Exendin-4 improves the oral glucose tolerance in diabetic rats: pancreas regeneration, better function of pancreatic islets, or impaired glucose uptake?

One major obstacle for successful clinical transplantation of isolated pancreatic islets (PI) is their limited survival in vivo. The aim of this study was to analyze the functional and morphological regeneration of PI in diabetic rats by Exendin-4 (Ex4) treatment in vivo. Male Wistar rats (n = 3/group) received 20 nmol/kg Ex4 i.p. for 20 days (day 0 to day +20). Diabetes was induced with 50 mg/kg streptozotocin i.v. on day -3 or on day +5. Diabetic and normal control rats received 0.9% NaCl i.p. instead. Body weight (BW), daily blood glucose (BG) and levels, oral glucose tolerance (OGT) were tested on day -5, day +10, day +20, and on day +22, ie, 48 hours after the last Ex4 injection. Histology of the pancreata ended the study on day +24. In vivo application of Ex4 could not prevent the development of diabetes. Injection of Ex4 led to a significant decrease in postprandial BG levels to 35% for 12 hours. Surprisingly, Ex4 increased postprandial BG levels up to 20% in normal rats. Ex4-treated rats showed better OGT than untreated controls. Interestingly, 48 hours after the last Ex4 injection on day +22 OGT was completely impaired. The Ex4-treated rats lost BW much faster than the untreated controls, and showed signs of gastroparesis at autopsy. Immunohistochemistry of the pancreata documented no signs of islet regeneration. Improvement of OGT in diabetic rats after Ex4 treatment may be explained by increased insulin release from the individual PI, which was confirmed by perifusion studies with isolated PI in vitro (data not shown). Yet, Ex4 may also exert an influence on the gastrointestinal tract as it delays the uptake of glucose during gastroparesis.

Use of the continuous glucose monitoring system in Goettingen Minipigs, with a special focus on the evaluation of insulin-dependent diabetes.

OBJECTIVES: Adult pig islet isolation has greatly improved in the past few years. Islet grafts may now be tested in large animals. Continuous Glucose Monitoring System (CGMS) was applied to diabetic Goettingen Minipigs (GMP) to improve the management of hyperglycemia and hypoglycemia and their welfare before transplantation. METHODS: GMP (25-35 kg) received a minipig diet once daily. Diabetes was induced by streptozotocin (STZ; 150 mg/kg intravenous [IV]; n = 5) or by surgical pancreatectomy (PGMP; n = 3). Interstitial glucose concentration (IGC) was monitored continuously with an implanted sensor; CGMS was calibrated using conventional blood glucose tests 3-4 times per day; CGMS data were fed into the monitor memory and analyzed using CGMS software. RESULTS: Glucose sensors were handled accurately. Diabetes occurred 2-3 days after STZ or immediately after pancreatectomy with basal C-peptide secretion of <0.4 ng/mL (measured using intravenous glucose tolerance test) and prompt loss of body weight. Insulin substitution was necessary to keep the GMP in good condition for up to 5-6 months, with stable body weight and normal behavior. Some GMP became hypoglycemic, which was only documented by CGMS, but not by conventional glucose assays. Tight glucose control and substitution of exocrine enzymes (Creon 25,000 E/d) reduced morbidity of the PGMP, which was then comparable with that of STZ-GMP. CONCLUSIONS: The CGMS, developed for humans, is equally suitable for the 2 GMP diabetes models. Close-meshed glucose monitoring and insulin treatment improved the general condition of the diabetic GMP, ie, the islet graft recipients, and will thus greatly add to posttransplantation success.

MHC class II-mediated antigen presentation by alloreactive rat CD4+ T cells does not induce regulatory properties.

