PVG Recipients Research Articles

Identification of miR-27b as a Novel Signature from the mRNA Profiles of Adipose-Derived Mesenchymal Stem Cells Involved in the Tolerogenic Response

Adipose-derived mesenchymal stem cells (adipose-derived MSCs, ASCs) possess the ability to differentiate into multiple tissue types and have immune-modulatory properties similar to those of MSCs from other origins. However, the regulation of the MSC-elicited immune-modulatory activity by specific microRNA (miRNA) mechanisms remains unexplored. Gene expression profiling with knowledge-based functional enrichment analysis is an appropriate approach for unraveling these mechanisms. This tool can be used to examine the transcripts and miRNA regulators that differentiate the rat tolerogenic orthotopic liver transplantation (OLT; DA liver into PVG) and rejection OLT (DA liver into LEW) models. In both models, the rejection reaction was observed on postoperative day 7∼14 (rejection phase) but was overcome only by the PVG recipients. Thus, the global gene expression patterns of ASCs from spontaneously tolerant (PVG) and acute rejecting (LEW) rats in response to LPS activation were compared. In this study, we performed miRNA enrichment analysis based on the analysis of pathway, gene ontology (GO) terms and transcription factor binding site (TFBS) motif annotations. We found that the top candidate, miR-27, was specifically enriched and had the highest predicted frequency. We also identified a greater than 3-fold increase of miR-27b expression in the ASCs of tolerant recipients (DA to PVG) compared to those of rejecting recipients (DA to LEW) during the rejection phase in the rat OLT model. Furthermore, our data showed that miR-27b knockdown has a positive influence on the allosuppressive activity that inhibits T-cell proliferation. We found that miR-27 knockdown significantly induced the expression of CXCL12 in cultured ASCs and the expression of CXCL12 was responsible for the miR-27b antagomir-mediated inhibition of T-cell proliferation. These results, which through a series of comprehensive miRNA enrichment analyses, might be relevant for stem cell-based therapeutic applications in immunosuppressive function using ASCs.

Immunological tolerance induced by liver grafting in the rat: splenic macrophages and T cells mediate distinct phases of immunosuppressive activity

In the rat combination DA into PVG, liver grafts are not rejected but induce donor-specific transplantation tolerance. We have examined the immunosuppressive properties of spleen cells from PVG recipients of DA liver grafts at various times post-grafting. The results indicate the development of two phases of cell-mediated suppressor activity, which appear to be mediated by separate spleen cell populations. Mitomycin-C-treated spleen cells taken from animals between 5 and 28 days post-grafting were able to suppress rat mixed lymphocyte reactions (MLRs). These 'early' suppressor cells were glass adherent and absent from populations purified by passage through nylon wool or G10 Sephadex columns. Suppression of MLR by purified glass adherent cells was not specific for either stimulator or responder haplotypes and was blocked by indomethacin. Nylon wool purified T cells were not suppressive at this time. Spleen cell suppressor activity declined to background levels after 35 days post-grafting. However, spleen cells from long-term surviving liver graft recipients (20 weeks or more) were again able to suppress MLR; the 'late' suppressor cells were nylon wool non-adherent and suppression was specific for the donor (DA) MHC type. We conclude that liver grafting in this combination generates early and late phases of suppression among spleen cells, that the early phase is produced by macrophages and mediated by prostaglandins and that the late phase is dependent on allospecific suppressor T cells.

Immunological Tolerance-Related Genes in a Spontaneous Tolerant Model of Rat Liver Transplantation Explored by Suppression Subtractive Hybridization

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgapl), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.

