Benfotiamine in diabetic polyneuropathy (BENDIP): results of a randomised, double blind, placebo-controlled clinical study.

AIM: Efficacy and safety of benfotiamine in treatment of diabetic polyneuropathy. METHODS: Double blind, placebo-controlled, phase-III-study. 181 patients were screened. 165 patients with symmetrical, distal diabetic polyneuropathy were randomised to one of three treatment groups entering the wash-out phase and 133/124 patients were analysed in the ITT/PP analysis: Benfotiamine 600 mg per day (n=47/43), benfotiamine 300 mg per day (n=45/42) or placebo (n=41/39). RESULTS: After 6 weeks of treatment, the primary outcome parameter NSS (Neuropathy Symptom Score) differed significantly between the treatment groups (p=0.033) in the PP (per protocol) population. In the ITT (intention to treat) population, the improvement of NSS was slightly above significance (p=0.055). The TSS (Total Symptom Score) showed no significant differences after 6 weeks of treatment. The improvement was more pronounced at the higher benfotiamine dose and increased with treatment duration. In the TSS, best results were obtained for the symptom "pain". Treatment was well tolerated in all groups. CONCLUSION: Benfotiamine may extend the treatment option for patients with diabetic polyneuropathy based on causal influence on impaired glucose metabolism. Further studies should confirm the positive experiences.

Metabolic effects of successful intraportal islet transplantation in insulin-dependent diabetes mellitus.

The intraportal injection of human pancreatic islets has been indicated as a possible alternative to the pancreas transplant in insulin-dependent diabetic patients. Aim of the present work was to study the effect of intraportal injection of purified human islets on: (a) the basal hepatic glucose production; (b) the whole body glucose homeostasis and insulin action; and (c) the regulation of insulin secretion in insulin-dependent diabetes mellitus patients bearing a kidney transplant. 15 recipients of purified islets from cadaver donors (intraportal injection) were studied by means of the infusion of labeled glucose to quantify the hepatic glucose production. Islet transplanted patients were subdivided in two groups based on graft function and underwent: (a) a 120-min euglycemic insulin infusion (1 mU/kg/min) to assess insulin action; (b) a 120-min glucose infusion (+75 mg/di) to study the pattern of insulin secretion. Seven patients with chronic uveitis on the same immunosuppressive therapy as grafted patients, twelve healthy volunteers, and seven insulin-dependent diabetic patients with combined pancreas and kidney transplantation were also studied as control groups. Islet transplanted patients have: (a) a higher basal hepatic glucose production (HGP: 5.1 +/- 1.4 mg/kg/ min; P < 0.05 with respect to all other groups) if without graft function, and a normal HGP (2.4 +/- 0.2 mg/kg/min) with a functioning graft; (b) a defective tissue glucose disposal (3.9 +/- 0.5 mg/kg/min in patients without islet function and 5.3 +/- 0.4 mg/kg/min in patients with islet function) with respect to normals (P < 0.01 for both comparisons); (c) a blunted first phase insulin peak and a similar second phase secretion with respect to controls. In conclusion, in spite of the persistence of an abnormal pattern of insulin secretion, successful intraportal islet graft normalizes the basal HGP and improves total tissue glucose disposal in insulin-dependent diabetes mellitus.

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)

Bovine islets of Langerhans. Potential source for transplantation?

Two methods are described for large-scale isolation of islets from the bovine pancreas. With the single-endpoint technique the average number of islets isolated from a 73.3-g pancreas sample was 150,560, representing an estimated total islet volume of 0.391 ml. Pancreases processed by the continuous digestion-filtration method yielded an average of 246,229 islets from a 93.9-g pancreas sample, representing an estimated total islet volume of 0.458 ml. Isolated bovine pancreatic islets demonstrated a well-preserved endocrine cell ultrastructure, responded to acute stimulation with propionic acid, and temporarily reversed experimental diabetes in mice.

Fish oil-enriched diet and reduction of low-dose streptozocin-induced hyperglycemia. Inhibition of macrophage activation.

