DA Liver Research Articles

Sex and line differences in biochemical indices and fat soluble vitamins sufficiency in rats on in vivo model of metabolic syndrome

Consumption of diets that are inadequate in energy value to the actual energy expenditure can lead to the development of metabolic syndrome (MS), which has consequences such as type 2 diabetes mellitus, non-alcoholic steatohepatosis, atherosclerosis, gout, allergic diseases. Experimental models of MS are needed to develop new approaches to its dietary and drug correction. The aim of the work was a comparative analysis of functional, biochemical and vitamin markers characterizing the effect of a diet with a high content of fructose (F) on males and females of various rat lines and the selection on this basis of an optimal in vivo MS model. Male and female rats of the outbred Wistar line (W) and the inbred Dark Agouti line (DA) were used in the work number of 16 individuals of each sex and line. The animals of the 1st (control) groups of each sex and line received a balanced semi-synthetic diet according to AIN93, and the animals of the 2nd (experimental) groups - the same diet and 30% solution of F instead of water in the regime of free access. Within 121 days, energy value of diets consumed, the increase in body weight and blood pressure were determined; relative mass of internal organs, biochemical parameters of blood plasma, content of fat-soluble vitamins A and E in blood plasma and liver were determined at withdrawal of animals from experiment. It was shown that, in spite of the increased energy value of the diet in the experimental groups throughout experiment, DA males and females practically did not respond to this by an increase in body weight gain, in contrast to W rats (in particular, females). Consumption of diets with F led to an increase in glucose level irrespective of gender and line, whereas triglyceride level (TG) significantly increased only in the case of W female. Addition of F caused in DA rats of both sexes an increase in the mass of the kidneys, as well as more pronounced, in comparison with W rats manifestation of markers of toxic effects on the liver (increases alanine aminotransferase and γ-glutamyltransferase activity, elevated urea and bilirubin level in blood plasma). In rats of both lines intake of F suppressed the accumulation of retinol palmitate in the liver in terms of its specific content. The total content of α-tocopherol in liver was significantly higher in W compared with DA. At the same time, α-tocopherol levels in blood plasma correlated with TG, and the α-tocopherol/TG ratio significantly decreased in female W receiving F, which were characterized by hyperlipidemia. Thus, the effect of F on W males and, in particular, females, basically corresponded to the classical picture of MS with body weight increasing, elevated blood pressure, glycemia and TG increase, whereas the toxic effect of F prevailed in DA liver and, possibly, kidneys without development of marked dyslipidemia and obesity.

Hepatic miR-301a as a Liver Transplant Rejection Biomarker? And Its Role for Interleukin-6 Production in Hepatocytes.

Acute rejection (AR) of liver transplantation remains a formidable challenge for diagnostic medicine and biomarker discovery. We characterized AR-related microRNAs (miRNAs) and the underlying AR mechanisms in liver transplantation. Using a rat model of orthotopic liver transplantation (OLT) as well as microarrays, we compared the miRNA expression profiles between naive and AR livers on day 7 after OLT with short- (<14 days, donor Dark Agouti [DA] liver into Lewis [LEW] recipient) and long-term (>60 days, donor DA liver into Piebald Virol Glaxo [PVG] recipient) survival fates. The microarray analysis revealed that the levels of miR-301a in the lethal AR livers were significantly higher than in naive and tolerogenic AR livers. The reduced expression of miR-301a in inflamed livers suggested a difference between AR and inflammation in terms of miR-301a-mediated molecular events. Overexpression of hepatic miR-301a induced IL-6 production in rat primary hepatocytes. Hepatocytes overexpressing miR-301a were capable of transferring miR-301a to cocultured splenocytes through exosomes. These splenocytes then showed overexpression of miR-301a and downregulation of protein inhibitor of activated STAT3 (PIAS3) expression, resulting in the induction of T helper 17 cell differentiation. In conclusion, this report raises the possibility that hepatic miR-301a might potentially prove value as a biomarker of liver transplant rejection. We call for future research on this molecular target and the attendant pathways as liver transplant rejection and its early diagnosis continue to be veritable healthcare challenges.