THE ROLES OF DONOR LEUKOCYTES AND SOLUBLE MHC CLASS I IN TRANSPLANTATION TOLERANCE

A275 Aims: Spontaneous induction of tolerance occurs in the DA (RT1a) to PVG (RT1c) rat model following orthotopic liver transplantation (OLT), while heart grafts, unless accompanied by a liver transplant, are acutely rejected in 8 days. The liver, unlike the heart allograft, is a source of both secreted soluble MHC molecules and resident leukocytes which may be responsible for spontaneous tolerance. In the present study, we measured levels of circulating soluble MHC class I molecules (sMHC) following OLT and tested whether sMHC and leukocytes could substitute for a liver transplant in protecting DA cardiac allografts from acute rejection in PVG recipients. Methods: PVG rats received either a heterotopic cardiac allograft or an OLT from DA donors. MHC class I heavy chains (RT1Aa, RT1A1c or RT1Au) and β2microglobulin were produced using a prokaryotic expression system, refolded around appropriate single peptides and purified by gel filtration using FPLC. Recombinant sMHC were continuously administered intravenously to graft recipients using osmotic mini infusion pumps. Allotype specific ELISA were developed and used to quantify levels of circulating MHC levels in the recipient sera. Results: Naïve DA rats had circulating levels of sRT1Aa of ∼350ng/ml while RT1A1c was <50ng/ml in naïve PVG rats. Following OLT, donor sMHC (RT1Aa) rise rapidly, peaking around 400ng/ml before returning to a plateau of ∼200ng/ml, 2 weeks after transplantation. Recipient RT1A1c levels also increased briefly to ∼100ng/ml following OLT. Infusion, at the time of transplant, of sRT1Aa alone failed to prolong cardiac allograft survival (median survival time, mst = 8d) despite serum levels of 2000ng /ml. However, combined administration of 10-150 million donor lymph node or spleen cells together with continuous sRT1Aa (serum levels >1000ng/ml) significantly prolonged allograft survival (mst = 12d versus 6.5d for cells alone). Similarly, pre-treatment with sRT1Aa alone also prevented rapid rejection (mst = 12d). Third party sMHC or cells were ineffective. Conclusions: These findings support the concept that soluble MHC class I may play a role in spontaneous liver tolerance. It is noteworthy that the sRT1Aa used in this study was of prokaryotic (not eukaryotic) origin and therefore lacked glycosylation and presented only a single peptide rather than a range of naturally selected peptides.

Selection of lowly immunogenic and highly tolerogenic donor and recipient allochimeric class I major histocompatibility complex proteins

Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1. An rat alloantigen. We examined the potential of alpha1h-RT1. An versus alpha2h-RT1. An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts. The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1. An AAs. Allochimeric and wild-type (WT)-RT1. An proteins were generated in an Escherichia coli expression system. A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1. An protein; alpha2h148-179n-RT1.Ac protein was ineffective. However, subcutaneous injection of 100 mug WT-RT1. An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation. The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

Effects of adenoviral up-regulation of bcl-2 on oxidative stress and graft coronary artery disease in rat heart transplants.

Bcl-2 has been shown to have antioxidant properties. Early oxidative stress is an important antigen-independent factor that contributes to the development of graft coronary artery disease (GCAD). We hypothesized that adenoviral up-regulation of bcl-2 would decrease early oxidative stress and inhibit GCAD after heart transplantation. PVG rat hearts were treated with adenovirus carrying the human bcl-2 gene (AdvBcl-2) or null adenovirus (AdvNull) then transplanted into the abdomens of PVG recipients. After 4 days of reperfusion to allow adenoviral gene expression, grafts were retransplanted into ACI rat recipients and reperfused for 4 or 8 hours or 90 days (cyclosporine A 7.5 mg/kg on postoperative day [POD] 0-9). Production of tumor necrosis factor (TNF)-alpha after 4 hours and oxidized glutathione (GSSG) after 8 hours indicated development of oxidative stress. 90-day allografts were assessed for GCAD by way of computerized morphometry. Over-expression of bcl-2 at the time of allograft reperfusion was confirmed by Western blotting. Whereas AdvNull-treated hearts demonstrated elevated TNF-alpha levels after 4 hours and increased GSSG after 8 hours of reperfusion, AdvBcl-2-treated hearts were no different from nontransplanted hearts. AdvBcl-2 treatment also resulted in decreased luminal narrowing and intima-to-media ratio at POD 90. Bcl-2 over-expression interrupts the development of oxidative stress in reperfused rat-heart allografts. Early up-regulation of bcl-2 also decreases GCAD, indicating the importance of early oxidative stress and the role that bcl-2 may play in the long-term function of heart transplants.

Regulation of matrix metalloproteinases and effect of MMP-inhibition in heart transplant related reperfusion injury.