Repeated low doses of streptozocin (STZ; 40 mg/kg, 5 injections/day) induce hyperglycemia in certain strains of mice after a latency of 1 wk. Omega-3 polyunsaturated fatty acids (omega 3FA) have been reported to suppress immune processes by blockade of the cyclooxygenase pathway of arachidonic acid metabolism. We investigated the effects of diets high in omega 3FA on the development of diabetes in the low-dose STZ-induced diabetes (LDSTZ-D) model. Male C57BL/6J mice were on a fish oil diet (FOD) as a source of omega 3FA 8 wk before STZ injection. Controls received laboratory chow only or a coconut oil diet (COD). Blood glucose levels in FOD mice were reduced (12.5 vs. 28 mM for COD mice, P less than .001) 60 days after STZ injection with a diet in which 20% of the calories were from fish oil. In FOD mice, immunohistology showed reduced numbers of class II antigen-expressing cells in pancreatic islets followed by a decreased extent of insulitis. FOD significantly decreased the number of Fc receptor-negative dendritic cells in cytospin preparations of islets isolated from diabetic mice. Interleukin 1-like activity of peritoneal exudate cell supernatants isolated from mice on FOD was reduced. FOD did not improve insulin secretion of isolated islets from LDSTZ-D mice. These data indicate a beneficial effect of FOD on the immune component of the mouse LDSTZ-D model.

Indications for clinical islet transplantation today and in the forseeable future--the diabetologist's point of view.

The treatment of patients with type 1 diabetes mellitus has to focus on short-term and long-term risks of the disease which means to avoid hyperglycemic or hypoglycemic coma as well as late complications. As we know from the DCCT study metabolic control substantially lowers the risk for retinopathy, nephropathy and neuropathy. We also know, that keeping the blood glucose in a nearly normal range inevitably is connected with a marked increase of severe hypoglycemia, an event which occurs more frequently when normoglycemia has been reached and the further slow decline of blood glucose is not recognized by the patient (autonomous neuropathy, hypoglycemia unawareness of other origin, long duration of diabetes etc.). Furthermore, counterregulatory hormones as glucagon and epinephrine may be lacking due to diminished or even lost alpha cells within the islets and as recently observed due to fibrosis of the adrenal medulla in long-term diabetes. The consequences of severe hypoglycemia are manifold: in the actual situation of unconsciousness the risk of heavy injuries and as long-term consequences irreversible brain damage may occur. Finally, the effort of the patient to reach normoglycemia includes the burden of an intensive blood glucose self-control day by day. This broad scenario of all the achievements and of all the problems connected with an intensified insulin treatment has to be regarded when the indication for an islet transplant will be discussed. From our point of view as clinicians it seems adequate not to give definite recommendations but to express our considerations for islet transplantation in patients with type 1 diabetes mellitus with the following list (table 1). It must be clearly stated, that at present transplantation of isolated islets by no means can serve as a treatment for a larger number of patients and this may hold through also for the foreseeable future. In this context, also the many contraindications should be summarized (table 2). Consequently we have to deal with several questions and problems which can be subdivided into those regarding the possible benefit for the patients from an islet graft (full success = insulin independence, partial success = lower exogenous insulin requirement due to additional endogenous insulin, measured by C-peptide levels, more stable glucose metabolism) and those regarding possible side effects (primary risk of implantation, threat for rejection of the primarily transplanted kidney). Furthermore, one may ask for risks when islets are transplanted alone (ITA). We therefore will address the following areas: 1. Simultaneous islet and kidney transplants 2. Islet transplants after kidney transplantation alone (IAK) 3. Islet transplantation after pancreas transplantation failure (P-failure) 4. Defect hypoglycemia counterregulation--life threatening hypoglycemia unawareness as indication for islet transplantation? 5. Autonomous cardiac neuropathy as indication for islet transplantation? 6. Significant clinical problems with exogenous insulin therapy as indication for islet transplantation?

Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice.