Activated CD4+ T cells have the capacity to express major histocompatibility complex (MHC) class II molecules and to present processed antigens to T cells. Because the role of MHC class II positive T cells in allograft rejection is unknown, the purpose of this study was to investigate their function as antigen-presenting cells (APCs) in the allogeneic immune response. For this, alloreactive CD4+ T cells were induced in Lewis rats by immunization with the allogeneic peptide P1. The P1-specific T cells are involved in the rejection of allografts from Wistar Furth rats. With monoclonal antibodies specific for the alphabeta T-cell receptor (clone R73) and MHC class II molecules (clone Ox6), the presence of antigen-specific T cells, with and without expression of MHC class II molecules, was demonstrated. Concerning their ability to bind these antibodies they were characterized as R73(pos), Ox6(pos) and R73(pos), Ox6(neg), respectively. The R73(pos), Ox6(pos) T cells loaded with P1 were indeed very effective in restimulating R73(pos), Ox6(neg) T cells but not vice versa. Further on, R73(pos), Ox6(pos) T cells, but not R73(pos), Ox6(neg) T cells, were able to activate naïve allogeneic T cells demonstrating their capacity to express co-stimulatory molecules. In addition, specific mRNA for CD86, MHC class II, and CIITA, the master regulator of MHC class II expression, were detectable in the R73(pos), Ox6(pos) T cells only. In conclusion, the R73(pos), Ox6(pos) T cells act as professional APCs with the possible biological capability of amplifying the local immune response to the allograft.

Immunoelectron microscopic localization of MHC structures in isolated pancreatic rat islets.

An immunogold-silver enhancement technique, which combines effective labeling of viable isolated islets with the ultrastructural resolution of cytological details, was applied in electron microscopy to identify major histocompatibility complex (MHC) structures on islet cells. Incubation of freshly isolated islets from CAP (RT1c) and LEW (RT1l) rats with OX18, an MHC class I antibody, showed strong positive reactivity in macrophages and/or dendritic-like cells (M0-DCs) and vascular endothelial cells (VEs) and a comparatively weaker reactivity in endocrine alpha-, beta-, and delta-cells. With MHC class II antibody OX6 (anti-I-A), M0-DCs were strongly labeled in both rat strains on the surface and on internal structures. Three of five particularly high titered batches of OX6 revealed MHC class II expression on VE and beta-cells. Four days of in vitro culture in combination with a high concentration of glucose and interferon-gamma induced strong enhancement of MHC class I structures and, to a lesser extent, class II structures on beta-cells.

New principle for large-scale preparation of purified human pancreas islets.

Because successful human islet transplantation requires large quantities of viable islets that must be separated from the highly immunogenic exocrine tissue and because handpicking is too time-consuming and laborious to be clinically relevant, a new approach for solving this problem has been established in rat models. It is based on the principle that magnetic microspheres (MMSs) coupled to lectins with binding specificity for the exocrine tissue portion are trapped in an electromagnetic field, thus providing effluent islets of a high degree of purity. In this study our aim was to adapt this principle to human islet preparations. In this context our prime interest was focused on a lectin suitable for human pancreatic tissue. Of 19 different lectins tested, only 1, Wisteria floribunda agglutinin (WFA), is suitable, as shown by immunofluorescence, MMS-lectin binding, and magnetic separation.

Measurement of rat insulin. Enzyme-linked immunosorbent assay with increased sensitivity, high accuracy, and greater practicability than established radioimmunoassay.

Total immunoreactive insulin (IRI) is conventionally determined by radioimmunoassays. IRI measurement in rats can be made more sensitive, accurate, and practical, as demonstrated by a new modified enzyme-linked immunosorbent assay (ELISA). It is characterized by indirect binding of an anti-insulin antibody by an antiglobulin antibody and uses the principle of competitive saturation. In this ELISA, IRI can be determined in a wide range of concentrations, corresponding to the standards. The standard curve ranges from 100 to 0.049 ng/ml IRI (1 ng/ml approximately 23.4 microU/ml approximately 172 pM rat insulin). The statistical analysis shows between- and within-assay coefficients of variation of less than or equal to 15%.

A comparison of the use of two immunomagnetic microspheres for secondary purification of pancreatic islets.

