Local autoantigen expression as essential gatekeeper of memory T-cell recruitment to islet grafts in diabetic hosts.

It is generally believed that inflammatory cues can attract noncognate, "bystander" T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8⁺ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts "invisible" to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206₋214-reactive CD8⁺ T cells into IGRP206₋214-competent and IGRP206₋214-deficient islet grafts in diabetic wild-type or IGRP206₋214(-/-) nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP206₋214-specific T-cells, respectively). All four host-donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP206₋214-specific T cells into IGRP206₋214(+/+) but not IGRP206₋214(-/-) grafts. In IGRP206₋214(-/-) hosts, there was no recruitment of IGRP206₋214-specific T cells, regardless of donor type. Graft-derived IGRP206₋214 activated naive IGRP206₋214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after ß-cell replacement therapy.

Immunogenicity of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

Kidney-derived mesenchymal stromal cells modulate dendritic cell function to suppress alloimmune responses and delay allograft rejection.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cells with immunoregulatory capacity that are present in most adult organs. We previously demonstrated that co-culture of C57BL/6 kidney-derived MSCs (KSCs) in syngeneic bone marrow-derived dendritic cell (DC) culture induced a DC phenotype (KSC-DC) with reduced major histocompatibility complex (MHC) class II/increased CD80 expression and ability to suppress T-cell responses. METHODS: To study their effects on allogeneic DCs, C57BL/6 KSCs were added to incipient BALB/c DC culture, with surface expression of MHC class II/CD80 measured by fluorescence-activated cell sorting. The ability to stimulate T-cell responses was then assessed in an allogeneic mixed leukocyte response. Next, we isolated either BALB/c (donor) or C57BL/6 (recipient) KSC-DCs from co-culture and measured the tempo of rejection after cotransplantation with islet grafts in a mouse model of islet transplantation. Finally, we measured the effects of KSC-DC stimulation on B-cell proliferation and IgM/IgG production in allogeneic cultures. RESULTS: C57BL/6 KSCs induced a BALB/c DC phenotype with significantly decreased MHC class II, increased CD80 expression, and decreased T-cell stimulatory capacity in the mixed leukocyte response (P<0.01 vs. control). Cotransplantation of donor (BALB/c) but not recipient (C57BL/6) KSC-DCs resulted in significant delay of rejection after islet transplantation (P<0.01 vs. control). Finally, stimulation by KSC-DCs resulted in significantly reduced B-cell proliferation and antibody production in allogeneic culture (P<0.01 vs. control). CONCLUSIONS: Our results highlight an important mechanism of MSC-based immunotherapy and its potential for use in clinical transplantation as prevention of rejection and possibly sensitization.

Perioperative infliximab application ameliorates acute rejection associated inflammation after intestinal transplantation.

As we have shown in the past, acute rejection-related TNF-α upregulation in resident macrophages in the tunica muscularis after small bowel transplantation (SBTx) results in local amplification of inflammation, decisively contributing to graft dysmotility. Therefore, the aim of this study is to investigate the effectiveness of the chimeric-monoclonal-anti-TNF-α antibody infliximab as perioperative single shot treatment addressing inflammatory processes during acute rejection early after transplantation. Orthotopic, isogenic and allogenic SBTx was performed in rats (BN-Lewis/BN-BN) with infliximab treatment. Vehicle and IV-immunoglobulin-treated animals served as controls. Animals were sacrificed after 24 and 168 h. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry, mediator mRNA expression by Real-Time-RT-PCR, apoptosis by TUNEL and smooth muscle contractility in a standard organ bath. Both, infliximab and Sandoglobulin® revealed antiinflammatory effects. Infliximab resulted in significantly less leukocyte infiltration compared to allogenic controls and IV-immunoglobulin, which was accompanied by lower gene expression of MCP-1 (24 h), IFN-γ (168 h) and infiltration of CD8-positive cells. Smooth muscle contractility improved significantly after 24 h compared to all controls in infliximab treated animals accompanied by lower iNOS expression. Perioperative treatment with infliximab is a possible pharmaceutical approach to overcome graft dysmotility early after SBTx.

Influence of immunosuppression on alloresponse, inflammation and contractile function of graft after intestinal transplantation.