The primary objective of this investigation was to determine the effect of endotoxin on islet xenograft survival within the first three days after transplantation. Pancreatic islets from Lewis rats were prepared under endotoxin-free conditions with Liberase (Boehringer) and purified by centrifugation on endotoxin-free Ficoll/Histopaque. After overnight incubation, with or without 10 microg/ml endotoxin, the islets were transplanted beneath the kidney capsule of normoglycemic C57Bl/6-mice. Three days later, kidneys were removed and their insulin content were measured. We could demonstrate significant differences (P<0.01) in insulin recovery between lipopolysaccharide-free and lipopolysaccharide-containing grafts. In case of endotoxin contaminated islets, we found only 13+/-2% (n=9) of the original insulin content, in contrast to 53+/-7% (n=9) when endotoxin-free islets where grafted. In experiments with islets isolated by use of conventional (lipopolysaccharide-containing) collagenase, and then cultured in endotoxin-free medium, insulin recovery three days after transplantation was 36+/-1% (n=13).

Glutamic acid decarboxylase antibodies are more frequent than islet cell antibodies in islet transplanted IDDM patients and persist or occur despite immunosuppression.

Pancreatic islet grafts transplanted into patients with autoimmune diabetes are potentially threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. In the present study we investigated the humoral autoimmune response directed to islet autoantigens by studying islet cell antibodies and glutamic acid decarboxylase (GAD 65) antibodies in twenty-one insulin-dependent diabetes-mellitus (IDDM) patients undergoing intraportal islet allotransplantation. Islet transplantation was performed according to the following recipient categories: Islet after kidney transplantation (n=10), simultaneous islet and kidney transplantation (n=6) and islet transplant alone (n=5). GAD 65 antibodies were detected in a radioligand GAD 65 antibody assay using recombinant, in vitro translated, human 35S-methionin labelled GAD 65 as tracer. Islet cell antibodies were determined by indirect immunofluorescence technique on human pancreas. In six out of twenty-one patients we observed GAD 65 antibodies before islet transplantation and the GAD 65 antibodies persisted despite immunosuppression. In contrast only two subjects were concordantly islet cell antibody positive and the titre decreased post transplantation. In addition we observed occurrence of GAD 65 antibodies in five subjects that were shown to be antibody negative before islet transplantation with three of them subsequently becoming positive for islet cell antibodies. The remaining ten patients were GAD 65 antibody and islet cell antibody negative before islet transplantation and remained negative thereafter. Interestingly none of the patients was exclusively positive for islet cell antibodies without being positive for GAD 65 antibodies. In summary we have demonstrated in twenty-one islet grafted individuals that humoral autoimmunity to islet antigens can persist or occur despite immunosuppression. Islet cell antibodies appear to be less frequent (5 out of 21, 23%) compared to GAD 65 antibodies (11 out of 21, 52%) suggesting that they are more affected by immunosuppressive therapy. We conclude that GAD 65 antibodies are a useful tool to further evaluate a possible link between persistent autoimmunity and early or late graft failure after islet transplantation.

Glucose sensitivity of porcine and human islets in vitro.

Preliminary experiments about the suitability of different commonly used culture media in our laboratory indicated, that prolonged exposure to high glucose concentrations during low temperature culture (LTC) impairs the viability of long term cultured human islets. As a consequence of the heterogeneity of tested media the present study was aimed to evaluate the influence of different glucose concentrations on survival, viability and in-vitro function of cultured human islets in order to optimize islet survival until transplantation and to compare species dependent differences in glucose sensitivity. Quantified aliquots of freshly isolated (digestion-filtration, ficoll gradient purification) islets from consecutively processed human (n=6) and porcine (n=11) pancreata were subjected to different glucose concentrations (human islets: 500, 750, 1000 and 2000 mg/l; porcine islets: 1000 and 2000 mg/l) in CMRL (22 degrees C) for 8-10 days. After LTC survival, viability and glucose-stimulated insulin release of incubated tissue was assessed. A reduction of glucose concentration promotes survival and viability of human islets but impairs in vitro function at the same time, presumably due to a reduced glucose oxidation as expressed by the significantly reduced stimulation index. In contrast to these findings in the human, elevated glucose concentration in porcine islet culture increases survival but reduces the glucose-stimulated insulin release and the viability of cultured islets. The contradiction of the results in regard to islet survival related to islet viability are still unclear in the pig and needs further evaluation.

Large variability of the intracellular ATP content of human islets isolated from different donors.

Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. The ATP content of cells can be reduced by several factors i.e. the nutritional donor status, storage technique, warm ischemia and cold ischemia time. This study investigates the intracellular ATP content of isolated human islets for the first time. Quantified samples of freshly isolated (digestion-filtration, continuous ficoll gradient purification) and cultured (22 degrees C, CMRL+10% FCS) islet equivalents (IEQ) of consecutively processed human pancreata from multiorgan donors (UW vascular flush) were shock frozen in liquid nitrogen and stored at -196 degrees C until rapid thawing, sonification and subsequent luminometric determination of ATP (Luciferin-Luciferase-reaction) and assessment of islet protein (IP). The ATP content was analysed for freshly isolated and subsequently 5+/-1 days cultured islets (n=10). The ATP content of freshly isolated human islets was 130.4+/-53.4 pg/microg IP (mean+/-SEM) corresponding to 20.7+/-6.3 pg/IEQ. After culture ATP content increased to 265.5+/-113.3 pg/microg IP (204.2+/-41.5%) corresponding to 43.7+/-15.3 pg/IEQ (216.1+/-34.9%; p<0.05). The coefficient of variation was 129.5%, 96.5% (fresh) and 135.0%, 111.0% (cultured) for ATP/microg IP and ATP/IEQ, respectively. The present data show that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22 degrees C culture suggesting that the capacity to produce ATP is maintained despite hypothermic environment. More data are necessary to clarify the relevance of intraislet ATP content for graft function and survival after islet transplantation.

Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll.

Endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. By the chromogenic Limulus amebocyte lysate assay we found rather high but variable endotoxin concentrations in the chemicals used for islet isolation, i.e. collagenase and Ficoll. Liberase, a special collagenase preparation from Boehringer, was nearly endotoxin-free. Correlating to the endotoxin content, collagenase and Ficoll had the capacity to induce interleukin-1beta release from human peripheral blood mononuclear cells. Because collagenase and density gradient media are needed in most cell isolation procedures from solid organs, each lot of these chemicals should be tested for endotoxin contamination. In pancreatic islet transplantation, the use of endotoxin-free chemicals may diminish early local inflammatory reactions at the graft site and thereby reduce the number of islets needed for successful islet transplantation.

Oxygen radical production in human mononuclear blood cells is not suppressed by drugs used in clinical islet transplantation.

Inflammatory islet damage mediated by cytokines and oxygen radicals may limit the success of clinical islet transplantation for treatment of insulin-dependent diabetes mellitus. In this study, we investigated whether drugs such as currently used in islet-transplanted patients inhibit the release of IL-1beta, TNFalpha, and superoxide from mononuclear blood cells in vitro. Methylprednisolone (10 microg/ml) inhibited the release of IL-1beta and TNFalpha, but had no effect on superoxide generation. Both pentoxifylline (66 microg/ml) and cyclosporin A (300 ng/ml) slightly inhibited TNFalpha release without affecting IL-1beta or superoxide generation. Nicotinamide (0.25 mM) did not interfere with the generation TNFalpha or superoxide and only slightly inhibited IL-1beta production. A combination of methylprednisolone, pentoxifylline, cyclosporin A, and nicotinamide (concentrations for each substance as described above) inhibited TNFalpha generation by 74+/-6% (mean value+/-SEM, mononuclear blood cells from seven diabetic patients) without affecting IL-1beta or superoxide generation. These data show that standard immunosuppressive therapy in islet transplanted patients may partially inhibit cytokine release but does not affect the generation of potentially islet-toxic superoxide from mononuclear cells.

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas.

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12-14 days culture at 22 degrees C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.

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.

Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients.

The macrophage derived cytokines interleukin-beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha), and the T-cell derived cytokine interferon gamma (IFNgamma) have been implicated to play an important role in early attack on islet cells during human islet transplantation (ITx). Therefore, the aim of this study was to investigate the influence of the current immunosuppressive induction therapy in clinical islet transplantation on mRNA expression of these cytokines in blood cells, compared to lipopolysaccharide (LPS) induced cytokine release in vitro and to plasma levels. The cytokine release correlated to lymphocyte counts and significantly decreased after ATG, and partially recovered 2 weeks after ITx. Unexpectedly, there was no correlation between mRNA expression for IL-1beta in total blood and the number of lymphocytes and monocytes remaining after anti thymocyte globulin (ATG)-therapy. Even when the blood was nearly totally depleted from mononuclear cells, high amounts of IL-1beta mRNA could be detected. However, IL-1beta secretion could not be stimulated in vitro. Our results show that application of ATG during ITx might contribute to graft survival during the early posttransplant period by suppression of the synthesis of monocyte derived cytokines IL-1beta and TNFalpha.

