摘要:

Background.The clinical importance of endothelial-cell (EC) antibodies (Abs) in allo-transplantation (Tx) has been reported. However, lack of a suitable method for isolation of donor-specific ECs has prevented routine detection of these Abs before Tx. We describe a quick and simple method for the direct isolation of ECs from whole blood, for routine crossmatching (XM) to detect anti-EC Abs.Methods.ECs were isolated using magnetic beads coated with Abs against the angiopoietin receptor Tie-2 that is expressed on EC precursors. A retrospective analysis of 50 previously well-characterized XM sera taken immediately before transplantation from patients with end-stage kidney disease were tested.Results.Tie-2+ cells expressed human leukocyte antigen (HLA) class I, class II, and other EC markers. Sera known to contain only EC-specific or EC- and monocyte (EM)-reactive Abs reacted positively with Tie-2+ cells but not with Tie-2- cells from the same individual. In addition, the Tie-2+ cells reacted with sera containing only HLA class I or class II Abs. In all, 3 of 25 sera from patients with stable graft outcome and no rejections reacted with Tie-2+ cells.Conclusions.For the first time, with use of a single-target cell population, the detection of clinically relevant donor-specific HLA class I, class II, EM, and EC-specific Abs can be performed routinely before Tx. This method is promising because it is quick, specific, and easy to perform on whole blood samples and can therefore be used to perform routine donor-specific EC crossmatching (ECXM) in the future. Routine use of ECXM will aid in identifying better donor-recipient combinations and thus have a greater impact on transplant survival as compared with lymphocyte crossmatch (LXM).