Immunomagnetic cell separation has been shown to be a highly attractive alternative to density-dependent methods for islet purification. There are two types of beads, magnetic inducible microspheres (MIMS) and Dynabeads, in this context. The aim of this study was to compare the two beads and ligands using the same method of purification. Two batches of collagenase were used. Using either monoclonal antibodies or lectins with a specificity for rat acinar tissue, the beads were used to immunomagnetically label pancreatic digest before magnetic separation. The results showed that both MIMS coated with a lectin and Dynabeads coated with an antibody removed 80% of the acinar contamination with a 70% islet yield. However, the MIMS were significantly less effective with the second enzyme, which produced larger acinar particles. In this study, although the MIMS produced the least nonspecific islet trapping, the Dynabeads coated with Leicester Department of Surgery no. 10 antibody were found to be the most efficient particle for immunomagnetic islet purification.

Apoptosis of T lymphocytes in liver and/or small bowel allografts during tolerance induction.

BACKGROUND: Apoptosis of parenchymal cells has been described during allograft rejection. Immunologically privileged tissue in the mouse has been found to prevent rejection by initiating apoptosis of infiltrating lymphocytes. The aim of this study was to investigate whether apoptosis may play a role in T-cell regulation during rejection and subsequent tolerance induction after liver transplantation (LTx) and combined liver/small bowel transplantation (LSBTx). METHODS: LTx and LSBTx (Brown Norway-->Lewis) were performed without immunosuppression. Cell migration, activation, and apoptosis were investigated by means of sequential histology, immunohistochemistry, and the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. Donor (Brown Norway) and third-party (Dark Agouti) cardiac allografts were transplanted into LSBTx recipients to determine specific tolerance. RESULTS: Transient acute cellular rejection occurred after LTx and LSBTx and was followed by specific tolerance. The kinetics of apoptosis were similar in liver allografts after LTx and LSBTx, but differed from the processes in small bowel allografts after LSBTx. Apoptosis of parenchymal cells in the grafted livers correlated directly with interleukin-2 receptor expression of the infiltrating T cells. During the late phase of rejection, a peak of apoptosis in the lymphocyte infiltrate was demonstrated, characterized as predominantly apoptotic CD8+ T lymphocytes. CONCLUSIONS: These results demonstrate that specific tolerance is achieved in both LTx and LSBTx after a transient rejection crisis. Apoptosis is involved in graft rejection and tolerance induction. Activation of T lymphocytes correlates with parenchymal cell apoptosis in the allograft. T-cell inactivation seems to result in apoptosis of cytotoxic T cells and tolerance, which appears to be unique to the liver allograft.

Tolerance of rat liver allografts induced by short-term selective immunosuppression combining monoclonal antibodies directed against CD25 and CD54 with subtherapeutic cyclosporine.

BACKGROUND: Our purpose was to develop and evaluate protocols for selective immunosuppression after liver transplantation using the monoclonal antibodies (mAbs) NDS-61, directed against the interleukin-2 receptor (CD25), and 1A29, directed against the intercellular adhesion molecule-1 (CD54), in combination with subtherapeutic cyclosporine (CsA). METHODS: Orthotopic rat liver transplantation (ORLT) was performed in a DA-to-LEW strain combination. Immunosuppression was administered from day 0 to +13. Functional parameters such as survival time, body weight, and serum bilirubin levels were measured and the liver grafts were evaluated histologically. RESULTS: A stepwise tapering of CsA from 3 to 0.25 mg/kg/day reduced the long-term survival rate. All animals died at a CsA dosage of 0.25 mg/kg/day, which was therefore defined as subtherapeutic. Monotherapy with the anti-CD25 mAb was performed at dosages of 600 and 1800 microg/kg/day. The lower mAb dosage resulted in a long-term survival rate of 12% and was defined as subtherapeutic. The combination therapy of CsA (0.25 mg/kg/day) and anti-CD25 mAb (600 microg/kg/day) produced a synergistic effect and led to a long-term survival rate of 84%. This survival rate was significantly higher than those after either CsA (P<0.005) or anti-CD25 mAb (P<0.001) monotherapy. Both dosages (10 and 30 microg/kg/day) of anti-CD54 mAb monotherapy as well as anti-CD54 mAb combined with a subtherapeutic dosage of CsA were ineffective in preventing acute allograft rejection. The addition of anti-CD54 mAb (30 microg/kg/day) to combined CsA plus anti-CD25 mAb therapy (triple therapy), however, increased the long-term survival rate to 100%. In the triple therapy group there was no rejection process in the liver allografts at any time, and donor-specific tolerance could be shown by donor-specific and third-party heterotopic heart transplantation. CONCLUSIONS: The synergistic action of subtherapeutic CsA plus anti-CD25 mAb NDS-60 could be demonstrated, whereas anti-CD54 mAb only had a positive effect in a triple therapy group. Triple therapy prevented both acute and chronic rejection and induced donor-specific tolerance.