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.

Lymphangiogenesis Associated With Acute Cellular Rejection in Rat Liver Transplantation

Lymphangiogenesis may be important for the cellular immune response in liver transplantation. In the present study, we examined lymphangiogenesis in liver allografts displaying acute cellular rejection (ACR), or long-term acceptance, or severe ACR plus antibody-mediated rejection (AMR). ACR and subsequent long-term graft acceptance developed in liver transplantations from DA to PVG rats without immunosuppression (mean survival time more than 90 days). Severe ACR and AMR developed in liver transplantations from DA to Lewis rats without immunosuppression (mean survival = 11 days). Normal DA donor livers before transplantation showed a small number of lymphatic vessels around portal veins. DA liver grafts in PVG showed ACR with lymphangiogenesis in portal areas and portal-portal bridging areas with cellular infiltration. Newly formed lymphatic vessels in ACR were characterized by proliferating endothelial cells with expression of the homeobox transcription factor PROX-1 and surrounded by discontinuous basement membranes. Thereafter, the infiltrates spontaneously disappeared, and the grafts survived more than 90 days. During the resolution of the cellular infiltration, expanded lymphatic vessels were packed with many lymphocytes. Thereafter, the number of lymphatic vessels decreased. In contrast, severe ACR and AMR in DA-to-Lewis transplantations showed lymphatic vessels disappeared with edema in the portal areas at day 11. In conclusion, lymphangiogenesis occurred during ACR. It may be involved in the resolution of ACR and reduction of inflammation. In severe ACR and AMR, lymphatic vessels were destroyed, which may be involved in persistent severe inflammation.

Reduced Expression of Fibroblast Growth Factor Receptor 2IIIb in Hepatocellular Carcinoma Induces a More Aggressive Growth

Fibroblast growth factor receptor 2 isoform b (FGFR2-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2-IIIb in hepatocellular carcinoma (HCC). FGFR2-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR2-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR2-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-2IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR2 expression in a subset of HCC cells. FGFR2-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR2-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR2-IIIb expression in HCC cells were not affected by keratinocyte growth factor or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR2-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.

Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts

BackgroundDonor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx).Methodology/Principal FindingsTolerance to Dark Agouti (DA; RT1a) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1l) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4+ T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4+Foxp3+ Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4+CD45RC− population whereas in which approximately 50% of these Tregs express CD25.Conclusions/SignificanceWe conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4+ T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4+CD45RC−Foxp3+Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection.

Induced Tolerance to Liver Allografts Involves Apoptosis and the Generation of Regulatory T Cells (141.40)

Abstract Post-transplant total lymphoid irradiation (TLI) is a non-myeloablative regimen that has been studied in rodent models for induction of tolerance to allografts. We show in a rat model of orthotopic liver transplantation that 88% of Lewis recipients of DA livers (n=16) survive more than 100 days (and accept donor skin grafts) after receiving post-transplant TLI alone whereas the control group (liver transplantation without host conditioning) rejected their liver allografts within 14 days. To establish the mechanism of this induced tolerance, lymphocytes were isolated from the livers of groups (n=3-5) of Lewis recipients of Lewis or DA liver grafts, on day 7 post-transplant, and analyzed for apoptosis. Apoptosis of T cells in allografts was increased by TLI treatment (Allo+TLI vs. Allo, CD4+ 41.5 % vs 13.4%, p=0.002, CD8+, 22.5% vs. 9.7% p=0.034). Syngeneic grafts from recipients treated with TLI had minimal apoptosis similar to the levels detected in normal livers. Further, intragraft CD4+CD25+FoxP3+ cells were increased in the TLI group in the first week post-transplant and remained elevated in the graft and in the spleen. Importantly, adoptive transfer of splenocytes from day &amp;gt;100 liver allograft recipients that received TLI prolonged the survival of donor, but not third-party, heart grafts in naïve recipients. Depletion of CD4+CD25+ cells from transferred splenocytes abrogated this prolongation. We conclude that post-transplant TLI significantly increases apoptosis of infiltrating T cells in the allograft during the first week post-transplant. We suggest that apoptosis of host lymphocytes helps to promote an environment whereby CD4+CD25+FoxP3+ T regulatory cells can develop and accumulate in the graft and periphery, and maintain graft non-responsiveness. Supported in part by NIH AI044095, AI037683

Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)

Abstract Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival. NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant. Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095

Inhibition of Semicarbazide-Sensitive Amine Oxidases Decreases Lymphocyte Infiltration in the Early Phases of Rat Liver Allograft Rejection

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation in vitro and in vivo. VAP-1 is also an ectoenzyme with semicarbazide-sensitive amine oxidase (SSAO) activity. In this study we investigated whether inhibition of SSAO influences the inflammatory infiltration in acute rat liver allograft rejection. BN recipients of DA liver allografts were treated with 50 mg/kg/d semicarbazide, an inhibitor of SSAO, or similar volumes of saline. 10 rats/group were followed for graft survival, and 10 rats/group were sacrificed on day 7 post-transplantation for histology and T-lymphocyte isolation. The area percentage of portal inflammatory infiltrates in the grafts was assessed from digital photomicrographs. The proportion of CD4-, CD8- and IL2-receptor positive lymphocytes in the graft was quantified with flow cytometry. On day 7, semicarbazide treatment significantly decreased the inflammatory infiltrate area in the grafts. CD4-, CD8- and IL2-receptor positive cells were equally affected. However, animal survival was not affected. Blockade of the enzymatic activity of VAP-1 has a significant effect on lymphocyte infiltration early in acute liver rejection. Later, activation of other adhesion pathways can by-pass the blockade caused by VAP-inhibition.

When flies and mice develop cancer

![][1] The Spanish National Cancer Institute (CNIO) Meeting on Development and Cancer took place between 4 and 6 February 2008 in Madrid, Spain, and was organized by K. Basler, G. Morata, E. Moreno and M. Torres. ![][2] Developmental biologists rightly argue that genes regulating developmental pathways are reiterated in cancer development. At the recent Spanish National Cancer Institute (CNIO) Meeting on Development and Cancer, novel concepts and processes that are considered to be common to both embryonic and cancer development were discussed. It is worth pointing out that tumour formation should be considered to be a form of aberrant organ development, in which many developmental pathways that control proliferation, differentiation and growth or morphogenesis are affected. This report is structured in three parts, describing the work presented at the meeting according to the stages of tumour development to which it pertains. First, the normal behaviour of cell populations that are the targets of transformation, in particular adult stem cells and the pathways that regulate their behaviour. Second, processes that occur at the early stages of cancer development before morphological malformations are detectable, in particular the process that is best known from studies in flies as ‘cell competition’. Third, developmental processes and pathways that are active in the growing tumour—which we consider to be an extrinsic organ—for example, the recruitment of blood vessels or the role of morphogens in controlling the size and shape of tumours, and the progression from hyperplasia to invasive tumours (Fig 1). Figure 1. Tumour development and genes involved in the different stages. Schematic representation of how stem cells (A) , by acquiring a mutation through cell competition ( B ) and by the selection of additional mutations, can give rise to the ‘tumour organ’ ( C ). The genes involved in these processes are listed on the right‐hand side, and most of them … [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif

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.

Tolerance to rat liver allograft after total lymphoid irradiation is mediated by CD4+CD25+ regulatory T cells