Metalloproteinases (MMPs) regulate extracellular matrix turnover and degrade basal lamina. Aim of the study was to examine the regulation of MMPs and the effect of an MMP inhibitor in transplant related ischemia/reperfusion injury. Heterotopic cardiac transplantation was performed after 4 h of ischemia in three groups of six rats: allografts (black hooded inbred strain, PVG donor/August Copenhagen Irish inbred strain recipient); allografts treated with a competitive MMP-inhibitor (Batimastat) 15 mg/kg every 24 h; isografts (PVG donor and recipient). Normal PVG hearts served as a control. Hearts were explanted after 72 h of reperfusion. Expression of MMP-2 and -9 was measured using gelatin zymography. Proteolytic activity was measured using a gelatinase activity assay. Myeloperoxidase activity and tumor necrosis factor-alpha (TNF-alpha) were measured as markers of inflammatory response. Immunostaining for collagen IV and laminin was used to study degradation of basal lamina. There was a significant increase of MMP-2 expression in allografts (2271+/-571 microg/ml) as compared to normal (683+/-139 microg/ml) and the Batimastat-treated (259+/-140 microg/ml, P<0.05) groups. Although pro-MMP-2 expression was equally high in the untreated iso- and allograft group (75+/-23 versus 62+/-30 microg/ml) MMP-2 expression in the isograft hearts was significantly lower (359+/-267 microg/ml) suggesting activation of the pro-form by an immunologic mechanism. Pro-MMP-9 levels were significantly higher in the untreated iso- and allograft groups as opposed to normal hearts and MMP-inhibited hearts. MMP-9 was completely inhibited by Batimastat treatment. Collagenolytic activity was lower in the treated group as compared to untreated allografts (538+/-140 versus 384+/-97 microg/ml, P<0.05), demonstrating effective inhibition of MMPs by Batimastat. In the treated group a lesser extent of basement membrane component alterations could be demonstrated by laminin and collagen IV staining. There was a significant reduction in myeloperoxidase activity (P=0.027) as well as lower TNF-alpha levels (ns) in the in the Batimastat treated group. Ischemia leads to an increase in MMP expression and degradation of basal lamina. This process is enhanced in allografts as compared to isografts suggesting additional activation of MMPs by immunologic mechanisms. MMP-inhibition is effective in preventing the proteolytic activity of MMPs and may alter the host rejection response by preserving extracellular matrix components and basement membranes.

LSF-1 may modulate the indirect allorecognition pathway to delay allograft rejection

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the non-rejector combination of DA donors into PVG recipients. In the present study, a polyclonal anti-LSF-1 antibody was used to detect the lymphoid cell populations expressing LSF-1 epitopes in several transplant models. In this study we examined cell samples acquired from rats that had undergone OLT in syngeneic, rejecting or non-rejecting combinations. Flow cytometry indicated that the polyclonal antibody reacts specifically with an epitope present on a CD4 CD11b positive cell of recipient origin. In the first 3 weeks after transplant there is a large increase in the number of these cells isolated from the spleens and livers of rats in tolerogenic OLT model, which correlates with prolongation of allograft survival. In the rejector and isograft models of OLT there is a minimal change in the expression of the LSF-1 N-terminal pep epitope. These results suggest that these recipient CD4 CD11b cells may have a critical role in the formation of transplant tolerance by interfering with the indirect pathway of allorecognition via as yet undetermined mechanisms. The increased expression of the LSF-1 N-terminal pep epitope on liver leukocytes 14 days after partial hepatectomy indicates a natural role for LSF-1 in the process of liver regeneration after insult or injury.

The fas and fas ligand pathways in liver allograft tolerance.

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

Identification of two down-regulated genes in rat liver allografts by mRNA differential display.

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

Identification of two down-regulated genes in rat liver allografts by mRNA differential display

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

Expression of clusterin in a rat tolerogenic OLT model

IN RAT orthotopic liver transplantation (OLT), PVG recipients of DA livers (tolerogenic model) survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart, and kidney. Many aspects of the mechanism regarding this naturally achieved tolerance have been discussed in our previous reports. However, the mechanism of liver allograft tolerance from the aspect of the complement system and its regulatory proteins has not yet been investigated. The complement system comprises a group of proteins that interact with other immune system molecules to provide many of the effector functions of humoral immunity and inflammation. Clusterin, which is a plasma glycoprotein, regulates the complement system by inhibiting the membrane attack complex (MAC) formation. Here, we studied the kinetics of clusterin expression after transplantation in the tolerogenic model (DA-PVG) compared with syngenic model (DA-DA) and acute rejector model (DA-LEW) by immunoblotting and Northern blotting.

Specific suppression of interleukin 2 biosynthesis by synthetic antisense oligodeoxynucleotides does not influence allograft rejection.