Persistence of stable intragraft cell chimerism in rat liver allografts after drug-induced tolerance.

BACKGROUND: Drug-induced tolerance of rat liver allografts is well documented. We analyzed cellular events during immunosuppressive therapy on day (d) 10 and in the late phase (d 100) after transplantation to assess for characteristics in the intrahepatic leukocyte (IHL) population in the phase of tolerance. METHODS: Lewis rats served as recipients of Dark Agouti rat livers. Temporary immunosuppression with either cyclosporine (CsA) monotherapy (3 mg/kg/d) or triple therapy that consisted of a subtherapeutic CsA dosage (0.25 mg/kg/d) and monoclonal antibodies directed against the interleukin-2 receptor (IL-2R, CD25) and the intercellular adhesion molecule-1 (ICAM-1, CD54) was administered from postoperative d 0 to d 13. Cell migration and cell activation within liver grafts was assessed by standard histology and flow cytometry. IHL apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL). RESULTS: Both CsA monotherapy and triple therapy prolonged liver allograft survival to more than 100 d and led to the induction of donor-specific tolerance. Untreated recipients rejected their allografts within 14 d. In both groups, donor-specific IHLs initially dropped to 18% to 25% on d 10, but they rebounded to as much as 40% on d 100 as a common characteristic of both groups. Within this population, donor-specific T cells were dominant. In both groups, increased numbers of activated (IL-2R+) CD8+ T lymphocytes were present on d 100. No accumulation of apoptotic IHL was observed on d 100. Their proportion was unchanged in the triple therapy group and slightly decreased in the CsA group compared to the syngeneic controls. CONCLUSIONS: The present study reveals that tolerant liver allografts are repopulated by donor-specific T lymphocytes. This phenomenon is independent of the type of applied immunosuppression. The persistence of activated CD8+ T cells in the phase of proven donor-specific tolerance on d 100 indicates that liver tolerance is associated with the state of a permanent intragraft immune activation. It seems that the coexistence of donor cells with infiltrating recipient cells within liver grafts, termed intrahepatic cell chimerism, is characteristic for tolerated liver allografts.

Videomicroscopic imaging of graft mucosa for monitoring immunosuppressive therapy after small intestinal transplantation in rats.

BACKGROUND: Early diagnosis of rejection and effective immunosuppressive treatment are essential after small intestinal transplantation. To date little is known about microscopic alterations of the intestinal mucosa of the graft during rejection. We attempted to determine whether videomicroscopic imaging of the graft mucosa is a suitable method for monitoring immunosuppressive therapy. METHODS: Real-time videomicroscopic imaging of an ileostoma was performed daily after allogeneic heterotopic small bowel transplantation in the rat (BN to LEW) with and without FK506 immunosuppression. Subsequently, the videomicroscopic findings were compared with the histologically determined grade of rejection. RESULTS: A semiquantitative staging system was established for the intravital mucosal changes during graft rejection. The earliest changes related to rejection appeared on POD 6 in the untreated allogeneic group. The mucosa developed patchy paleness and the mucosal architecture was interrupted in places. The crypts were slightly widened and their color turned dark red (stage I). These alterations spread progressively over the mucosa on POD 7 (stage II). On POD 9 the mucosa appeared pale, the villi were shortened, and the crypts appeared wide and rounded (stage III). In the animals treated with FK506 similar changes were observed, but with a delayed onset. When FK506 was administered as antirejection therapy at the onset of rejection, a temporary improvement of mucosal alterations was observed (stage II --> stage I). The video-microscopic stages correlated with the histological grade of rejection. CONCLUSIONS: The introduction of videomicroscopy has made computer-based high resolution imaging of mucosal microarchitecture possible. With videomi-croscopy beginning rejection can be detected, although it can still be reversed with antirejection therapy. This is a new noninvasive technique that might be of high clinical relevance.