In small bowel transplantation (SBTx), graft manipulation, ischemia/reperfusion injury and acute rejection initiate a severe cellular and molecular inflammatory response in the muscularis propria leading to impaired motility of the graft. This study examined and compared the effect of tacrolimus and sirolimus on inflammation in graft muscularis. After allogeneic orthotopic SBTx, recipient rats were treated with tacrolimus or sirolimus. Tacrolimus and sirolimus attenuated neutrophilic, macrophage and T-cell infiltration in graft muscularis, which was associated with reduced apoptotic cell death. Nonspecific inflammatory mediators (IL-6, MCP-1) and T-cell activation markers (IL-2, IFN-gamma) were highly upregulated in allogeneic control graft muscularis 24 h and 7 days after SBTx, and tacrolimus and sirolimus significantly suppressed upregulation of these mediators. In vitro organ bath method demonstrated a severe decrease in graft smooth muscle contractility in allogeneic control (22% of normal control). Correlating with attenuated upregulation of iNOS, tacrolimus and sirolimus treatment significantly improved contractility (64% and 72%, respectively). Although sirolimus reduced cellular and molecular inflammatory response more efficiently after 24 h, contrary tacrolimus prevented acute rejection more efficiently. In conclusion, tacrolimus and sirolimus attenuate cellular and molecular inflammatory response in graft muscularis and subsequent dysmotility of the graft after allogeneic SBTx.

Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice.

Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte-associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.

Autoimmunity to vimentin potentiates graft vasculopathy in murine cardiac allografts.

BACKGROUND: There is increasing evidence for a role for autoimmunity in transplant rejection. It has previously been shown that autoantibodies to vimentin (Vim) accelerate acute rejection of murine cardiac allografts. We have investigated whether autoimmunity to Vim contributes to development of cardiac allograft vasculopathy (CAV). METHODS: Two well-established minor mismatch murine models of CAV were used, transplantation of 129/sv hearts into T-cell-depleted C57Bl/6 (B6) recipients and transplantation of FVB hearts into nonimmunosuppressed DBA/1 recipients. Recipients were immunized with recombinant mouse Vim in complete Freunds adjuvant, and controls received hen egg lysozyme 2 weeks before transplantation. T cell and antibody responses to Vim were assessed by ELISPOT and ELISA, respectively. CAV within transplanted hearts was assessed by quantitative morphometry of occluded vessels, presence of smooth muscle cells, deposition of C3d, and confocal microscopy. RESULTS: Allografts were harvested from B6 recipients at days 30 and 45 and from DBA/1 recipients at days 18 and 35. At all days, there was significantly more intimal occlusion of arteries of Vim -immunized mice than controls. There was significantly more smooth muscle cell alpha actin in vessels from Vim-immunized mice, and more C3d deposited in hearts from Vim-immunized mice. Confocal microscopy demonstrated colocalization of Vim with C3d on endothelial cells, leukocytes, and platelets in allogeneic but not syngeneic hearts. Serum from Vim-immunized mice, but not controls, caused platelet/leukocyte conjugation when added to mouse leukocytes. CONCLUSION: The autoimmune response to Vim accelerates CAV progression in these minor-mismatched models.

Speckle-tracking 2-dimensional strain echocardiography: a new noninvasive imaging tool to evaluate acute rejection in cardiac transplantation.

BACKGROUND: There remains no reliable non-invasive method to detect cardiac transplant rejection. Recently, speckle-tracking 2-dimensional strain echocardiography (2DSE) was shown to be sensitive in the early detection of myocardial dysfunction in various models of cardiomyopathy. We aim to determine if 2DSE-derived functional indices can detect cardiac transplant rejection. METHODS: Heterotopic rat cardiac transplantation was performed in histocompatible isografts or histoincompatible allografts. Histologic rejection scores were determined. Short-axis, mid-left ventricular (LV) echocardiography was performed on Day 6 after transplantation. Conventional measures of function were measured, (including LV fractional shortening and ejection fraction) as well as 2DSE parameters. RESULTS: Despite class IIIB rejection in allografts and no rejection in isografts, there was no difference between isografts vs allografts in fractional shortening (15% +/- 3% vs 12% +/- 3%) or ejection fraction (36% +/- 5% vs 26% +/- 6%; both not significant). In contrast, 2DSE revealed decreases between isografts and allografts in global radial strain (12.6% +/- 5.6% vs 1.1% +/- 0.2%, p < 0.05), peak radial systolic strain rate (3.10 +/- 0.74/s vs 0.54 +/- 0.13/s, p < 0.001), and peak circumferential systolic strain rate (-1.99 +/- 0.55 vs -0.43 +/- 0.11/s; p < 0.01). CONCLUSIONS: Systolic strain imaging using 2DSE differentiates myocardial function between experimental cardiac transplant rejection in allografts and non-rejection in isografts. Therefore, 2DSE may be useful in early non-invasive detection of transplant rejection.

Deficiency of Atf3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngeneic mouse transplantation model.