Interleukin (IL)-2 supplementation can reverse both blood transfusion-induced tolerance to kidney allografts and spontaneous tolerance to liver allografts in rats. Moreover, IL-2 expression is frequently suppressed in models of allograft tolerance. The failure of IL-2 biosynthesis might therefore play a critical role in tolerance induction. Three antisense oligodeoxynucleotides (AS-1, AS-2, AS-3) to rat IL-2, and a control oligo (C-1) consisting of a scrambled version of AS-1, were evaluated for gene-specific suppression of IL-2 biosynthesis in vitro and in vivo, and for their effects on kidney allograft survival. Reverse transcriptase-polymerase chain reaction and IL-2 protein assays were used to assay concanavalin A-driven IL-2 biosynthesis by lymph node lymphocytes in vitro. PVG recipients of Dark Agouti kidney allografts were treated with the oligos. Graft survival and IL-2 biosynthesis by reverse transcriptase-polymerase chain reaction in spleen and graft biopsy specimens were assessed. The AS-1 oligo, but not the AS-2, AS-3 or C-1 oligos, suppressed concanavalin A-driven IL-2 biosynthesis for the 4 days of culture. This effect was dependent on delivery of the AS-1 oligo with lipofectamine. Supplementation with exogenous IL-2 reversed the suppression of lymphocyte proliferation in AS-1-treated cultures. Administration of AS-1 intravenously at 10 mg/kg/day to PVG recipients of Dark Agouti kidney allografts suppressed IL-2 (but not IL-6, interferon-gamma, or tumor necrosis factor-alpha) synthesis in the grafts of seven of nine rats, as measured in biopsy specimens taken at days 2-7. By contrast, all nine control grafts strongly expressed IL-2. However, neither graft histopathology nor graft survival was affected. Antisense oligonucleotides can powerfully suppress IL-2 biosynthesis in vitro and in allograft recipients in vivo, but this does not affect kidney allograft rejection.

Malononitrilamides and tacrolimus additively prevent acute rejection in rat cardiac allografts

The novel immunosuppressive agents malononitrilamides (MNA) 279 and 715 are derivatives of A77 1726, the primary metabolite of leflunomide. The effects of these agents have been previously demonstrated in rat skin and cardiac allo- and xenotransplant models. The aim of this study was to evaluate the combination of MNA and tacrolimus in a high-responder rat cardiac allotransplant model. Graft survival was evaluated following 10 days of post-transplant oral therapy of MNA or tacrolimus alone and the drugs combined in PVG recipients of DA grafts. An iso-effect curve of single and combined drugs was constructed. Histological changes in grafts were evaluated at 10 days. MNA (20 mg/kg) or tacrolimus (2.4 mg/kg) alone prolonged graft survival with median survival of 14 and 13.5 days, respectively. Combined therapy of MNA (10 mg/kg) and tacrolimus 1.6 mg/kg likewise resulted in a median survival of 13.25 days and an iso-effect curve for these doses was constructed. Another iso-effect curve for median graft survival of 18–19 days, including MNA (10 mg/kg) + tacrolimus (3.2 mg/kg) in combination, MNA (30 mg/kg) alone and tacrolimus (4.8 mg/kg) alone, was constructed and both isoboles showed a straight line, demonstrating additive effects (zero interaction). In addition, histological analysis of grafts confirmed the benefit of the drug combination. No additional toxicity was noted with combined therapy. Optimal doses of MNA or tacrolimus had comparative effects on graft survival and histological changes, and a combination of the two drugs was beneficial with respect to both these parameters. The iso-effect curves verified additives effects of the drug combination.

A COMPOSITE GRAFT OF ISLET PLUS DONOR LYMPH NODE CELLS COULD NOT INDUCE DONOR SPECIFIC TOLERANCE IN A "LOW RESPONDER" RAT COMBINATION WITH CLASS I MHC DISPARITY