Induction of specific tolerance by 15-deoxyspergualin (DOS) treatment after rat skin and kidney transplantation in rats. Analysis of effectivity of various protocols of DOS application.

The purpose of this study was to analyse the effect of various protocols of 15-deoxyspergualin (DOS) application on skin or kidney graft survival. Following rat strain combinations were used: AS-->LEW (MHC identical/non-MHC-different) and DA-->LEW (MHC-different/non-MHC-different). Reference DOS dose was 2.5 mg/kg, i.p. It was shown that the effect of DOS depended on multiple factors, such as: type of tissue or organ, onset of treatment, drug dose and length of drug application. In skin transplantation graft survival was 32-34 days in AS-->LEW and 24-26 days in DA-->LEW. Kidney graft survived more than 150 days. DOS prolonged skin survival when the application was started earlier than day 8, whereas kidney graft survived only when DOS treatment was started not later than 3-4 days after transplantation. In skin transplantation a dose of 0.3 mg/kg had a small effect-prolongation graft survival up to 4 days. Higher doses induced longer graft survival, however, maximal survival of allogeneic skin was 22 days. In kidney transplantation a dose of 0.3 mg/kg led to prolonged graft survival-up to 150 days. Doses of 2.0-2.5 mg/kg were able to induce specific tolerance. The optimal skin or kidney graft survival was obtained when DOS was applied for 14 days. Shorter than 12-day treatment with DOS led to a shorter graft survival. When donor was pretreated with DOS prolongation of non-allogeneic graft survival was observed. Our results showed that short-term application of DOS is safe and effective. To obtain optimal DOS effect the drug application must be started directly after transplantation.

In vitro analysis of the mode of action of the immuno-suppressive drug 15-deoxyspergualin.

The purpose of the study was to analyse the action of the immunosuppressive drug 15-deoxyspergualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-1; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in "suppressor" MLC, 16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.

Influence of a rat kidney perfusion using the anti-MHC class II monoclonal antibody and reduced dose of 15-deoxyspergualine on the allogeneic graft survival.

One of the main factors inducing rejection of the allogenic graft are the donor MHC-class II antigens. In this study, the allogenic rat renal graft survival after the blockage of MHC-class II positive cells was analyzed and compared with the effectiveness of the recipient treatment with 15-deoxyspergualine (15-DOS). It was found that the DA (RT1 a) rat kidney perfusion with the anti-MHC class II monoclonal antibody (MoAb 29A1--Kiel) allowed to prolong survival of the graft in the LEW (RT1 1) recipient (9.6 +/- 0.8 vs. 7.7 +/- 0.5 days). Our another study demonstrated that the 14 day treatment of the LEW recipient with 15-DOS at the dose of 0.5 mg/kg body weight can induce tolerance to the grafted kidney from the DA strain. The dose of 0.2 mg/kg body weight of 15-DOS prolonged the graft survival only to a small extend (16.5 +/- 0.5 days). In contrast, the combination of the graft pretreatment with MoAb 29A1 with the application of the reduced dose of 15-DOS to the LEW recipient allowed to further prolong the graft survival (97.4 +/- 59.0 days, n = 5). In 3 cases, the long-time (close to 150 days) graft survival was obtained. The above presented results suggest that the blockade of the MHC-class II antigens can reduce the immunogenicity of the graft. Although this procedure was not sufficient to induce tolerance, it allowed to minimize the immunosuppressive treatment.

Cell migration between graft and host--an analysis with monoclonal antibodies after allogeneic rat kidney transplantation.