AIMS/HYPOTHESIS: Islet transplantation is a potential therapeutic option for type 1 diabetes. However, the need for multiple donors per patient and heavy immunosuppression of the recipients limit its use. The goal of this study was to test whether the gene encoding activating transcription factor 3 (ATF3), a stress-inducible pro-apoptotic gene, plays a role in graft rejection in islet transplantation. METHODS: We compared wild-type (WT) and Atf3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation and hypoxia. We also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. RESULTS: ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets compared with KO islets. ATF3 upregulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (1) apoptosis (Noxa [also known as Pmaip1] and Bnip3), and (2) immunomodulation (Tnfalpha [also known as Tnf], Il-1beta [also known as Il1b], Il-6 [also known as Il6] and Ccl2 [also known as Mcp-1]). In vivo, Atf3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in the recipients and the reduction of the following variables in the KO grafts: caspase 3 activation, macrophage infiltration and expression of the above apoptotic and immunomodulatory genes. CONCLUSIONS/INTERPRETATION: ATF3 plays a role in islet graft rejection by contributing to islet cell death and inflammatory responses at the graft sites. Silencing the ATF3 gene may provide therapeutic benefits in islet transplantation.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells.

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Detection of citrate synthase autoantibodies in rats with chronic allograft nephropathy.

UNLABELLED: Citrate synthase (CS) is the one of the key enzymes in the citric acid cycle and an important mitochondrial autoantigen. The autoimmune responses against CS have not been studied in chronic allograft nephropathy (CAN). This study investigated the role of specific CS autoantibodies in rats bearing renal allografts affected with CAN. METHODS: Fisher344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. Lewis-to-Lewis and Fisher344-to-Fisher344 kidney transplantations were also performed as autologous control groups (each n = 9). All the allograft recipients given cyclosporine (10 mg/kg(-1)d(-1) x 10 d) were divided into four groups (each n = 9): (1) vehicle: normal saline orally; (2) cyclosporine: 6 mg/kg(-1)d(-1); (3)FK506: 0.15 mg/kg(-1)d(-1); (4) mycophenolate mofetil (MMF): 20 mg/ kg(-1)d(-1). At 4, 8, and 12 weeks posttransplantation, the animals were sacrificed to harvest sera and renal allografts. The serum creatinine (SCr) was measured and pathological changes assessed according to Banff 97 criteria. IgM and IgG isotypes of CS antibodies were detected in all recipient sera by enzyme linked immunosorbent assays. RESULTS: Both IgM and IgG isotype CS autoantibodies were observed in the sera of all the recipients before and after transplantation, but the levels of IgM CS autoantibody were obviously higher than IgG isotype in all the blood samples. It was stable not only in autologous but also in allograft groups. In both autologous groups, the SCr and IgM and IgG isotype CS autoantibodies showed no obvious change before and after transplantation, and no typical CAN occurred. The values of IgG isotype of CS autoantibody (DeltaOD) at 4, 8 and 12 weeks were stable. At 4 weeks, the values of SCr, Banff score, and IgG isotype CS autoantibody (DeltaOD) were not significantly different (P > .05) among the allograft groups. At 8 and 12 weeks, with progression of CAN in vehicle, cyclosporine and FK506 groups' values of SCr, Banff score, and IgG (DeltaOD) also increased dramatically (P = .005) in all three groups when compared with the baseline and 4 week values, but the differences among the three groups were not significant (P > .05). At 8 and 12 weeks, the MMF group suffered mild-to-moderate CAN, but the values of SCr and Banff score were significantly lower than those in the other three groups. MMF significantly inhibited the formation of IgG (DeltaOD) when compared with the other three groups (P = .02). CONCLUSION: This study suggested that the IgG isotype of CS autoantibody contributes to CAN after kidney transplantation. The IgM isotype is physiological. MMF significantly inhibited the formation of IgG isotype CS autoantibody, which may be related to its effects to alleviate CAN.

Immunological behavior of enhanced green fluorescent protein (EGFP) as a minor histocompatibility antigen with a special reference to skin isograft and specific regulation of local graft-versus-host reaction (GvHR).

Although enhanced green fluorescent protein (EGFP) is widely used as a molecular tag in cell biology, it has become evident that immunogenicity of transgenic or transduced EGFP is important when it applies to transplantation model. Indeed, it appears that applications of EGFP-expressing cells, tissues and organ transplantation are limited in vivo due to the ultimate rejection of the graft. Nevertheless, the immunological behavior of transduced EGFP, in particular, as a minor histocompatibility antigen is not fully understood. Thus employing two strains of EGFP transgenic (Tg) rats generated by the same vector construct, e.g., EGFP-F344 Tg (RT11) and EGFP-DA Tg (RT1a), and its F(1) hybrid with a non-transgenic rat, behavior of EGFP-transgenic antigen(s) was examined by in vivo assays, such as EGFP-transgenic test skin grafts or regulation of EGFP-transgenic lymphocytes. In the latter system, EGFP-specific, T-cell-mediated immune regulation of local graft-versus-host reaction (GvHR) was further investigated with a special reference of in vivo cytotoxic assay, i.e., elimination of colored lymphocytes with either EGFP-incompatible or CFSE-labeled sex-mismatched lymphocytes. We provide evidence that differential immunological behavior of EGFP-transgenic minor histocompatibility antigen was observed in vivo. Thus, immune responses to EGFP-minor histocompatibility antigen(s) were not always accompanied with the rejection of test skin isograft. It only becomes apparent for EGFP-specific elimination and suppression of both systemic and local GvHR induced by EGFP-transgenic T lymphocytes after EGFP-specific sensitization. However, this was not the case where test skin isografting was applied even under extensive sensitization protocols. These findings demonstrate that minor histocompatibility antigen specific immune elimination of EGFP-transgenic T lymphocytes or regulation of local GvHR provides more sensitive and better immune assay systems in vivo than classical test skin isograft systems.

