Isolation of porcine pancreatic islets for xenotransplantation.

This chapter deals with a technique for isolating intact islets of Langerhans from the pig pancreas based on our experience performing approximately 750 isolations. The procedure we describe involves identification of an optimal donor pancreas, purification and in vitro culture of islets, diabetes induction in recipients, and transplantation of islets and their immunomodulation. Besides the sophistication of the technical equipment employed, the major factors influencing the isolation outcome are the pig breed, the number and morphology of the islets in the donor pancreas, the quality of the collagenase/neutral protease, and the skill of the team members.

Goettingen Minipigs (GMP): Comparison of Two Different Models for Inducing Diabetes.

PURPOSE: Preclinical experiments on large animals are indispensable for evaluating the effectiveness of diabetes therapies. Miniature swine are well suited for such studies due to their physiological and pathophysiological responses. METHODS: We compare two methods for inducing diabetes in Goettingen minipigs (GMP), in five with the beta cell toxin streptozotocin (STZ) and in five other GMP by total pancreatectomy (PE). Glucose homeostasis was assessed with the intravenous glucose-tolerance test (IVGTT) and continual monitoring of interstitial glucose levels. At conclusion of the observation period, the pancreata were examined histologically. Three non-diabetic GMP served as control group. RESULTS: The IVGTT revealed markedly diabetic profiles in both GMP groups. STZ-GMP were found to harbor residual C-peptides and scattered insulin-positive cells in the pancreas. PE-GMP survived the total pancreatectomy only with intensive postoperative care. CONCLUSIONS: Although both methods reliably induced diabetes in GMP, the PE-GMP clearly had more health problems and required a greater expenditure of time and resources. The PE-GMP model, however, was better at eliminating endogenous insulin and C-peptide than the STZ-GMP model.

Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function.

OBJECTIVE: The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPR(dn)) in pancreatic islets. RESEARCH DESIGN AND METHODS: GIPR(dn) transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and beta-cell proliferation and apoptosis were quantified to characterize this novel animal model. RESULTS: Eleven-week-old GIPR(dn) transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic beta-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPR(dn) transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPR(dn) transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, beta-cell proliferation was reduced by 60% in 11-week-old GIPR(dn) transgenic pigs, leading to a reduction of beta-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. CONCLUSIONS: The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of beta-cells, and physiological expansion of beta-cell mass.

Analysis of parathyroid graft rejection suggests alloantigen-specific production of nitric oxide by iNOS-positive intragraft macrophages.

BACKGROUND: During acute rejection of organ or tissue allografts T cells and macrophages are dominant infiltrating cells. CD4-positive T cells are important for the induction of allograft rejection and macrophages are important effector cells mediating cytotoxicity via production of nitric oxide (NO) by the inducible NO-synthase (iNOS). In the present study we analysed whether the destruction of primarily nonvascularised parathyroid allografts is also mediated by iNOS-positive macrophages. METHODS: Hypocalcaemic Lewis rats received parathyroid isografts (from Lewis donors) and allografts (from Wistar Furth donors), respectively, under the kidney capsule. Levels of serum calcium above 2 mmol/L correlated with normal parathyroid function and below 2 mmol/L with parathyroid rejection. Accelerated parathyroid allograft rejection was induced by immunisation of Lewis recipients with the allogeneic peptide P1. RESULTS: Determination of serum calcium levels is a useful parameter to control parathyroid graft function, and therefore to determine allograft rejection. Macrophages positive for both major histocompatibility complex (MHC) class II molecules and costimulatory molecules accumulated in iso- and allografts, but iNOS-positive macrophages were only detectable in allografts in the presence of activated CD4-positive T cells. These results confirm a cooperation between activated T cells and intragraft macrophages to induce macrophage iNOS expression. Recipients immunised with the allogeneic peptide P1 demonstrated accelerated rejection of allografts (mean+/-SD: 9.2+/-0.9 days) in contrast to nonimmunised animals (mean+/-SD: 15.8+/-1.8 days). Allografts of P1-immunised animals were infiltrated faster by activated CD4-positve T cells and, in addition, the infiltrates of iNOS-positive macrophages were stronger than those in allografts of nonimmunised animals. CONCLUSIONS: Intragraft iNOS-positive macrophages seem to be able to produce cytotoxic NO involved in the killing of allogeneic cells during the alloimmune response against primarily nonvascularised parathyroid organ grafts. Infiltrates of iNOS-negative macrophages found in parathyroid isografts were caused by antigen-independent inflammation triggered by surgically induced injury. The absence of activated T cells in isografts and their presence in allografts underlines their importance in inducing macrophage iNOS expression.

