based on histological findings have suggested recurrent autoimmune insulitis despite immunosuppressive therapy both in clinical pancreas and in islet transplantation. In the present study, in 23 islet-grafted patients with IDDM receiving standard immunosuppressive therapy, we demonstrate that progressive impairment of islet graft function occurs significantly earlier in those individuals positive for autoantibodies as a typical stigma of diabetes-associated autoimmunity that is well established in the prediabetic periods of IDDM. Intraportal infusion of allogeneic islets was performed in 23 C-peptide-negative IDDM patients, according to the clinical transplantation categories defined as islet after kidney (IAK) or simultaneous islet and kidney (SIK). Complete islet graft failure was defined as the 1st day of permanent C-peptide negativity in the serum (<0.2 ng/ml) and C-peptide negativity in the urine (<2 microg/dl). The median observation period following islet transplantation was 12 months (range 1-50) with a cumulative follow-up of 336 months. Islet cell antibodies (ICAs) and GAD65 antibodies were monitored before and regularly after islet transplantation. Kaplan-Meier survival analysis and log-rank statistics revealed a significant (P < 0.05) difference in cumulative islet graft survival depending on the presence of islet cell and/or GAD65 antibodies. These results strongly suggest that recurrent autoimmunity directed to transplanted beta-cells contributes to islet graft failure despite sustained immunosuppression. For successful clinical islet transplantation in the future, new immunosuppressive therapies are needed to prevent both alloimmunity and autoimmunity.

Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?

The remarkable difference in success rates between clinical pancreas transplantation and islet transplantation is poorly understood. Despite the same histocompatibility barrier and similar immunosuppressive treatments in both transplantation procedures, human intraportal islet transplantation has a much inferior success rate than does vascularized pancreas transplantation. Thus far, little attention has been directed to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. We have tested this hypothesis in vitro with human islets and in vivo with porcine islets. Human islets were exposed to nonanticoagulated human ABO-compatible blood in surface-heparinized polyvinyl chloride tubing loops. Heparin and/or the soluble complement receptor 1 (sCR1) TP10 were tested as additives. Adult porcine islets were transplanted intraportally into pigs, and the liver was recovered after 60 min for immunohistochemical staining. Human islets induced a rapid consumption and activation of platelets. Neutrophils and monocytes were also consumed, and the coagulation and complement systems were activated. Upon histological examination, islets were found to be embedded in clots and infiltrated with CD11+ leukocytes. Furthermore, the cellular morphology was disrupted. When heparin and sCR1 were added to the blood, these events were avoided. Porcine islets retrieved in liver biopsies after intraportal islet allotransplantation showed a morphology similar to that of human islets perifused in vitro. Thus, exposure of isolated islets of Langerhans to allogenic blood resulted in significant damage to the islets, a finding that could explain the unsatisfactory clinical results obtained with intraportal islet transplantation. Because administration of heparin in combination with a soluble complement receptor abrogated these events, such treatment would presumably improve the outcome of clinical islet transplantation by reducing both initial islet loss and subsequent specific immune responses.

Metabolic effects of restoring partial beta-cell function after islet allotransplantation in type 1 diabetic patients.

