摘要:

Background.Pancreatic islet transplantation (PIT) is an attractive alternative for patients with type I diabetes mellitus. PIT is not yet an effective clinical reality due in part to the high incidence of rejection and early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. In the present study, the effects of PIT on glycemic parameters were assessed in spontaneously diabetic primates.Methods.Five insulinopenic nonhuman primates (threeMacacca fascicularis,oneCeropithecus aethiops, and oneMacacca mulatta) were studied. All required twice-daily treatment with 4–10 U of insulin. For immunosuppression, the animals received anti-CD3-immunotoxin (100 &mgr;g/kg/initially infused 2 hr before transplantation and again on day +1), cyclosporine (CsA) (20 mg/kg/i.v./2 hr before transplantation), cyclosporine microemulsion (Neoral) 60 mg/kg/b.i.d. on days +1 to +3 with dose adjusted by blood levels, and methylprednisolone (15 mg/kg day 0 to +3). Three recipients were given islets from a single donor (M mulatta). The islets were prepared by a semiautomated technique using Liberase. A mean of 13,136 islet equivalents/kg was infused into the portal vein. Two animals (M fascicularis and M mulatta) were used as a diabetic, nontransplanted control. Several metabolic parameters were evaluated.Results.All monkeys that underwent transplantation experienced reversal of diabetes mellitus with normalization of all diabetic glycemic parameters. In the nontransplanted primates given the same immunosuppression but no PIT, diabetic metabolic parameters were unchanged after 9 months of follow-up. In contrast, all three PIT recipients established fasting and nonfasting euglycemia within 1–2 weeks, and none required exogenous insulin after day 10. Normal intravenous glucose tolerance tests were observed at day 15, and no significant differences in the glucose disappearance rate (Kg) were observed at days 15, 45, 190, and 365 days after transplantation. The acute insulin response to glucose indicated no significant reduction of functional islet mass.Conclusions.PIT in severely insulinopenic type I diabetes mellitus primates resulted in restoration of normal glycemic parameters and durable islet mass. Operational tolerance was achieved with only 4 days of drug administration, sparing the animals from chronic exposure to potentially diabetogenic immunosuppressive drugs. These results offer an exciting new potential for type I diabetes mellitus treatment.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem.Methods.To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later.Results.One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20×106OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy. Survival was significantly prolonged in 90% of hepatectomized rats receiving OUMS-29 cells.Conclusions.A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.The major problem of xenotransplantation is, that hyperacute xenograft rejection (HXR) causes graft failure within minutes or a few hours because of natural antibodies and activation of the complement system. As a preclinical model we transplanted pig hearts orthotopically into baboons. To prevent HXR after orthotopic xenotransplantation (oXHTx), the immunoglobulins (Ig) and natural antibodies were adsorbed to reusable Ig-Therasorb\Rimmunoadsorption (IA) columns.Methods.We performed three oXHTx of landrace pig hearts into baboons (19±6.8 kg), using extracorporeal circulation (ECC) connected to the IA unit. After separating the recipient’s blood into plasma and cellular fraction by a plasma filter, plasma flow was directed to the Ig-Therasorb column coated with polyclonal sheep-antibodies against human IgG, IgM, and IgA. Intraoperative treatment consisted of 4 cycles of IA. For a control, we transplanted one pig heart into a baboon (16.9 kg) without applying IA. Perioperatively, serum concentrations of Ig, anti-pig-antibodies, complement and cardiac enzymes were determined. Tissue samples of myocardium were collected at the end of the study for immunohistochemical examinations, light microscopic examination (LM) and electron microscopic examination (EM). For cardiac monitoring after oXHTx, we used ECG, echocardiography, and invasive measurement of cardiac output. To prevent a mismatch of donor and recipient heart size, the donor pig had a 30–40% lower body weight than the recipient baboon.Results.Four cycles of IA removed >80% of IgG, IgM, and IgA from plasma. The graft of the control animal failed after 29 min. The first oXHTx with IA was intentionally terminated after 100 min, the second oXHTx after 11 hr and the third oXHTx after 21 hr. All xenografts showed no histological signs of HXR. After weaning off ECC, these donor hearts worked in sinus rhythm without electrocardiographic ST-segment elevation. An excellent cardiac output was measured by echocardiography and thermodilution (2 L/min). Serological parameters indicating cardiac damage were significantly lower after IA if compared with the