Inhibitory effects of rat bone marrow-derived dendritic cells on naïve and alloantigen-specific CD4+ T cells: a comparison between dendritic cells generated with GM-CSF plus IL-4 and dendritic cells generated with GM-CSF plus IL-10.

BACKGROUND: Unlike mouse immature bone marrow (BM)-derived dendritic cells (DC), rat immature BMDC have not been thoroughly characterised in vitro for the mechanisms underlying their suppressive effect. To better characterise these mechanisms we therefore analysed the phenotypes and immune inhibitory properties of rat BMDC generated with GM-CSF plus IL-4 (= IL-4 DC) and with GM-CSF plus IL-10 (= IL-10 DC). RESULTS: Both IL-4 DC and IL-10 DC exhibited lower surface expression of MHC class II and costimulatory molecules than mature splenic DC. They had a strong inhibitory effect on responsive T cells in vitro and despite their weak function as antigen-presenting cells they induced anergic T cells. However, only anergic T cells induced by IL-4 DC had a suppressive effect on responsive T cells. Induction of suppressive/tolerogenic T cells by IL-4 DC required direct contact between antigen-specific T cells and IL-4 DC. In addition, IL-4 DC and IL-10 DC prolonged allograft survival in an antigen-specific manner. CONCLUSION: A unique phenotype of immature BMDC was isolated from the cultures. The mechanisms underlying the suppressive effect may be caused by their inability to deliver adequate costimulatory signals for T-cell activation. In addition, IL-4 DC but not IL-10 DC induce anergic T cells with suppressive function. This indicates that IL-4 DC and IL-10 DC may differ in the quality of their costimulation although no differences in the surface expression of costimulatory molecules were found.

Investigation of the regeneration potential of the recurrent laryngeal nerve (RLN) after compression injury, using neuromonitoring.

INTRODUCTION: The aim of this study was to investigate the regeneration potential of RLN after the compression of the nerve, without disrupting its continuity, using neuromonitoring. METHODS: In the first operation, the RLN and nervus vagus of adult Goettingen minipigs were dissected free, and the neuromonitoring parameters (amplitude, threshold and lag time of signal) were measured. Injury of the RLN was induced using a "bulldog" clamp. When the signal was no longer detectable, after the 15 min regeneration phase, the operation was finished. The neuromonitoring studies (see above) were repeated in a second operation 6 months later. RESULTS: (1) After the first operation, acute clamping of the RLN led to a reduction in the amplitude of the neuromonitoring signal; the lag time and the threshold of signal remained. Complete restitution of the signal was observed during the first regeneration phase. Repeated clamping led to complete disappearance of the signal. (2) During the second operation, i.e., after 6 months of regeneration, the neuromonitoring signals of both RLN and nervus vagus were detected in 93% of the GMP. No statistical differences (p = 0.17) were noticed between the amplitude of the RLN before the nerve injury (first operation) and after nerve regeneration (second operation). A significant increase in the lag time (p < 0.0005) was shown for both RLN and nervus vagus. CONCLUSIONS: The acute compression of RLN can only be detected by observing the amplitude of the neuromonitoring signal. Restitutio ad integrum is possible after a short clamping period but it is important to preserve the RLN continuity.

Xenogeneic islet transplantation of microencapsulated porcine islets for therapy of type I diabetes: long-term normoglycemia in STZ-diabetic rats without immunosuppression.