Successful intraportal islet transplantation normalizes glucose metabolism in diabetic humans. To date, full function is not routinely achieved after islet transplantation in humans, with most grafts being characterized by only partial function. Moreover, the duration of full function is variable and cannot be sufficiently predicted with available methods. In contrast, most grafts retain partial function for a long time. We hypothesized that partial function can restore normal protein and lipid metabolism in diabetic individuals. We studied 45 diabetic patients after islet transplantation. Labeled glucose and leucine were infused to assess whole-body glucose and protein turnover in 1) 6 type 1 diabetic patients with full function after intraportal islet transplantation (FF group; C-peptide > 0.6 nmol/l; daily insulin dosage 0.03 +/- 0.02 U x kg(-1) body wt x day(-1); fasting plasma glucose < 7.7 mmol/l; HbA1c < or = 6.5%), 2) 17 patients with partial function (PF group; C-peptide > 0.16 nmol/l; insulin dosage < 0.4 U x kg(-1) body wt x day(-1)), 3) 9 patients with no function (NF group; C-peptide < 0.16 nmol/l; insulin dosage > 0.4 U x kg(-1) body wt x day(-1)), and 4) 6 patients with chronic uveitis as control subjects (CU group). Hepatic albumin synthesis was assessed in an additional five PF and five healthy volunteers by means of a primed-continuous infusion of [3,3,3-2H3]leucine. The insulin requirement was 97% lower than pretransplant levels for the FF group and 57% lower than pretransplant levels for the PF group. In the basal state, the PF group had a plasma glucose concentration slightly higher than that of the FF (P = 0.249) and CU groups (P = 0.08), but was improved with respect to the NF group (P < 0.01). Plasma leucine (101.1 +/- 5.9 micromol/l) and branched-chain amino acids (337.6 +/- 16.6 micromol/l) were similar in the PF, FF, and CU groups, and significantly lower than in the NF group (P < 0.01). During insulin infusion, the metabolic clearance rate of glucose was defective in the NF group versus in the other groups (P < 0.01). Both the basal and insulin-stimulated proteolytic and proteosynthetic rates were comparable in the PF, FF, and CU groups, but significantly higher in the NF group (P = 0.05). In addition, the PF group had a normal hepatic albumin synthesis. Plasma free fatty acid concentrations in the PF and FF groups were similar to those of the CU group, but the NF group showed a reduced insulin-dependent suppression during the clamp. We concluded that the restoration of approximately 60% of endogenous insulin secretion is capable of normalizing the alterations of protein and lipid metabolism in type 1 diabetic kidney recipients, notwithstanding chronic immunosuppressive therapy. The results of the present study indicate that "success" of islet transplantation may be best defined by a number of metabolic criteria, not just glucose concentration/metabolism alone.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets.

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft. With improved isolation methods based on the original procedure published by Ricordi et al. yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation.

BACKGROUND: Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). METHODS: Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM. RESULTS: Compared to unstored (429200 +/- 86700 IEQ) and OLM-stored pancreases (338600 +/- 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 +/- 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 +/- 0.4) was significantly decreased after TLM storage (1.8 +/- 0.2, P < .05) but not after OLM storage (2.3 +/- 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. CONCLUSIONS: This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Influence of neutral protease activity on human islet isolation outcome.

UNLABELLED: Observations in rat pancreata have revealed that enzymatic islet release is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. Since no information is available about the effect of NP activity on islet release from the human pancreas, the present study evaluated the effect of various NP concentrations on the outcome of human islet isolation. METHODS: Following intraductal collagenase distension, pancreata obtained from adult multiorgan donors were digested using 2000 PZ-U of purified Serva collagenase NB 1 supplemented with 2.6 (n = 10) or 4.5% (DMC-U/PZ-U) (n = 10) of NP. RESULTS: Increasing NP from 2.6% to 4.5% reduced the amount of undigested tissue from 22 +/- 2 to 17 +/- 2 g (P < .05) while simultaneously increasing the volume of digested tissue (26 +/- 2 vs 40 +/- 3 mL, P < .01). Increased NP concentrations increased the islet yield prepurification (459,800 +/- 22,900 vs 587,600 +/- 69,000 IEQ, P < .05), but simultaneously affected islet purification, resulting in equal islet yields (345,700 +/- 31,200 vs 391,500 +/- 35,400 IEQ, NS) and less purity (70 +/- 6 vs 49% +/- 5%, P < .01). A NP concentration of 4.5% reduced the stimulation index (4.7 +/- 1.2 vs 2.0 +/- 0.5, P < .01) and viability (100 +/- 1 vs 95% +/- 3%, P < .05). CONCLUSIONS: Although increased NP activity seems to improve islet release from adult human pancreata, it significantly affects islet viability and function. The reduction in purity reflected damage to acinar tissue by increased NP activity presumably affecting islet integrity.