control experiment. Macroscopically, the xenograft of the control animal showed massive hemorrhage in comparison with the almost inconspicuous grafts after IA. The myocardium of the IA group demonstrated fewer deposits of Ig and complement components compared with the control animal.Conclusion.Baboons do not hyperacutely reject a porcine xenograft after antibody depletion by the Ig-Therasorb column. In our experiment only 4 cycles of immunoapheresis effectively prevented HXR after oXHTx of baboons. The Ig-Therasorb column is a reusable device, which can be handled easily in combination with the ECC. IA must be tested in oXHTx long-term survival experiments, especially in combination with transgenic pig organs, which could be a reliable preclinical approach for future clinical xenotransplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.It is crucial for clinical islet transplantation to find a procedure to improve the success rate of insulin independence after islet transplantation. In the present study, we determined whether hepatocyte growth factor (HGF) has a favorable effect on amelioration of hyperglycemia in streptozotocin (STZ, 200 mg/kg)-induced diabetic mice (C57BL/6) receiving a marginal mass of intrahepatic islet isografts.Methods.Isolated syngeneic islets were transplanted into the liver of recipients. HGF with dextran sulfate (DS) was administered intraperitoneally once a day at day 0, 2, 4, 6, and 8 relative to islet transplantation. DS has been known to enhance the effect of HGF.Results.It was found that the number of 250 islets was a marginal mass as donor islets in this model, in which 2 out of 14 diabetic mice receiving 250 islets became normoglycemic by 90 days after transplantation. The treatment with HGF (100 &mgr;g) in conjunction with DS (200 &mgr;g) produced normoglycemia in all mice (n=5). Morphological study as well as intraperitoneal glucose tolerance test revealed the beneficial effects of HGF. To our surprise, six out of nine mice receiving 250 islets and treated with DS alone became normoglycemic. Additional anti-HGF antibody treatment (100 &mgr;g, day −1, 0, 2, 4, 6, and 8) abolished the effects of DS, indicating that the effect by DS is mediated via the endogenous HGF. The effects of DS were not observed when the renal subcapsular space was the site of islet transplantation. There was a significant increase in plasma HGF levels in mice after the intrahepatic grafts but not the renal subcapsular one.Conclusions.These findings demonstrate that HGF is essential for amelioration of hyperglycemia in STZ-induced diabetic mice when a marginal mass of islets was grafted into the liver. As the liver is the site of clinical islet transplantation and the inability to achieve insulin independence after transplantation is a major obstacle for successful transplantation, HGF may facilitate to overcome such an important issue for clinical islet transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.As T-cell receptor-major histocompatibility complex (MHC) class I/self peptide interaction regulates T-cell development in the thymus, we reasoned that presentation of peptides by self dendritic cells (DC) to developing T cells in the thymus might induce acquired thymic tolerance. This hypothesis is based on the finding that intrathymic injection of allopeptides in the adult animal induces acquired tolerance. To examine this hypothesis, we studied the effects of intrathymic (IT) injection of a single immunodominant Wistar-Furth (WF) MHC class I (RT1.Au) peptide-pulsed host DC on islet allograft survival in the WF-to-ACI rat combination.Methods.Bone marrow-derived ACI DC expressing MHC class I and II, OX62, and ED2 present allopeptides to naive and specifically peptide-primed syngeneic T cells in mixed lymphocyte reaction. Host DC pulsed with RT1.Aupeptide 5 (residues 93–109) were injected into the thymus of streptozotocin-induced diabetic ACI that were transplanted 7 days later with donor-type (WF) or third-party (Brown Norway [BN]) islets.Results.Whereas IT injection of 300 &mgr;g of peptide 5 alone led to normoglycemia and permanent islet survival in three of six diabetic ACI recipients, similar treatment combined with simultaneous intraperitoneal injection of 0.5 ml of anti-lymphocyte serum (ALS) on day −7 led to 100% permanent islet allograft survival (>200 days) compared to a mean survival time of 15.0±2.3 days in controls treated with ALS alone. In contrast, similarly prepared animals rejected the third-party (BN) islets in an acute fashion. To address the question of indirect allorecognition in acquired thymic tolerance, we examined the effect of peptide-pulsed host DC on graft survival. Whereas IT injection of peptide-pulsed host DC alone resulted in permanent islet survival in two of five animals, IT injection of peptide-pulsed host DC combined with 0.5 ml of ALS induced 100% donor-specific permanent islet allograft survival in the WF-to-ACI rat combination. These results suggest that thymic DC take up, process, and present the administered peptide to the developing T cells by the indirect allorecognition pathway in the induction of acquired thymic tolerance.Conclusion.We have demonstrated a novel approach to inducing transplant tolerance to islet allografts with IT injection of allopeptide-pulsed host DC. This finding suggests that immunization strategies using DC expressing MHC allopeptides or peptide analogue might be potentially useful in the treatment of autoimmune diabetes mellitus.