We have previously determined that post transplant total lymphoid Irradiation (TLI) induces tolerance in an experimental model of liver transplantation. The purpose of this study is to characterize the mechanism of TLI induced tolerance.TLI significantly prolonged survival in Lewis recipients of fully‐histoincompatible DA liver allografts with 88% of the recipients surviving long‐term (&gt;100 days). CD4+CD25+Foxp3+ T cells were markedly increased in the liver grafts, PBMC, and spleen by day 35 post‐transplant and remained high in the spleen &gt;100 days post‐transplant. Splenocytes from long‐term surviving recipients have a reduced alloresponse in MLR. Moreover, isolated splenic CD4+CD25+ T cells inhibited the MLR of naive Lewis splenocytes to DA splenocytes. Importantly, adoptive transfer of whole splenocytes into naive Lewis recipients of DA heart grafts significantly prolonged cardiac graft survival (MST whole splenocytes 33 days vs. control 18.5 days:p&lt;0.01), whereas depletion of CD4+CD25+ T cells from splenocytes completely abrogated the prolongation of cardiac allograft survival (MST 16 days; p&lt;0.01, vs. whole splenocytes).TLI induces tolerance to liver allografts via a mechanism involving CD4+CD25+Foxp3+ T cells that accumulate in the liver early after transplant and remain in the spleen &gt;100 days. Studies using TLI to induce tolerance in clinical liver transplantation are currently underway.

Rapamycin, But Not Cyclosporine or FK506, Alters Natural Killer Cell Function

Infiltration of natural killer (NK) cells into solid organ allografts is observed in clinical and experimental transplantation. Studies suggest a role for NK cells in acute and chronic rejection of solid organ allografts; however, the effects of immunosuppressive agents on NK cells are not clearly established. Rat NK cell lines were analyzed for proliferation and cytotoxicity in the presence of cyclosporine, FK506, or rapamycin. Lewis recipients of DA liver allografts received immunosuppressive agents after transplantation. NK cells demonstrated robust function both in the absence and presence of cyclosporine and FK506. In contrast, rapamycin significantly inhibited proliferation and cytotoxicity of NK cells. NK cell numbers remained stable in graft recipients treated with cyclosporine and FK506, whereas there was a significant decrease in NK cells in rapamycin-treated recipients. These data indicate that immunosuppressive drugs have differential effects on NK cell function that may impact the immune response of transplant recipients.

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.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Role of Antinuclear Antibodies in Experimental and Clinical Liver Transplantation

Objective We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation. Materials and Methods We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT. Results In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers. Conclusions Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

Gene array analysis of a rat model of liver transplant tolerance identifies increased complement C3 and the STAT-1/IRF-1 pathway during tolerance induction

This study aimed to define the molecular mechanism during induction of spontaneous liver transplant tolerance using microarrays and to focus on molecular pathways associated with tolerance by meta-analysis with published studies. The differences in the early immune response between PVG to DA liver transplant recipients that are spontaneously tolerant (TOL) and PVG to Lewis liver transplants that reject (REJ) were examined. Spleens from TOL and REJ on days 1 and 3 were compared by 2 color microarray. Forty-six of 199 genes differentially expressed between TOL and REJ had an immunological function. More immune genes were increased in TOL vs. REJ on day 1, including STAT-1, IRF-1 and complement C3. Differential expression of selected genes was confirmed by quantitative RT-PCR. The results were compared to two published high-throughput studies of rat liver transplant tolerance and showed that C3 was increased in all three models, while STAT-1 and IRF-1 were increased in two models. The early increases in immune genes in TOL confirmed previous reports of an active early immune response in TOL. In conclusion, the increase in STAT-1, IRF-1 and complement component C3 in several models of liver transplant tolerance suggests that the STAT-1/IRF-1 apoptotic pathway and C3 may be involved in the tolerogenic mechanism.

The persistence of regulatory cells developing after rat spontaneous liver acceptance