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas.

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Expression of complement regulatory proteins on islets of Langerhans: a comparison between human islets and islets isolated from normal and hDAF transgenic pigs.

BACKGROUND: The expression of regulators of complement activity (RCAs) on islet cells may be of great importance for protecting them against complement-mediated lysis in the immediate posttransplant period after intraportal islet transplantation. We examined porcine and human islet cells for expression of RCA. We also examined to what extent human decay accelerating factor (hDAF) is expressed on adult and fetal islet cells isolated from hDAF transgenic (TG) pigs having a high transgene expression on endothelial cells. Moreover, the susceptibility of the various types of cells to lysis in human serum and blood was investigated. METHODS: Adult human islets (n=5), normal adult and fetal porcine islets (n=9 and n=8, respectively), and islets from adult and fetal hDAF TG pigs (n=5 and n=6, respectively) were examined. With islet single-cell suspensions and flow cytometry, adult human islet cells were examined for expression of hDAF (CD55), hCD59, and human membrane cofactor protein (hMCP; CD46), while porcine islet cells were examined for expression of pCD59 and pMCP. Islet cells from hDAF TG pigs were also examined for hDAF expression. Porcine peripheral blood lymphocytes, normal and hDAF TG porcine endothelial cell lines, a human endothelial cell line, and the human cell line U937 served as controls. Islet cytotoxicity was assayed after incubation of the islet cells with fresh human serum. Furthermore, adult islets from normal control pigs and hDAF TG pigs were exposed to fresh human blood in vitro for 60 min, and the inflammatory reaction elicited was compared between the different types of islets. RESULTS: All human islet cell preparations expressed hCD59, two of five expressed hMCP, but none expressed hDAF. Porcine islet cells expressed both pCD59 and pMCP. Normal adult porcine islet cells exposed to fresh human serum resulted in 74+/-5.4% cell lysis (mean+/-SEM, n=16). In comparison, only 1.3+/-2.8% (n=20, P<0.001) of human islet cells were lysed in the human serum. One islet cell preparation from an hDAF TG pig expressed small amounts of hDAF. This preparation from hDAF TG pigs bound significantly less C3c than did normal control islets (mean fluorescence ratio 16+/-2.2 and 58+/-4.3, respectively; P=0.046) and were partially protected from cell lysis in fresh human serum (47+/-10% and 78+/-18% cell lysis, respectively; P=0.046). The other four preparations from hDAF TG pigs were negative for hDAF and were equally susceptible to lysis as normal control islets. All fetal pancreatic islet cells from hDAF TG pigs analyzed were negative for hDAF expression. When exposed to fresh human blood in vitro, adult and fetal islets from hDAF TG pigs elicited equally strong inflammatory changes as did the normal control islets. The inflammatory changes were characterized by activation of the complement and coagulation systems, resulting in islet damage with "dumping" of insulin into the blood. CONCLUSIONS: Porcine and human islet cells express species-restricted complement regulatory proteins, with the human islet cells expressing mainly hCD59. A low expression of hDAF was detected on islet cells from one of five hDAF TG pigs. These islet cells displayed reduced islet cell cytotoxicity in fresh human serum. We conclude that protection from complement-mediated lysis will be important in the context of intraportal pig-to-human islet transplantation, and expression of a human RCA on islet cells should be beneficial in this context.

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.

A minimally invasive technique for intrathymic cell transplantation in the dog.

As an alternative to drug immunosuppression, attempts at inducing donor-specific tolerance by intrathymic (IT) inoculations to transplant recipient of donor origin alloantigenic products has proven very promising. Using fiber optic thoracoscopy, a technique for the study of this phenomena was developed for the dog. We show an approach to the dog thymus using fiber optics for injection of bone marrow (BM) cells as the tolerogen. Bone marrow was retrieved from the donor beagles and purified using an automated Ficoll-Paque gradient technique. The purified cellular suspension was injected into the thymus through a small intercostal incision with the use of an injection needle port guided by the use of a rigid fiberoptic scope. To demonstrate engraftment, supravital staining with Fluorescein Diacetate of the BM cells was performed prior to inoculation. Immunofluorescence of cryostat sections obtained at necropsy confirmed the presence of viable BM cells up to several days after transplantation. Results of this study show that the thoracoscopic approach to the thymus can be safely and effectively used for IT inoculation studies in dogs.