Critical islet mass for successful porcine islet autotransplantation.

A major reason for the failure of clinical islet transplantations may be a limited islet mass. The aim of this study was to determine the critical islet mass necessary for normalization of glucose metabolism in a porcine model. Diabetes was induced by total pancreatectomy. The splenic lobe of the pancreas was intraductally distended with UW-solution containing 2.67-3.33 mg/ml collagenase, and the distended pancreas was digested in a continuous digestion filtration device. The islets were purified on a isoosmotic Ficoll-sodium-diatrizoate gradient. The survival period of the diabetic recipients in group 2 and 3 receiving, respectively, a low (2.14+/-0.39 microL/kg body weight) and a high (4.99+/-0.83 microL/kg body weight) islet mass was significantly prolonged compared to that of diabetic recipients in group 1 receiving no islet transplantation. However, the survival period of the recipients in group 2 was not significantly different to that in group 3. Three recipients of an islet mass of >5 microl/kg body weight became normoglycemic (fasting blood glucose <100 mg/dl) for more than two months. Furthermore, the glucose and insulin release reactions to the glucose challenge were comparable to that before pancreatectomy. Contrarily, another five diabetic recipients of an islet mass of <4 microL/kg body weight became a fasting blood glucose level of <200 mg/dl. The glucose and insulin release reactions to the glucose challenge were improved only, but not normalized compared to that before pancreatectomy. The data presented in this study demonstrate that metabolic normalization in pancreatectomized diabetic minipigs can be established by autotransplantation of an islet mass of >5 microl/kg body weight.

Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation.

Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3+/-8.0% for vitrified and 51.8+/-3.0% for conventionally cryopreserved islets; the recovery rate was 84.8+/-12.2% and 92.8+/-12.4%, respectively. Insulin recovery after transplantation was 25.6+/-7.3% for fresh and 24.1+/-7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.

Isokinetic gradient centrifugation prolongs survival of pig islets xenografted into mice.

Highly purified porcine islets were prepared by isokinetic gradients performed subsequently to isopycnic gradients. This additional purification step separates ductal, vascular, and lymphoid tissue effectively from endocrine tissue. Although ductal, vascular, and lymphoid tissue comprises only a minor contamination of the islet suspensions, a significant prolongation of the survival of porcine islets xenografted into streptozotocin diabetic C57BL/6 mice can be achieved by the elimination of the non-endocrine tissue. Rejection after islet transplantation is delayed from 2.2+/-0.4 days (n=27) to 13.1+/-2.1 days (n=36), respectively, when conventionally purified and highly purified islets are compared. Irrespective of the purification state, pretreatment of islets by low temperature culture had no effect on xenograft survival.

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation.

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4+/-30.1x10(6)) compared with empty capsules (41.4+/-19.7x10(6)) and non-encapsulated porcine islets (18.1+/-3.3x10(6)). The percentage of CD 3 positive T-lymphocytes rose to 44.5+/-11.5% in case of microencapsulated xenografts compared with 19.2+/-8.2% for non-encapsulated xenografts and 4.9+/-2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2+/-8.9% vs 15.2+/-7.0% for free islets and 4.9+/-1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Insulin-specific IgG and IgE antibody response in type I diabetic subjects exclusively treated with human insulin (recombinant DNA).

Bovine and porcine insulins elicit specific antibody response in diabetic subjects after a few months of treatment. In seven type I diabetic individuals who were exclusively treated with human insulin (recombinant DNA) each month, sera were examined for the development of insulin-specific IgG and IgE antibodies. In all patients (except one), IgG antibodies occurred after 2 mo and tended to further increase in concentration after 5-6 mo. IgE antibodies could be detected after 1 mo with a further increase after 2-3 mo and a marked decline thereafter. No patient exhibited allergic symptoms. The results indicate that physicochemical properties of insulin preparations used for treatment and the route of administration are of more importance for immunogenicity than species differences of insulin.