In order to analyse migration patterns of donor MHC class II cells out of transplanted kidney and accumulation of host cells within the graft, immunomorphological studies were performed using monoclonal antibodies in rat allogeneic kidney transplantation model. To answer the question of how many donor cells migrate out of the renal cortex MRC 0 x 3 monoclonal antibody (MoAb) against LEW MHC class II antigens was used. In the grafts explanted after 4,24 48 and 73 h, a slow reduction of donor class II cells was observed and some areas in cortex showed only very few, if any, donor cells. At the same time, starting from day 2 after transplantation accumulation of donor cells was found in perivascular spaces. Spleen sections stained at 24, 48, 72 and 96 h after transplantation revealed donor cells present in recipient's spleen. They were detected up to day 3 after surgery. Their numbers, however, decreased after day 2. After 2 and 3 days, accumulations of recipient's cells between tubules were detected. It was found that many cells in infiltrations were stained with anti-T lymphocyte MoAb. Expression of class II antigen on rat kidney cells increases significantly from the day 4 after transplantation.

Hematological effects of the new immunosuppressive drug 15-deoxyspergualin.

Since systematic hematological studies on blood and bone marrow changes after treatment with 15-Deoxyspergualin (DOS) are lacking, a quantitative assessment was performed fourteen or twenty eight days after intraperitoneal application of DOS to rats. Further observations done 7 and 14 days after discontinuation of DOS administration allowed analysis of bone marrow regeneration. DOS induced lymphocytopenia, granulocytopenia and anemia with a decrease of bone marrow cellularity due to suppression of cell maturation. The effect was dose-dependent and bone marrow as well as blood changes were observed in animals treated with doses from 0.5 to 10.0 mg/kg DOS. Within 14 days after termination of the treatment, rapid recovery with normalization of all hematological parameters was observed. In the light of our data, these hematological side effects may not be a major disadvantage, if DOS is used in doses below 2.5 mg/kg, and for a course of therapy which is limited to 7 to 14 days.

Graft-versus-host reaction in small bowel transplantation and possibilities for its circumvention.

To describe GVHR in small bowel transplantation and its underlying mechanisms and to find methods for circumventing that response, accessory small bowel transplantation was carried out in the rat model. Animals not treated with cyclosporine, irradiation, or removal of the mesenteric lymph nodes of the graft died within 22 days postoperatively due to graft versus host disease. Mesenteric lymph nodes of the graft and recipient spleen and peripheral lymph nodes showed strong immunologic stimulation histologically and high antihost T-cell-mediated cytotoxic antihost reactivity. Seventy-one percent of the animals that had received 15 mg of cyclosporine per kilogram body weight orally survived 150 days after transplantation. After donor irradiation with 50 rads, 77 percent of the recipients survived 120 days. After microsurgical removal of the mesenteric lymph nodes of the graft, 89 percent survived 120 days. We conclude that GVHR plays an important role in small bowel transplantation and that the experimental regimens of donor, graft, and recipient treatment described herein have proved their efficacy for circumventing GVHR.

Highly efficient isolation of porcine islets of Langerhans for xenotransplantation: numbers, purity, yield and in vitro function.

UNLABELLED: Xenogeneic transplantation of porcine islets of Langerhans is regarded as a potential future treatment for diabetes mellitus. Despite considerable biotechnological progress, however, it is still very difficult and often unreliable to isolate sufficient numbers of highly purified, intact islets from the porcine pancreas with good in vitro function. OBJECTIVE: Of this study was to describe an efficient and reliable method to isolate sufficient numbers of highly purified islets of Langerhans with good in vitro function from adult as well as from young hybrid pigs. METHODS: Islets were isolated from the pancreas of young (4-6 months) hybrid pigs and old (2-3 years) retired breeders using Liberase PI and digestion-filtration. Average islet size was detected by dithizone staining of tissue sections prior to isolation; only organs with an average islet size > or = 200 microns were used. Density gradient purification with OptiPrep was performed in a COBE 2991 cell processor. Viability was investigated using fluorescence staining. Perifusion studies were carried out to asses in vitro function of isolated islets. RESULTS: Islets were successfully isolated from young hybrid pigs (3,671 +/- 598 IEQ/g) and old retired breeders (5,182 +/- 545 IEQ/g). After purification islet purity was 92% for retired breeders and 87% for young hybrid pigs. Yield after purification was still not satisfactory: 64% for retired breeders (3,209 +/- 444 IEQ/g) and 44% for young hybrid pigs (1,669 +/- 386 IEQ/g). Viability of isolated islets was 80-95%. Perifusion studies of porcine islets showed sufficient insulin release upon glucose challenge; however, the level of insulin release depended on the density of islets within the perifusion chamber. Low temperature culture (24 degrees C) prior to perifusion studies had no detrimental effect on insulin release. Long-term culture over 11 days was followed by a dramatic loss of islet function. CONCLUSIONS: If xenograft rejection can be overcome and the risk of xenosis can be minimised, sufficient numbers of purified porcine islets with good in vitro function can be isolated to serve as a potential source for islet transplantation in diabetic patients.