PURPOSE: Rejection and possible infection with porcine pathogens are obstacles in clinical xenogeneic transplantation of porcine pancreatic islets (PPI) to treat diabetic patients. A solution to this problem could be microencapsulation of the PPI. However, isolation and microencapsulation are highly demanding tasks with considerable risks of damaging the PPI. Thus, it is not surprising that the long-term function (>200 days) of microencapsulated PPI (mPPI), transplanted to diabetic rats, has been observed only in a few cases. METHODS: Diabetes was induced in Wistar rats with streptozotozin (STZ 60 mg/kg body weight). Animals with consecutive blood glucose levels >300 mg/dl for more than 2 days were considered diabetic. PPI were isolated from brain-dead hybrid pigs (age 6-7 months or 2-3 years) using the Ricordi-technique and Liberase(PI). After in vitro culture PPI were microencapsulated with highly purified barium-alginate and 1,000 mPPI of 300-500 microm Ø were transplanted under the left kidney capsule and/or into the peritoneal cavity of STZ-diabetic rats (n = 15) without immunosuppression. Daily, later weekly, blood glucose level and body-weight were measured. RESULTS: mPPI showed normal glucose tolerance in vitro and also in vivo. Normoglycemia occurred between day 1 and 15 after transplantation. Four mPPI grafts functioned for more than 230 days, the longest now for >550 days. Three rats are currently normoglycemic for >40 days. Six rats lost xenograft function after 12-20 days, due to inflammatory reactions at the site of the grafts. Two xenografts failed to induce normoglycemia, because the capsules did not contain enough viable PPI. CONCLUSIONS: Microencapsulated xenogeneic islets can induce long term normoglycemia in rats without immunosuppression. However, very often the grafts fail to control the blood glucose level adequately. The reasons for these failures are currently under investigation. Nevertheless, our results are very promising and might lead the way towards preclinical trials in non-human primates.

No transmission of porcine endogenous retroviruses (PERVs) in a long-term pig to rat xenotransplantation model and no infection of immunosuppressed rats.

BACKGROUND: Xenotransplantation from pig to humans may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) that are present in the genome of all pigs and that infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential to perform in vivo infection studies in animal models. MATERIAL/METHODS: To study PERV transmission in rats, rat primary cells and cell lines were treated in vitro with virus from different sources. Based on the assumption that susceptible cell lineages not yet tested in vitro could be present in the animal, PERV was inoculated into naïve and immunosuppressed animals. To investigate PERV transmission in a long-term exposure experiment, sera from animals grafted with pig Langerhans islet cells were tested in a Western blot assay for antibodies against PERVs. The animals were treated with streptozotocin to induce diabetes and microencapsulated and non-microencapsulated pig islet cells were applied without immunosuppression. RESULTS: No productive infection of a few selected rat primary cells or cell lines was observed in vitro. PERV-specific antibodies were found in none of the animals and no integration of PERV into rat cells of different organs was observed, indicating that infection had not occurred. CONCLUSIONS: This report demonstrates a lack of infection of rats in vivo even during immunosuppression or long-term exposure (up to 460 days) to a functioning xenotransplant. This report also shows that rats possibly due to a low receptor concentration on their cells are not a suitable animal model to study PERV transmission in vivo.

Neoadjuvant therapy of gastric cancer with the human monoclonal IgM antibody SC-1: impact on the immune system.

Adjuvant therapies for minimal residual disease are a promising approach to improve the poor survival rates after surgery of gastric tumors. A pilot study of a neoadjuvant therapy was performed using a human monoclonal IgM antibody (SC-1) specifically inducing apoptosis in signet ring cell stomach carcinomas. However, scarce information exists on how such a treatment affects the immune system, in particular what are the effects of apoptosis induction and infusion of large amounts of IgM. Thus, the leukocyte composition (CD3, CD4, CD8, CD19, CD16+56, CD14) and several cytokines (TNF-alpha, IL6, IL12, IFN-gamma, GM-CSF, Neopterin) before and after SC-1 application were measured and compared to results of patients that underwent surgical removal of gastric carcinoma without antibody treatment. After SC-1 application, an increase in TNF-alpha and a decrease of lymphocytes and CD3+ T-cells but in the range obtained in healthy individuals was observed before surgery. After surgery, the IL6 levels increased and the TNF-alpha levels remained at the elevated level. Furthermore, there was a significant drop in lymphocytes and CD3+ T-cells. These effects were due to the surgical treatment. Other parameters did not show significant changes. It seems that the application of an apoptosis-inducing antibody prior to surgery of gastric tumors has mild if any effect on the immune system. Therefore, from an immunological point of view, the treatment with this monoclonal antibody is extremely safe.