408 We previously reported that WF(RT1U) pancreas transplantation, but not islet transplantation protected recurrence of IDDM by anti-ICAM-1/LFA-1 mAbs in diabetic prone BB rats (RT1U). We have recently reported that class I MHC disparate PVG.R1 (RT1.A'B'D'C') pancreaticoduodenal grafts could be rejected in diabetic PVG (RT1.A'B'D'C') recipients by streptozotocin, while PVG.R1 liver, heart, and kidney grafts could survive indefinitely without immunosuppression. Purpose: This study was to examine that passenger leukocytes have a potencial to induce donor specific tolerance or not, in this "low responder" combination with MHC class I disparity. Materials and Methods: PTx was performed using a modification of S. Lee's method. Approximately overnight cultured 1500 islets were implanted into the subcapsular space of left kidney. Rejection was confirmed by hyperglycemic relapse over 300 mg/dl and by histological examination. PVG recipients were divided into the following 6 groups. Mixed lymphocyte reaction (MLR) and limiting dilution assay to estimate frequencies of IL-2 producing helper (fThp) and cytotoxic (fTcp) T cell precursors to PVG.R1 alloantigens were adopted for cellular immuity.□Flow cytometoric analysis (FACS) to detect antidonor antibodies was performed. Results: Only one case was rejected within 57 days in PTx (n=7) and 3 cases were rejected in ITx (n=7) within 26, 20, 19 days post grafting. Tolerant rats which PTx or ITx grafts survived more than 100 days, were challenged with donor (R1) and 3rd party (BUF) skin grafts to examine the alloantigen specificity. PTx rats rejected BUF skin grafts within 15 days, but interestingly accepted R1 skin (>100 days). Tolerant ITx rats (group 2) rejected both R1 and BUF skin grafts in 13 and 10 days, respectively. Tolerant ITx rats□(group 3.4) transplanted copmosite graft of islet plus with donor R1 ileoceacal lymph node cells, however, are prolonged only donor R1 skin than ITx (group 2). Tolerant ITx rats (group 5) transplanted copmosite graft of islet plus with BUF ileoceacal lymph node cells, however, are prolonged only donor R1 skin than ITx (group 2). Tolerant ITx rats (group 6) transplanted copmosite graft OF islet plus R1 spleen cells, however, are prolonged only donor R1 skin but could not induced tolerance. Conclusion: Donor specific tolerance was obtained by PTx, but not ITx. Immunoreguratory passenger leukocytes might be necessary for the induction of tolerance in ITx in this combination. It was not necessary for donor ileocecal lymph node to prolong graft survivals. (Table)Table

Telomerase Activity in Liver Allografts in Rats

296 Alteration of post-transplant cellular senescence following rejection may be associated with post-transplant carcinogenesis. Telomere and telomerase are involved in the central timing mechanisms for cellular aging. Here, we studied telomerase activity in livers from acute rejector OLT (DA-LEW) rats, non-rejector OLT (DA-PVG) and syngeneic OLT (DA-DA) rats. At the age of 5 months, donor livers were harvested from DA (RT1a) rats and orthotopically transplanted into LEW (RT11), PVG (RT1c) or DA recipients. All OLT (DA-LEW) rats die from acute rejection within 14 days. In OLT (DA-PVG), rejection occurs but is naturally overcome without any immunosuppression. Syngeneic OLT (DA-DA) rats are controls where no rejection occurs and rejection-related factors are eliminated. At various days after OLT, animals were sacrificed and the liver used for a telomerase study. As a strain control, the livers from age-matched DA rats were also used. Hepatic telomerase activity was measured by a semiquantitative Telomerase PCR ELISA (Boehringer Mannheim). As a positive control for the telomerase assay, rat derived liver tumor cell line or drug-induced hepatoma was used. At day 7 when the most serious rejection was observed in OLT (DA-LEW) and OLT (DA-PVG), the telomerase activity in livers (average OD value: 2.65/OLT(DA-LEW), 3.02/OLT(DA-PVG)) was much higher than that (0.54) of day 7 livers of syngeneic OLT (DA-DA) rats. Telomerase activity in OLT (DA-PVG) livers decreased after post-operative 40 days when the tolerance was established. However, longer OLT (DA-PVG) and OLT (DA-DA) survived, higher the telomerase activity increased. The intensity of the telomerase activity in OLT (DA-PVG) and OLT (DA-DA). We conclude that telomerase activity in liver allografts might reflect the immune response and alteration of cellular senescence following OLT.

Decreased expression of the interleukin 2 receptor on CD8 recipient lymphocytes in intestinal grafts rendered tolerant by liver transplantation in rats

Background—In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel...

Analysis of Immunosuppressive Proteins in Serum of Liver-Transplanted Rats by Using an Anti-LSF-1 Affinity Column

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation was performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 μ g single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days;n= 7 vs 6.5 days;n= 5, respectively,P= 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days;n= 7) or those which received only Intralipos (median = 10.5 days;n= 5,P= 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum.