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Diagnostic algorithm and management of immune-mediated complications associated with subcutaneous insulin therapy.

Immune mediated complications associated with subcutaneous insulin therapy such as insulin neutralizing antibodies and/or skin reactions are rare conditions since human insulin is in general use. Nevertheless, if it occurs, a stepwise diagnostic approach is essential for differential diagnosis and consecutive treatment of these complications. Here we suggest a diagnostic algorithm to deal with e.g. insulin antibody formation of the IgG and/or IgE type and/or severe skin reactions resulting in poor metabolic control and often "brittle diabetes" in affected patients. This diagnostic algorithm includes step 1: Intradermal skin testing with positive and negative controls, additives and different insulin preparations; step 2: Quantification of insulin specific IgG and IgE in the serum, and step 3: Analysis of the time dependent binding/dissociation curves of the insulin neutralizing antibodies in an ex vivo/in vitro assay to assess the clinical significance of these antibodies. Based on 158 insulin treated control subjects and four patients with typical symptoms and signs representing the spectrum of immune-mediated complications subsequent to subcutaneous insulin therapy we demonstrate that the proposed stepwise approach leads to a definite diagnosis as a prerequisite for individual and successful therapy.

Islet autoantibodies as potential markers for disease recurrence in clinical islet transplantation.

At present, indications for clinical islet transplantation exist almost exclusively in type-1 diabetic patients with end-stage renal disease receiving the islets either simultaneously with or after an already established kidney graft. This implies, that islet transplantation is performed in type-1 diabetic patients with long disease duration. The fate of the islet allograft is determined by a combination of immunological effector mechanisms. Beside early non-specific inflammation and alloreactivity, chronic autoimmunity may contribute to islet graft failure. The immunologic characterization of the prediabetic state has considerably progressed, whereas, the nature of autoimmunity years and decades after the onset of diabetes is largely unknown. Islet autoantibodies as surrogate markers for islet autoimmunity are well established in prediabetic periods of type-1 diabetes. In contrast, only few data exists in the setting of long-term type-1 diabetic patients undergoing islet transplantation. This article reviews the original data from the Giessen islet transplantation project and the pertinent literature with respect to islet autoimmunity and disease recurrence. It is demonstrated, that autoimmunity may persist in an individual with type-1 diabetes for decades after diabetes onset and that autoimmune responses to transplanted islets are resistant to the immunosuppressive drugs currently used. It is suggested from pilot trials, that type-1 diabetic patients with persistent autoantibodies and individuals, in whom autoantibodies become detectable after the transplantation are at higher risk for early islet graft failure potentially due to recurrent autoreactivity directed to the islet graft.

The influence of donor specific vertebral body derived bone marrow cell infusion on canine islet allograft survival without irradiation conditioning of the recipient.

In recent studies in rodents, it was shown, that donor specific tolerance towards islet allografts without irradiation therapy of the recipient is induced by bone marrow cell infusion in combination with temporary immunosuppression. In the present study, the effect of donor specific bone marrow cell (DBMC) infusion at the time of intrahepatic islet allotransplantation without irradiation conditioning of the recipient was investigated in the canine model, paralleling ongoing clinical trials. It was observed, that unfractionated bone marrow cells given simultaneous to islet allografts led to higher frequencies of rejection periods and decreased islet allograft survival, when administered to recipients immunosuppressed with Cyclosporine A only. In contrast, an additional short inductive treatment of the recipient with an anti-dog-T-lymphocyte monoclonal antibody (5G2) abrogated the enhanced immunogenicity of the unfractionated bone marrow preparation, prolonging islet allograft survival with no rejection episodes observed during the immunosuppressive treatment with Cyclosporine. The composition of bone marrow cells might have contributed to the higher immunogenicity, since the percentage of MHC-class II antigen bearing cells is similar to man, but significantly higher than compared to rodents. It is therefore suggested, that further studies should encompass both timing of bone marrow cell infusion, appropriate immunosuppression and strategies to functionally inactivate mature MHC-class-II positive cells prior to DBMC infusion.