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.We examined the role of nitrosative stress in allograft destruction.Methods.Rats undergoing cardiac transplants received NOX-100, a water-soluble nitric oxide (NO) scavenger with antioxidant properties, with or without low-dose cyclosporine (CsA). Graft survival, NO production, and nuclear factor kappa B (NF-&kgr;B) activity were studied.Results.Using NOX-100 daily until rejection prolonged graft survival (11.6±0.6 vs. 7.4±0.2 days;P<0.05). Daily low-dose CsA (2.5 mg/kg im) for 7 days or until rejection also prolonged survival (12.6±0.5 and 21.6±1.6 days, respectively;P<0.01 vs. Controls). Low-dose CsA for 7 days and NOX-100 for 30 days prolonged graft survival (45.0±4.7days;P<0.01 vs. all groups.). NOX-100 had no effect on whole blood CsA levels. Combination therapy until Day 100 resulted in 1 graft loss at Day 116 and indefinite survival in 3 animals (>300 days), which accepted a second WF strain heart without further immunosuppressive therapy but promptly rejected a third party (ACI) cardiac allograft. NOX-100 and CsA reduced nitrate and nitrite, and combination therapy completely normalized NO through to Day 30. Electron paramagnetic resonance spectroscopic analysis demonstrated reduction of signals for nitrosylmyoglobin and nitrosylheme with NOX-100 and elimination of signals with CsA alone or combination therapy. Activity of myocardial NF-&kgr;B decreased with monotherapy vs. untreated allografts. Combination therapy resulted in further inhibition of NF-&kgr;B up to Day 30. The extent of graft survival correlated with the extent of NO scavenging and NF-&kgr;B inhibition. Short-term combination therapy had no effect on graft lymphocytic infiltrate on Days 15, 20, and 30.Conclusion.These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.Organ xenografts are fulminantly rejected by antibody-mediated vascular rejection. Surrogate tolerogenesis (ST), the induction of tolerance within the donor, is effective with aorta xenografts. This preliminary study assesses the effect of ST on preformed antibodies and rejection of porcine heart xenografts.Methods.Tolerance to the donor pig was induced by infusing recipient marrow into fetal pigs. Later, pig splenocytes were transfused and heterotopic pig hearts transplanted using chimeric or nonchimeric pigs. Anti-pig antibodies were assessed.Results.With ST alone, xenografts developed cellular rejection at 4–6 days, whereas control grafts developed vascular rejection at 3–4 days (cellular vs. vascular,P<0.03). There was a reduction in preformed antibodies (P<0.03). ST combined with moderate cyclosporine prevented rejection at 9+ and 25 days in sensitized recipients compared with vascular rejection at 0.5–2 days for controls (P<0.07).Conclusions.ST seems to provide protection against vascular rejection. The cellular rejection seems sensitive to cyclosporine.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.The immunosuppressive effect and other properties of a novel immunosuppressant, FTY720, have been studied mostly in the experimental transplantation of various extrahepatic organs. In this experiment, we evaluated the antirejection potency and adverse effects of this agent on liver grafts using a canine liver transplantation model.Methods.Forty-eight orthotopic liver transplantations were performed by the standard technique under a veno-venous bypass. Liver recipients were divided into two studies: a single-dose study with FTY720 at various doses and a combined dose study with conventional immunosuppressants (cyclosporine or tacrolimus) alone and combined with FTY720. Survival, biochemical and hematological tests, blood levels of immunosuppressants, and postmortem histology were determined.Results.The median survival of untreated control animals was 9 days, whereas treatment with FTY720 at a dose of 0.1 mg/kg/day prolonged graft survival to 49.5 days. FTY720 at 1 mg/kg/day showed a slight but insignificant prolongation to 16 days, but when the dose was increased to 5 mg/kg/day, the graft was rejected at 10 days. The combination of FTY720, 0.1 mg/kg/day, with a subtherapeutic dose of cyclosporine, 5 mg/kg/day, prolonged median animal survival from 40 days with cyclosporine alone to 74 days. A combination of FTY720 (0.1 mg/kg/day) with tacrolimus (0.5 mg/kg/day) compromised animal survival, reducing survival from 83.5 days with tacrolimus alone to 30.5 days due to infectious complication and emaciation by overimmunosuppression. No