E. COLI-PRODUCED SOLUBLE MHC CLASS I PROTEINS INDUCE POTENT ALLOANTIGEN-SPECIFIC IN VIVO EFFECTS

116 Because this method provides rapid production, we employed an E. coli protein expression system to produce soluble rat heavy chain class I MHC proteins. A PCR-based method with designed primers was used to introduce a six Histidine (6-His) tag and a stop codon after the α3 domain into RT1.An and RT1.Ac cDNAs. These constructs were expressed in bacterial pET-23a(+) vector with an ampicillin resistance gene and an isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible viral T7 promoter. The produced proteins were solubilized in 6M urea and purified on a Ni2+ affinity column. Untreated Lewis (LEW, RT1.Al) recipients rejected Brown-Norway (BN, RT1.An) heart allografts at a mean survival time (MST) of 7.5±0.5 days. Although subcutaneous (SC) immunization at day -4 with 1 µg of RT1.An was ineffective (8.0±1.0 days; NS), either 50 µg or 100 µg RT1.An protein induced accelerated rejection of BN heart allografts at 5.0±0.0 days (p<0.05) and 5.0±0.0 days, (p<0.05), respectively. This effect was antigen-specific; immunization of LEW rats with self-RT1.Al protein failed to induce accelerated rejection of BN heart allografts (MST 8.0±1.0 days; NS). In another strain combination, untreated PVG (RT1c) recipients rejected BN heart allografts at 9.1±1.0 days. Although SC administration (day -7) with 10 µg RT1.An was ineffective (10.0±0.0 days, NS), either 50 µg or 100 µg of RT1.An induced accelerated rejection of BN heart allografts at 7.8±0.6 days (p=0.004) and 6.1±1.0 days (p=0.0004), respectively. Immunization of the PVG rats with self-RT1.Ac protein failed to cause accelerated rejection of BN heart allografts (8.0±0.0, NS). In another control experiment, immunization with RT1.An did not induce an accelerated rejection of third-party control LEW heart allografts (11.0±1.0 days, NS). In a tolerogenic protocol, 50 µg RT1.An injected by portal vein (PV) at the time of transplantation to PVG rats in combination with a 7-day course of cyclosporine (CsA, 4 mg/kg/day) prolonged the survival of BN heart allograft to a MST of 33.0 days (13×3, 19, 20, >50, >100 days; p=0.07). Administration of RT1.An protein alone produced a MST of 9.75±0.25 days, and CsA alone, a MST of 11.0±1.0 days. Thus, the bacterial expression system may be used to produce class I MHC proteins that are active in vivo in immunogenicity and graft prolongation protocols.

Contrasting outcomes of donor-specific blood transfusion: effectiveness against cell-mediated but not antibody-mediated rejection.

Giving recipients a prior donor-specific blood transfusion (DST) is effective in prolonging organ allograft survival in some inbred strains but not in others. The present investigation analyzed two such contrasting strains of rats in an attempt to define the basis for this variation. The survival of fully mismatched Dark Agouti (RT1a) cardiac allografts was significantly prolonged (from 7 to 44 days, median survival times) in PVG (RT1c) rats given a prior (-14 day) DST, whereas it shortened survival in the high-responder PVG-RT1u strain. Injecting PVG recipients with blood from strains bearing defined differences indicated that each disparity contributed to the increased survival time in an incremental way: blood and heart matched at the MHC class I (A) and/or class II (B/D) loci had a major influence on survival; class I-like (C) and non-MHC antigens made only minor contributions. MHC disparities had contrasting effects in RT1u rats. Blood transfusions from Dark Agouti or PVG-R8 (AaB/DuCu) rats induced accelerated rejection and anti-Aa alloantibody formation; transfusing PVG-R23 (AuB/DaCa) blood, a class II and class I-like difference, induced indefinite R23 heart allograft survival. Although produced in high titer, anti-class II antibody was not able to induce rejection in RT1u rats. Specific anti-Aa alloantibody was able, after passive transfer, to destroy class I-disparate allografts in both RT1u nude and PVG nude recipients. However, under normal circumstances, acute rejection in the PVG strain occurred in the absence of anti-Aa antibodies, presumably by a cell-mediated mechanism. Anti-class I alloantibody, when produced, seemed to override the unresponsiveness induced by DST. The results indicated that DST was effective only when rejection was induced by a cell-mediated response. The two contrasting response patterns in animals may reflect the experience of transplant patients who either benefit from DST or become sensitized instead.