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.There is marked heterogeneity in blood concentrations of tacrolimus following standard body-weight-based dosing. This is most apparent in black patients, who have a higher dose requirement when compared with other ethnic groups. Differences in intestinal P-glycoprotein and hepatic and intestinal cytochrome P4503A activity have been postulated as contributing to this problem.Methods.The dose-normalized blood concentrations of tacrolimus at 3 months after renal transplantation were related to CYP3AP1 and multiple drug resistance (MDR)-1 genotypes determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis.Results.We found that a single nucleotide polymorphism in the CYP3AP1 pseudogene (A/G44) that previously has been noted to be more common in African Americans and strongly associated with hepatic CYP3A5 activity correlated well with the tacrolimus dose requirement. A weaker association was found for a polymorphism in the MDR-1 gene, which influences intestinal P-glycoprotein expression.Conclusions.The CYP3AP1 genotype is a major factor in determining the dose requirement for tacrolimus, and genotyping may be of value in planning patient-specific drug dosing.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets.Methods.We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry.Results.The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2.Conclusions.Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.Polyomavirus (PV) nephropathy has been attributed to reactivation of BK virus (BKV) or more rarely JC virus (JCV). The simian virus (SV) 40 is PV that was likely introduced into the human population through contaminated vaccines. The purpose of this study was to identify and characterize the PV that is associated with PV nephropathy.Methods.The clinical diagnosis of PV nephropathy (PVN) was made in patients with acute deterioration in renal function whose renal biopsies showed typical viral cytopathic changes in tubular epithelial cells and staining for PV T antigen. Polymerase chain reaction (PCR) amplification of DNA from peripheral blood mononuclear cells (PBMC), urinary cells, and renal biopsy tissue was performed using specific primers for the transcription control regions of BKV, JCV, and SV40, respectively.Results.Six cases of PV nephropathy were identified in 91 renal transplant recipients (7%). Immunosuppressive therapy was modified in all patients. Renal function stabilized or improved in four patients and deteriorated in two patients, and one patient has lost his allograft, after follow-up from 2 to 25 months. PCR detection demonstrated BKV genome in three of five PBMC samples, six of six urinary cell samples, and two of four renal biopsies. SV40 genome was detected in two of five PBMC samples, one of six urinary cell samples, and two of four renal biopsies. Infectious SV40 and BKV was demonstrated in CV-1 co-cultures using urine from one patient. JCV was not detected in any PVN sample. Co-infection with BKV and SV40 was found in two PVN patients. Urine samples obtained 12 months after transplant from 26 transplant recipients without PVN on simultaneous protocol renal biopsy were analyzed by PCR; BKV genome was demonstrated in 5 of 25 samples, JCV genome was demonstrated in 3 of 25 samples, and SV40 genome was demonstrated in 0 of 25 samples.Conclusion.The authors report molecular evidence that co-infection with BKV and SV40 occurs in renal transplant patients with PVN, suggesting that SV40 may contribute to PVN after renal transplant.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.Ischemia-reperfusion injury is associated with an increased risk of acute rejection, delayed graft function, or chronic graft dysfunction. Mitochondria play a central role in this process.Methods.Using an autotransplantation pig kidney model, both early (40 min and 7 days) and late (2–16 weeks) changes in renal function and morphology were determined after different periods of cold ischemia in University of Wisconsin or Euro-Collins solutions. We have also investigated the expression of the peripheral-type benzodiazepine receptor (PBR), which is also critical in maintaining outer mitochondrial membrane stability.Results.Function of the kidneys was better preserved after 1 hr and 24 hr than after 48 hr and 72 hr in Euro-Collins and University of Wisconsin solutions. Medulla injury was reduced in 1 hr-preserved and 24 hr-preserved groups. PBR was found to be present in epithelial cells of the deep cortical and outer medulla in both normal human and well-preserved pig kidneys. PBR expression was modulated by ischemia-reperfusion injury and the concurrent tubular injury and repair processes.Conclusion.This study indicates that PBR expression correlates with the quality of kidney preservation and might serve as an index of kidney and mitochondria viability. Moreover, these data suggest that PBR might be involved in membrane biogenesis during reperfusion. In addition, considering the identical localization of PBR in human and pig kidneys, these findings could have a direct application in human clinical settings of kidney pathology.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.Emerging data place extracellular matrix (ECM) proteins as important elements in lymphocyte positioning and effector function in alloreactive responses. Using a non-vascularized model of allogeneic heart transplantation in Swiss mice, we have observed a correlation between the cellular infiltration and ECM deposition towards the interior of the graft during the kinetics of rejection.Methods.To confirm the importance of ECM during the rejection process in this model, we treated the transplanted animals with local injections of antilaminin monoclonal antibody and analyzed, by histology and immunohistochemistry, the grafts on day 15, which corresponds to the peak of cellular infiltration and ECM deposition.Results.The treatment with mAb antilaminin decreased the cellular infiltrate and ECM deposition within the grafts, as compared to controls. Moreover, we found a diminished IFN-&ggr;, TNF-&agr; and IL-2 deposition in the transplant area, and a reduced co-localization of these cytokines with laminin. By contrast, the antilaminin treatment increased tenascin deposition, a molecule with immunosuppressive properties, and also caused an increase in apoptosis of the cellular infiltrate.Conclusions.These data hallmark the importance of laminin, in distinct aspects concerning the events leading to allograft rejection, and also reinforce this molecule as a potential target for immune intervention in organ transplantation.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.The recent results of clinical islet transplantation have improved substantially with the introduction of a more potent but less diabetogenic immunosuppressant protocol. The successful development of this protocol was based in part on the outcomes of studies reported herein, addressing the diabetogenic potential of a series of immunosuppressant agents used alone or in combination in a canine islet autograft model. Although it is recognized that failure to achieve long-term insulin independence in human islet allotransplantation has been multifactorial, with low engraftment mass, acute or chronic rejection, autoimmune recurrence, loss of islet-acinar integrity, heterotopic site, denervation, and insulin resistance all being implicated to varying degrees, avoidance of diabetogenic immunosuppression has been pivotal to the enhanced outcomes of clinical islet transplantation. We here explore the effects of clinically relevant doses of cyclosporine or tacrolimus when given alone or in combination with glucocorticoids on long-term canine islet autograft function.Method.Dogs (n=8) underwent total pancreatectomy, islet isolation, and intrasplenic autotransplantation and were normoglycemic with stable long-term graft function 3 months to 8 years posttransplant. The frequently sampled intravenous glucose tolerance test (FSIGT) was performed predrug (baseline), at 1 month of therapy (on drug), and again 1 month after withdrawal of therapy (postdrug).Results.Monotherapy treatments with low- or high-dose prednisone, Neoral, or tacrolimus had minimal impact on islet autograft function. The combination of Neoral and prednisone led to a marked impairment in glucose decay (25% decline from 1.77±0.2 to 1.24±0.2,P<0.05), without significant change in insulin responsiveness or glucose effectiveness. However, insulin sensitivity was markedly impaired while on therapy (7.10±1.2 to 3.10±0.5,P<0.01). Importantly, glucose decay and insulin sensitivity failed to return to baseline after withdrawal of therapy. The combination of tacrolimus and glucocorticoids led to permanent and irreversible diabetes in all recipients (n=6,P<0.001). Similar treatment of healthy control dogs led to a 44% decrease in glucose decay (P<0.01).Conclusions.Immunosuppression must be specifically tailored for islet transplantation and be glucocorticoid free if insulin independence is to be sustained clinically.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.Using a rat (Lewis-Wistar Furth) abdominal heterotopic transplantation model, we reported previously that the expression of cyclooxygenase (COX)-2 is increased in parallel with that of nitric oxide synthase (NOS)-2 during cardiac allograft rejection.Methods.To investigate effects of COX-2 inhibition in this model, allograft recipients were treated orally (PO) with 5 mg/kg per day of the tetra substituted furanone selective COX-2 inhibitor 5,5-dimethyl-3-(3 fluorophenyl)–4-(4 methylsulfonal) phenyl-2 (5H)–furanone (DFU) in 1% methyl cellulose solution.Results.In the treated animals, allograft survival was increased from 6.3±0.5 to 12.6±2.6 days (P=.001). At days 3 and 5 posttransplantation, there were reductions in the extent of the inflammatory infiltrate, endovasculitis, myocardial edema, and cardiomyocyte damage in rejecting allografts. The mean numbers of apoptotic cardiomyocytes determined with the terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling (TUNEL) technique were significantly reduced in DFU-treated grafts compared with untreated controls (P<0.05). At day 3 posttransplantation, prostaglandin E2 synthesis by myocardial slices incubated with 100 &mgr;M bradykinin was reduced from 1097±156 to 153±63 pg/mg of protein in the treated allografts (P<.005). At day 5, COX-2 protein and mRNA together with COX-2, NOS-2, and nitrotyrosine immunostaining in damaged cardiomyocytes were diminished in treated versus control grafts.Conclusion.The data indicate that the inhibition of COX-2 prolongs allograft survival and reduces myocardial damage and inflammation during acute cardiac allograft rejection.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Background.We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals.Methods.Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day −2. Cyclosporine was administered for 30 to 60 days beginning on day −1. A total of 1 to 2×1010/kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine.Results.Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected.Conclusions.These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.

ISSN:0041-1337    

出版商:OVID    

年代:2002

数据来源: OVID