Expression pattern of extracellular matrix proteins in the pancreas of various domestic pig breeds, the Goettingen Minipig and the Wild Boar.

UNLABELLED: In spite of progress in biotechnology, isolation of porcine pancreatic islets remains a difficult task with unpredictable results. One reason could be the lack of knowledge as to the expression of extracellular matrix proteins in porcine exocrine and endocrine tissues, particularly in "islet capsules". Such proteins are subject to digestion by proteases, yet they might have a protective function for the fragile islets. OBJECTIVE: Of our study was a detailed histological analysis of the extracellular matrix proteins in various pig breeds. A broad panel of commercial, human-specific antibodies were used, since antibodies against porcine tissue were not available. METHODS: Frozen pancreatic tissue section of 7 domestic pig breeds, the Goettingen Minipig and the Wild Boar were stained with antibodies against collagen types I, II, III, IV, V, VI, VII, IX, laminin, fibronectin, vitronectin and elastin. Binding of antibodies was detected by immunoperoxidase and evaluated microscopically. Human and rat tissue was treated in the same way. RESULTS: (1) With the exception of anti-collagen type II, type VII and vitronectin, all antibodies revealed distinct binding patterns in the pancreas of the different pig breeds. However these antibodies bound on human cartilage and skin. (2) Collagen types I, III, IV, laminin and fibronectin are expressed on porcine pancreatic "islet capsules". (3) Expression levels of these proteins on "islet capsules" vary in the different pig breeds. However, no significant differences could be found in the expression pattern of collagen types I, III, IV, laminin and fibronectin, comparing domestic, experimental and wild type pigs. (4) Older individuals (Goettingen Minipig) appear to express higher levels of proteins on "islet capsules" than younger ones. CONCLUSIONS: Antibodies with specificity for human extracellular matrix proteins can be used successfully in the porcine pancreas. Thus, analysis of the structure and composition of porcine pancreatic tissue can be performed even without pig-specific antibodies. Particularly, the effects of various proteases and collagenases on the pancreatic tissue can now be monitored by immunohistochemical analysis allowing a rational design of protease mixtures for the isolation of pancreatic islets.

[PAUL procedure. A new biocompatible concept for the therapy of congenital abdominal wall defects]. / PAUL-Procedure. Biokompatibles Konzept zur Therapie kongenitaler Bauchwanddefekte.

Treatment of small congenital abdominal wall defects (e.g. omphalocele and gastroschisis) can be performed by direct closure. In large defects non-resorbable artificial materials (e.g. Gore-Tex) are necessary to close the fascia. The aim of this study was to find out whether a new procedure, the PAUL pocedure, might be suitable for the treatment of large abdominal wall defects. A full thickness abdominal wall defect was created in young Wistar Rats. These defects were then closed by implantation of a 1x2 cm sized piece of PTFE (Dual-Mesh), a polypropylene mesh (Prolene(R)) or by using the PAUL procedure. Over a period of 6 weeks no wound infections or hernias were monitored. In contrast to PTFE the PAUL procedure showed only minimal adhesion to the intestine and a high stability of the implanted material. A xenogenic extracellular matrix, such as that used in the PAUL procedure, may induce an immune response, which is comparable with a remodeling reaction rather than rejection. Based on these good results a large animal model study (Goettinger mini-piglets) was performed. No wound infections or hernias could be observed throughout the experiment. Control laparoscopy after 3, 6, 9, and 12 months showed only minimal adhesion to the intestine. Our results indicate that the PAUL procedure can be used easily and successfully for the therapy of congenital abdominal wall defects.