Preoperative evaluation of microencapsulated human parathyroid tissue aids selection of the optimal bioartificial graft for human parathyroid allotransplantation.

Allotransplantation of microencapsulated parathyroid tissue is a promising approach to the treatment of permanent hypoparathyroidism. Preoperative assessment of the quality of microencapsulated parathyroid tissue could facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation. Parathyroid tissue from patients with secondary hyperparathyroidism (n = 15) was processed mechanically or enzymatically (collagenase type II). Tissue particles and single cells/cell clusters were routinely microencapsulated with amitogenic Ba(2+) alginate. Parathyroid secretion dynamics in response to stimulation of nonencapsulated and microencapsulated parathyroid tissue with Ca(2+) were evaluated in a perifusion system. The stability of the different types of microcapsule was assessed using an osmotic pressure test. Mechanical cutting of parathyroid tissue led to peripheral necrosis of tissue particles and impaired their vitality. Collagenase digestion, in contrast, resulted in single cells and cell clusters without peripheral necrosis. The quality of microencapsulation of single cells/cell clusters was significantly better than that of tissue particles (deformed and imperfect capsules). Microencapsulation itself did not decrease cell vitality. Nonencapsulated and microencapsulated tissue particles and single cells/cell clusters from different donors maintained their own levels of response to stimulation with low Ca(2+). Microcapsules containing tissue particles showed poor stability compared with those containing single cells/cell clusters. Preoperative evaluation of microencapsulated parathyroid tissue can disclose differences in vitality and function and thus facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation.

Surface analysis of an encapsulation membrane after its implantation in mini-pigs.

The biocompatibility of membranes aiming at being a part of a bioartificial pancreas has been tested. For that purpose, we have studied a polycarbonate membrane surface after its implantation in mini-pigs. The membranes were made hydrophilic by an argon plasma surface treatment followed by a dipping in a hydrophilic polymer solution. Two polymers were tested: polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC). To test their biocompatibility, an encapsulation device for pig Langerhans islets, with external membranes treated as described above, was implanted in different mini-pigs. The pigs received no further treatment. The devices were explanted after in vivo exposure and the membranes were analysed by XPS (x-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). After this time, the substrate with the PVP or HPMC treatment was still detected on the different samples. The surface treatment signal, however, was attenuated. This is explained by the detection of other components partly covering the surface. XPS and ToF-SIMS analyses revealed the presence of biological molecules on the two faces of the membrane: the outside face in contact with the biological environment and the inside face in contact with the device. ToF-SIMS images show the inhomogeneity of the biological molecules on the membrane surface. In conclusion, biological molecules adhered to the encapsulation membrane surface after implantation but the surface treatments remained unaltered.

A new biocompatible material (Lyoplant) for the therapy of congenital abdominal wall defects: first experimental results in rats.

Congenital abdominal wall defects are impressive and dramatic malformations. Common surgical therapy for omphalocele and gastroschisis is to place the herniated viscera back into the abdomen and to close the fascia. Small defects can be closed directly by surgical treatment. In large defects, resorbable and non-resorbable artificial materials are necessary to close the fascia. The aim of this study is to find out whether new biocompatible materials might be suitable for the treatment of such abdominal wall defects. A median laparotomy was performed in young Wistar rats with a body weight of 75-100 g. Then a full thickness defect was created by excising a 1.5 x 2.5 cm segment including fascia, muscles and peritoneum. These defects were then closed by implantation of a PTFE mesh (Dual-Mesh, n = 6), a PPP mesh (Prolene, n = 6) or a new biocompatible mesh (NBM; Lyoplant, n = 6). Each rat was examined daily after treatment. Bodyweight was determined and the possible development of a hernia was monitored. After 6 weeks, the abdomen was opened again. Adhesions to the intestine were measured and the abdominal wall was removed for histological and tensiometric examination. (1) Compared to the untreated controls, all animals showed physiologic growth and normal bodyweight curve. (2) Only in one rat (Prolene) did an abdominal hernia develop. (3) In contrast to PTFE and PPP mesh, NBM showed only minimal adhesion to the intestine. (4) Tensiometry revealed high stability for non-resorbable materials. However, the characteristics of NBM were very similar to untreated abdominal wall. Our initial results indicate that biocompatible materials can also be used for the therapy of congenital abdominal wall defects.