evident drug-induced side effects were observed.Conclusions.FTY720 has a potent immunosuppressive effect when used alone at 0.1 mg/kg/day in canine liver transplantation. FTY720 is a promising candidate for future clinical application in orthotopic liver transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.In recent years, hepatic support systems using xenogeneic cells have been developed to support patients in fulminant hepatic failure. The extent to which xenogeneic hepatocytes metabolize and excrete human organic anions is unclear. In these studies we examined the ability of the ex vivo porcine liver to clear human bile acids during extracorporeal liver perfusion (ELP).Methods.Four patients with fulminant hepatic failure underwent extracorporeal liver perfusion with 9 porcine livers. The venovenous circuit was designed as previously described (NEJM,1994,331:234) as were the immunologic features (Transplantation 1994,58:1162). Bile from the porcine liver and serum samples were collected hourly during perfusion. Three bile acids (glycocholic, glycodeoxycholic, taurodeoxycholic acid) were selected as markers for human bile and three (glycohyocholic, glycohyodeoxycholic, and glyco-3&agr;-hydroxy-6-oxo-5&bgr;-cholanoic acid) for markers of pig bile. Bile acids from both serum and bile were processed and analyzed through high performance liquid chromatography. The Students’ttest was used for statistical analysis.Results.The mean duration of perfusions was 4.1±1.5 hr. The mean total bile acid clearance from serum (243±44 &mgr;mol/h) was similar to the total bile acid biliary excretion (286±84 &mgr;mol/hr,P=0.06). After 1 hr of perfusion, bile samples demonstrated a predominance of pig bile salts (65%). After 3 hr of perfusion, human bile acids made up 85% of total biliary bile acids. Pig bile acids appeared in patients’ sera after 1 hr of perfusion, and after 3 hr, 35% of serum bile salts were pig-specific.Conclusions.Porcine livers perfused with human blood can clear the serum of potentially toxic human bile acids and excrete them into bile. Simultaneously, the percentage of pig-specific bile acids in patient serum increases during xenogeneic perfusion for unknown reasons. The relative hepatic uptake of bile acid from serum is similar to bile acid excretion in bile. Further development of systems using porcine livers or hepatocytes is warranted.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background.Unlike simple cold storage (CS), pulsatile machine preservation (MP) of kidneys for transplantation permits pharmacologic manipulation of the perfusate and aids in the pretransplant assessment of the kidney graft. These characteristics of MP may have importance in the era of increasing use of extended criteria donor kidneys. The overall aim of this article is to critically assess practices at our preservation unit with respect to graft function. Specific aims are to (1) compare the influence of MP versus CS on graft function, (2) determine which pretransplant variables have significance in pretransplant assessment, and (3) determine whether pharmacologic manipulation during MP is advantageous.Methods.There were 650 consecutive kidneys preserved in our laboratory between January 1, 1993 and March 1, 999, by either MP or CS. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer-MPS or Belzer II solution. Perfusion parameters and electrolytes were measured serially during pulsatile perfusion. All CS kidneys were stored in University of Wisconsin solution. All kidneys obtained from donors exhibiting extended criteria features underwent pretransplant frozen section biopsies. Transmission electron microscopy (EM) was performed on a subset of kidneys undergoing pharmacologic manipulation. Four agents were assessed prospectively for their ability to influence MP characteristics when added to perfusate: PGE1, trifluoperazine, verapamil, and papaverine.Results.MP was associated with improved immediate, 1- , and 2-year graft function and reduced length of initial hospital stay when compared with CS grafts. Changes in the machine perfusion variables flow and resistance, and the [Ca++] in perfusate, were significantly associated with delayed graft function (DGF) after the transplant. Biopsy information was not predictive of DGF. The addition of PGE1to perfusate improved MP characteristics, reduced the release of [Ca++] into perfusate, and ameliorated mitochondrial ischemic injury in transmission EM images. Early graft function was improved in the presence of PGE1+MP, compared with function in the presence of other pharmacologic agents or CS alone.Conclusions.MP is associated with improved early and long term renal function. Moreover, PGE1augments MP in improving graft function. The combination of MP+PGE1may be important in optimizing the ability to use extended donor criteria kidneys and, thereby, improve the overall efficiency of cadaveric renal transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2000

数据来源: OVID