[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.

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.

[Obtaining of mesenchymal progenitor cells from the human umbilical cord]. / Gewinnung mesenchymaler Progenitorzellen aus der humanen Nabelschnur.

BACKGROUND: Mesenchymal progenitor cells (MPCs or mesenchymal stem cells, MSC) have the capability for differentiation into various lineages of mesenchymal tissue. MPCs are widely distributed in a variety of tissues in the adult human body and also present in the fetal environment. However, MPCs are a rare population in these tissues. In this study we evaluated the possibility that MPCs or cells with MPC-like potency are present in the umbilical cord (UC). METHODS: Term UCs were collected and stored in sterile saline solution. The UCs (10 cm) were cut into 1 cm length, the vessels were striped manually and the tissue immersed in an enzyme cocktail for 3 h at 37 degrees C. The isolated umbilical cord mesenchymal progenitor cells (UCMPCs) were pelleted by low speed centrifugation, suspended and cultured. RESULTS: (1) Umbilical cord mesenchymal progenitor cells (UMPCs) could be isolated in sufficient quantities and (2) could be cultured easily. (3) These cells demonstrated a fibroblast-like phenotype. (4) They could be expanded in culture and induced to form several different types of cells. (5) In immunochemistry these cells express mesenchymal markers (CD 13, CD 105) but not haematopoetic lineage markers (CD 14 and CD 34). CONCLUSION: Our observation suggested that MPCs are present in human umbilical cord. Instead, it should be considered a valuable resource for the isolation of potent cells for cell-based therapies, especially in general and pediatric surgery.

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.

Immunogenicity of parathyroid allografts in the rat: immunosuppressive dosages effective in passenger leukocyte-rich small bowel transplants are not effective in parathyroid gland transplants with few passenger leukocytes.

BACKGROUND AND AIMS: The passenger leukocytes harboured within an allograft induce a massive allo-immune response that leads to allograft rejection if not countered by immunosuppression. We compared the response to short-term immunosuppression of parathyroid gland transplants possessing few passenger leukocytes with that of passenger leukocyte-rich small bowel transplants. METHODS: Heterotopic parathyroid and orthotopic small bowel transplantation was performed in a Wistar Furth-to-Lewis rat strain combination. Immunosuppression with cyclosporine A (CsA) was administered in different dosages for 14 days. Dysfunctional allografts were examined immunohistologically. RESULTS: CsA more effectively suppressed the immune response provoked by immunogenic small bowel grafts than that induced by less-immunogenic parathyroid grafts. Immunosuppression with 20 mg/kg per day induced long-term survival in the small bowel (165+/-21 days) but not in the parathyroid (28+/-3 days). All rejected grafts featured massive cellular infiltration by activated T cells as a sign of immune rejection. CONCLUSION: Immunosuppressive dosages effective in passenger leukocyte-rich small bowel transplants were not as effective in parathyroid gland transplants harbouring few passenger leukocytes. In spite of the paucity of passenger leukocytes in parathyroid grafts it is more difficult to control by immunosuppression the immune response to them than that to the passenger leukocyte-rich small bowel.

Influence of donor MHC class I antigen expression on graft survival after rat parathyroid allotransplantation.

BACKGROUND AND AIMS: We investigated the influence of donor MHC antigen expression on graft survival after parathyroid transplantation in three different strain combinations. METHODS: MHC class I and II expression on parathyroid tissue of Lewis (LEW), Dark Agouti (DA), and Wistar-Furth (WF) rats was first analysed semiquantitatively by immunohistochemistry. Additionally, five groups were transplanted: (1) LEW to LEW, (2) DA to DA, (3) LEW to DA, (4) WF to LEW, and (5) DA to LEW. METHODS: MHC class I expression was strong in DA, moderate in WF, and weak in LEW rats; MHC class II expression was negative in all three strains. In the interstitium of all investigated tissue specimens, the proportion of MHC class II-expressing cells was low. RESULTS: After syngeneic transplantation, graft survival could be documented over the whole observation period. A mean graft survival of 20 (+/-2) days was observed following transplantation from LEW to DA, grafts in the group WF to LEW were rejected after 13 (+/-1) days, and graft function lasted 8 (+/-2) days in the group DA to LEW. The number of intragraft leukocytes expressing MHC class II molecules was equal in all groups, whereas increased levels of MHC class I on rat parathyroid tissue before transplantation resulted in a more rapid rejection. CONCLUSION: These results demonstrate that immunogenicity of rat parathyroid tissue seems to be determined by the amount of MHC class I expressed on donor parenchymal cells.

Orthotopic liver/small bowel transplantation in rats: a microsurgical model inducing tolerance.

Liver cirrhosis in patients with short bowel syndrome is successfully treated in humans by simultaneous liver/small bowel transplantation. However, until now, a clinically relevant experimental rat model for this procedure has not existed. We therefore established a protocol that, for the first time in rats, allows the simultaneous transplantation of arterialized liver and small bowel into an orthotopic position. Short-term immunosuppression induced not only allograft acceptance but tolerance (as demonstrated by indicator heart/skin transplantation). The immunosuppressive dose required to achieve this result was dramatically less than that of protocols for successful small bowel transplantation alone. Immunohistochemistry detected a transient rejection crisis before tolerance. During this crisis, apoptotic recipient-type T lymphocytes, mainly CD8+ cells, accumulated in the liver but not in the small bowel allograft. The initiation of T-cell apoptosis is one possible explanation for the specific immunosuppressive effect of the liver allograft, which also supports the simultaneously transplanted small bowel allograft in our model.

Rat parathyroid allotransplantation: Influence of MHC antigen expression on graft survival.

MHC antigen expression in parathyroid tissue and its influence on graft survival after allogeneic transplantation were investigated using a heterotopic rat transplantation model. MHC class I and II expression in parathyroid tissue of Lewis (LEW), Dark Agouti (DA), and Wistar-Furth (WF) rats was analysed semi-quantitatively by using immunohistochemistry. MHC class I expression was strong in DA, moderate in WF, and weak in LEW rats parenchyma, whereas MHC class II expression was negative. In the interstitium of all investigated tissue specimens, the proportion of MHC class II-expressing cells was low. Additionally, four groups were transplanted: 1) LEW to LEW, 2) DA to DA, 3) LEW to DA, and 4) WF to LEW. After syngeneic transplantation, graft survival could be documented over the whole observation period. A median graft survival of 20 (+/-2) days was observed after transplantation from LEW to DA, whereas grafts in the group WF to LEW were rejected after 13 (+/-1) days. The number of intra-graft leucocytes expressing MHC class II molecules was the same in all groups, whereas increased levels of MHC class I in parathyroid tissue before transplantation resulted in a more rapid rejection. These results indicate that immunogenicity of rat parathyroid tissue might be determined by the amount of MHC class I expressed in donor parenchymal cells. Further experiments are necessary to validate this observation.

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.