Interaction of polyelectrolytes and their composites with living cells.

Since the layer-wise polyelectrolyte deposition offers the opportunity to modify surfaces for biomedical applications, interactions and toxicity between polyelectrolytes and living cells become interesting. The aim of the present work is to determine the different factors such as contact area, charge, and transplantation site that influence the cell reaction to a specific polymer. We found that toxicity is influenced by all these factors and cannot be tested easily in a model.

Differentiation of in vitro-modified human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells.

BACKGROUND & AIMS: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. METHODS: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions. RESULTS: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed beta cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1. CONCLUSIONS: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration.

Intragraft distribution of lymphocytes expressing beta7 integrins after small bowel transplantation in mice.

Lymphocytes with activated beta7 integrins (alphaEbeta7 and alpha4beta7) contribute to inflammatory reactions in the small bowel. Since the selective recruitment of lymphocytes to the lymphoid compartments of the small bowel is controlled by distinct adhesion molecule interactions, a compartment-dependent use of beta7 integrins may influence the rejection response within intestinal transplants. To further delineate the nature of beta7 integrin-mediated graft infiltration, we analysed their expression on T lymphocytes in the heterotopically transplanted small bowel of BALB/c and C57BL/6 mice. Lymphocytes isolated from the epithelium, lamina propria (LP), Peyer's patches (PP), and mesenteric lymph nodes (MLN) were analysed by three-color fluorescence flow cytometry using monoclonal antibodies (mAb) to integrin the subunits, lymphocyte markers, and MHC I of the donor and recipient strains. On postoperative day 5 (POD) after allogeneic small bowel transplantation (SBT), 43% of intraepithelial lymphocytes (IEL) and 63% of LP, 93% of MLN, and 93% of PP lymphocytes were of host origin. In the MLN and PP of allografts, a major infiltrating lymphocyte population consisted of CD8+ cells with increased expression of alpha4beta7 and decreased expression of L-selectin, an adhesion molecule profile characteristic of intestinal effector cell phenotypes. An increase in alpha4beta7 levels was also found on CD8+ host lymphocytes in the LP. The integrin profile of a number of host IEL suggests an ongoing transition from the phenotype of graft infiltrating lymphocytes with high levels of alpha4beta7 and low levels of alphaepsilonbeta7 to that of resident IEL with high levels of alphaepsilonbeta7 and low levels of alpha4beta7. The importance of beta7-mediated lymphocyte trafficking to the graft is attested by the significant reduction in the host lymphocyte population in the LP, PP, and epithelium following the administration of a beta7-blocking mAb to allograft recipients. In conclusion, while the infiltration patterns of lymphocytes may vary between the lymphoid compartments of intestinal allografts, host CD8+ lymphocytes with high levels of alpha4beta7 constitute a major effector cell population that affects the entire graft.

Allogeneic core amino acids of an immunodominant allopeptide are important for MHC binding and TCR recognition.

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.A(u)) and recipient (RT1.A(l)) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response.

Comparison of in vivo lymphocyte proliferation between allogeneic and xenogeneic heart transplantation in mice.

There are controversial in vitro data comparing the strength of the cellular immune response between allogeneic and xenogeneic stimulator/responder combinations. The present study therefore compares in vivo lymphocyte proliferation using heart transplantation (HTx) models in mice. Heterotopic HTx into BALB/c mice was performed using donor organs from mice (BALB/c and C57BL/6) or Lewis rats. Intraperitoneally given bromodeoxyuridine (BrdU) was incorporated into the DNA and was subsequently analyzed by flow cytometry. On postoperative days 3 and 5, proliferation of splenocytes, CD4(+) T-lymphocytes, and CD19(+) B-lymphocytes was significantly higher after xenogeneic than after allogeneic and isogeneic HTx. No significant difference was observed when proliferation of CD8(+) lymphocytes was determined. The increased in vivo proliferation after xenotransplantation may reflect an earlier and probably stronger cellular immune response compared to allogeneic transplantation. The higher CD4(+) lymphocyte proliferation underscores the importance of this cell population in xenograft rejection.

Short-term immunosuppression after rat parathyroid allotransplantation.