Pancreatic islet and stem cell transplantation: new strategies in cell therapy of diabetes mellitus.

Long-term studies strongly suggest that tight control of blood glucose can prevent the development and retard the progression of chronic complications of type 1 diabetes mellitus. In contrast to conventional insulin treatment, replacement of a patient's islets of Langerhans either by pancreas organ transplantation of by isolated islet transplantation is the only treatment to achieve a constant normoglycemic state and avoiding hypoglycemic episodes, a typical adverse event of multiple daily insulin injections. However, the expense of this benefit is still the need for immunosuppressive treatment of the recipient with all its potential risks. Islet cell transplantation offers the advantage of being performed as a minimally invasive procedure, in which islets can be perfused percutaneously into the liver via the portal vein. As of June 2003, 705 pancreatic islet transplants worldwide have been reported to the International Islet Transplant Registry (ITR) at our Third Medical Department, University of Giessen/Germany. Data analysis shows at 1 year after adult islet transplantation a patient survival rate of 97%, a functioning islet graft in 54% of the cases, whereas insulin independence was meanwhile achieved in 20% of the cases. However, using a novel protocol established by the Edmonton Center/Canada, the insulin independence rates have improved significantly reaching meanwhile a 50-80% level. Finally, the concept of islet cell or stem cell transplantation is most attractive since it offers many perspectives: islet cell availability could become unlimited and islet or stem cells my be transplanted without life-long immunosuppressive treatment of the recipient, just to mention 2 of them.

Can the density of native pancreatic tissue slices predict human islet isolation and purification outcome?

INTRODUCTION: With currently available technology, the outcomes of human islet isolation and purification are still inconsistent, in part due to a lack of control of the pancreas donor and the procurement conditions. Using a single donor pancreas, the critical islet mass for establishing insulin independence of approximately 5000 engrafted islet equivalents (IEQ)/kg of recipient weight can only be retrieved from about one third of isolations. The purpose of this study was to analyze whether successful islet isolation and purification outcomes might be predicted from the density of native pancreatic tissue. METHODS: Tissue slices (TS) were obtained from the neck of 9 nondistended human donor pancreata. The density of the TS was determined using gravity sedimentation in continuous density gradients under either iso-osmolar or hyperosmolar conditions. Correlation coefficients were calculated with regard to the density of isolated exocrine and endocrine tissue, donor age, body mass index (BMI), cold ischemia time (CIT), IEQ prepurification and postpurification, IEQ recovery, and purity. RESULTS: (1) There was no change in density over time for TS in 300 mOsm/kg (mean, 1.079 +/- 0.0019 g/cm(3)) (2) In 500 mOsm/kg, there was a significant increase in density from 1.086 +/- 0.0021 g/cm(3) to 1.092 +/- 0.0021 g/cm(3) over time. (3) Density of isolated exocrine and endocrine became more distinct with lower density of TS (r = -0.776; P < .05). (4) Donor age, BMI, recovery of IEQ from gradients, and number of IEQ after purification did not correlate significantly with TS density. (5) In contrast, a significant inverse correlation existed betwen TS and CIT (r = -0.829; P < .05), and between TS versus IEQ number prior to purification (r = -0.867; P < .05). CONCLUSION: No homogeneous distribution of pancreas tissue density was seen among 9 consecutive human organs. Taken together, the density of native pancreas TS is not a suitable sole predictor for successful islet isolation and purification.