We have previously reported that the spontaneous acceptance of Lewis (LEW, RT1(l)) to Dark Agouti (DA, RT1(a)) rat orthotopic liver transplant (OLT) is eliminated by donor gamma-irradiation. The acceptance of the irradiated LEW liver is also reestablished in a naïve rat after the adoptive transfer of T regulatory (T-reg) cells from a LEW to a DA liver-tolerant long-term (>60 days) survivor (LTS) into a naïve DA rat. However, little is known about the growth conditions required to maintain T-reg function. In this study, we examined the need for continued donor-specific alloantigen stimulation for the maintenance and function of T-reg cells. Splenocytes (SCs; 1.5 x 10(8) cells) from a LEW liver allograft-tolerant LTS DA recipient were adoptively transferred fresh or after in vitro stimulation into another naive DA rat on day 1, 4, or 7 before an irradiated (1000R) LEW liver transplant. For in vitro alloantigen stimulation, SCs from LEW to DA LTS were co-cultured with mitomycin-C (MMC)-treated naïve LEW (donor alloantigen-specific) or Brown Norway (BN) (RT1(n); third party) SCs for 72 hours. Graft rejection, as defined by death of the recipient, was confirmed histologically. All LEW liver grafts were accepted spontaneously by DA recipients for more than 60 days (n=32), while all irradiated LEW livers were acutely rejected (n=9; mean survival time [MST]=12.8 +/- 4.0 days). When LTS DA SCs were adoptively transferred into a naive DA rat 1 day before OLT, all irradiated LEW grafts were accepted greater than 60 days (n=9). However, when fresh LTS DA SCs were transferred to a new naïve DA rat on 4 or 7 days before OLT, all irradiated LEW liver grafts were acutely rejected (MST=10.2 +/- 0.5 days [n=4] and MST=13.5 +/- 5.0 days [n=4], respectively). When LTS DA SCs were stimulated in vitro before adoptive transfer, irradiated LEW liver grafts after 4 days (n=5) were then accepted. In vitro culture of LTS DA SCs with MMC-treated BN SCs (third-party) for 72 hours before adoptive transfer resulted in 3 of 5 irradiated LEW livers at day 4 being accepted (n=5). The maintenance of T-reg function requires continuous LEW donor-specific alloantigen stimulation.

Posttransplant Interleukin-4 Treatment Converts Rat Liver Allograft Tolerance to Rejection

Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection. DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition. IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition. IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.

POST-TRANSPLANT IL-4 TREATMENT CONVERTS RAT LIVER ALLOGRAFT TOLERANCE TO REJECTION

O391* Aims: Our previous studies showed that liver transplant rejection in the fully mismatched PVG to Lewis combination was associated with more IL-4 mRNA in the graft on days 5-7 than spontaneous tolerance of transplanted liver in the PVG to DA combination. There was also deposition of IgG1 in the rejected grafts which suggested that IL-4 might be inducing rejection of the liver mediated by its promotion of the IgG1 antibody response. The aim of this study was to investigate the effect of IL-4 treatment on liver transplant tolerance. Methods: DA (RT1a) rats were recipients of orthotopic PVG (RT1c liver transplants and DA liver transplants were syngeneic controls. Untransfected CHO supernatant or supernatant from IL-4-transfected CHO cells (0.5 ml, 30,000 U) was injected i.p. on days 3-7. Samples were taken at day 7 or post-mortem from IL-4-treated animals for analysis. Cellular infiltrate and antibody deposition was analysed by immunoperoxidase staining of frozen tissue sections. Results: IL-4 treatment (Group 4) significantly reduced survival of liver allografts compared with untreated animals (Group 2)(p=0.004). Pathology analysis of group 4 animals showed that death was due to liver transplant rejection with a heavy infiltrate of mononuclear cells, disruption of portal tract areas and infarction. T cell infiltrate in group 4 was significantly higher compared with group 2 (695±75 vs. 372±15, p=0.002). There was also a much more extensive monocyte/macrophage infiltrate and more MHC class II expression in Group 4 compared to Group 2. In contrast, there was no evidence of IgG1 deposition within the graft in IL-4-treated animalsFigureConclusions. IL-4, a Th2 cytokine that has previously been thought to inhibit delayed-type hypersensitivity (DTH) responses such as rejection, was found to promote rejection of liver allografts in association with an increased DTH response in the graft. IL-4 increased the number of T cells within the graft but did not induce a graft-specific antibody response, which raises the possibility that it might be inducing rejection by promoting T cell survival. This could be mediated by its known function of signalling through the common gamma chain of the cytokine receptor to prevent T cell apoptosis, a function that it shares with the cytokines IL-2, IL-7, IL-9, IL-15 and IL-21.