The aim of this study was to evaluate whether short-term postoperative immunosuppression is able to sufficiently prolong graft survival after experimental allogeneic parathyroid transplantation. Heterotopic parathyroid transplantation was performed in 6 groups: 1) syngeneic control Lewis (LEW) to LEW; 2) allogeneic control Wistar-Furth (WF) to LEW; 3-5) WF to LEW plus short-term immunosuppression, postoperative days 1-13 (cyclosporine 5/10/20 mg/kg); and 6) WF to LEW plus 10 mg/kg CyA from preoperative day 7 to postoperative day 7. Graft function was examined up to 60 days; histological and immunohistological examination was performed on all grafts with impaired function. Graft function after syngeneic transplantation was indefinite, while recipients of allogeneic grafts turned hypocalcemic after 13 +/- 2 days. With immunosuppression, graft function was 21 +/- 2 days (groups 5 and 6) and 28 +/- 3 days (groups 3 and 4). Histologically, a cellular infiltrate responsible for graft destruction was found. The results show that indefinite parathyroid allograft survival cannot be achieved by short-term immunosuppression alone. Whether the combination of an additional graft pretreatment and immunosuppression has an impact on graft function will be further examined.

Monoclonal antibody against beta7 integrins, but not beta7 deficiency, attenuates intestinal allograft rejection in mice.

BACKGROUND: beta7 integrins mediate homing and retention of lymphocytes to the normal and inflamed small bowel in a tissue-selective fashion. In the present study, we investigated the expression of beta7 integrins after small bowel transplantation (SBT) and tested the effects of blocking beta7-mediated pathways by using monoclonal antibody (mAb) or knockout mice. METHODS: Heterotopic SBT from BALB/c to C57BL/6 (B6) was used as a surgical model. Expression of beta7 integrins was measured on recipient lymphocytes (CD4 and CD8 ) in spleen, blood, and mesenteric lymph nodes (MLN) using flow cytometry. To analyze the effects of blocking beta7 integrins on graft survival, either beta7-deficient B6 or wild-type B6 mice that were treated with mAb against beta7 were studied. RESULTS: After allogeneic SBT, there were markedly increased levels of alpha4beta7 recipient CD8 lymphocytes in MLN, blood, and spleen as early as 3 days postoperatively. In contrast, alpha4beta7 integrin levels in isograft recipients were similar to those of normal mice. Mean survival time of intestinal allografts was not affected by beta7 deficiency (7.3+/-1 days) compared with wild-type mice (7.5+/-0.8 days). However, mAb against beta7 integrins significantly prolonged graft survival (12.8+/-1 days) compared with treatment with control mAb (7.3+/-1 days, <0.001). Histologic changes of SBT rejection were significantly attenuated when mice were given mAb against beta7. CONCLUSION: As indicated by the increased levels of alpha4beta7 CD8 lymphocytes, activation of this integrin contributes to the immune response in SBT rejection. Furthermore, blocking beta7 integrins with mAb provides a suitable target for immunosuppressive therapy. The discrepancy in survival data obtained by mAb and beta7 deficiency may be a result of the more rapid activation of compensatory mechanisms in the knockout mice.

Hierarchical immunogenicity of donor MHC class I peptides in allotransplantation.

The recognition of major histocompatibility complex (MHC) allopeptides by recipient MHC class II-restricted CD4(+) T cells via indirect pathway is a prerequisite for the generation of an immune response to the allograft. We tested 13-mer to 24-mer peptides from the MHC class I molecule for their possible immunogenicity in a fully MHC-mismatched rat strain combination. Our results confirm the hierarchical distribution of the immunogenicity of donor MHC class I peptides in the T cell alloactivation via indirect pathway. In addition, we show that allopeptide-induced immune response is critical for acute rejection of heart allografts. Among the seven allopeptides tested, peptide P1 was identified as immunodominant; it induced the greatest T cell proliferation and cytokine production in vitro as well as a significant reduction in allograft survival time. The TCR repertoire of T cells involved in the in vitro and in vivo responses induced by the dominant allopeptide P1 was found to be limited to the Vbeta10 and Vbeta 19 gene families. The identification of dominant allopeptides should greatly facilitate characterization of the specific T cell population responsible for allograft rejection and may be used to modulate the alloimmune response through antigen-specific therapy.