Primary graft dysfunction in lung transplantation: the role of CD26/dipeptidylpeptidase IV and vasoactive intestinal peptide.

BACKGROUND: Enzymatic activity inhibition of CD26/dipeptidylpeptidase IV (CD26/DPP IV) attenuated short-term post-Tx (transplantation) ischemia-reperfusion injury after 18-hr-ischemia. Here, we investigated the effect of intragraft CD26/DPP IV catalytic inhibition on primary graft dysfunction during 7 day post-Tx, following extended ischemia. METHODS: A syngeneic rat (LEW [Lewis abstract]) orthotopic lung Tx model was used, grafts exposed to 18 hr cold ischemia before Tx. Controls were flushed and preserved in Perfadex, and harvested after 1 day (CON1) or 7 day (CON7) post-Tx. Investigational groups IN1, IN3, and IN7 grafts were perfused with and stored in Perfadex + inhibitor (AB192) and harvested at 1, 3, and 7 days post-Tx, respectively. Blood gas analysis, peak airway pressure (PAwP), wet/dry weight ratio, myeloperoxidase thiobarbituric acid reactive substances (TBARS), and staining for vasoactive intestinal peptide (VIP) were analyzed. RESULTS: IN1 versus CON1 showed preserved histology, increased pO2 (P<0.01), lowered PAwP (P<0.01), less edema (P<0.05) and decreased TBARS (P<0.05). Survival was better for IN7 versus CON7 (P<0.01). The course of AB192-perfused grafts from 1 to 7 days displayed improved values for pO2 (P<0.01), PAwP (P<0.01), edema (P<0.05), TBARS (P<0.05), and myeloperoxidase (P<0.05). Compared with controls, VIP was preserved during 18 hr ischemia in alveolar macrophages (P=0.0001) and respiratory epithelial cells (P=0.001). CONCLUSIONS: Perfusion with an inhibitor of CD26/DPP IV enzymatic activity significantly reduced the incidence and severity of pulmonary primary graft dysfunction and enabled recovery after extended ischemia. This is the first report that CD26/DPPIV inhibitor treatment increases local pulmonary VIP levels, which correlate with preserved ventilatory function and pulmonary structural integrity.

Effects of systemic immunosuppression on islet engraftment and function into a subcutaneous biocompatible device.

The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P < .05 with IS; P < .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P < .01, Tac; P < 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.

Investigation of antibody-mediated rejection in composite tissue allotransplantation in a rat limb transplant model.

BACKGROUND: Despite the widely accepted implication of antidonor antibodies and complement in solid organ transplantation, their role in reconstructive allotransplantation is not clear. The aim of this study was to analyze the humoral immune response using a rat orthotopic limb transplantation model. METHODS: We used the Brown Norway to Lewis rat orthotopic hind-limb transplant model: Group 1, isografts; group 2, allografts with daily continuous cyclosporine treatment to prevent acute rejection; and group 3, allografts undergoing multiple episodes of acute rejection. Samples were taken at 30, 60, and 90 days. Serum was analyzed by FACS for antidonor antibodies. Tissue deposition of antibodies and complement was investigated by immunofluorescence. RESULTS: By day 90, animals in group 3 had undergone 19 (+/-3.2) acute rejection episodes. There was no difference in the occurrence of serum antidonor antibodies between the three groups at any time point. However, at 90 days, anti-third-party antibodies were significantly greater among group 3. There was no difference in antibody or complement deposition in muscles between the 3 groups. CONCLUSION: Despite the increased antibody against a third party after multiple rejection episodes in this animal model, there was no clear evidence of an antibody-mediated alloresponse in limb transplantation.

Donor preconditioning with a calcineurin inhibitor improves outcome in rat syngeneic kidney transplantation.

Ischemia-reperfusion injury (IRI) in the early posttransplant period affects immediate graft function and late allograft dysfunction. This study determines the influence of pharmacologic preconditioning with a calcineurin inhibitor on IRI in a syngeneic F344 rat kidney transplant model. Donor rats were pretreated with one dose of cyclosporine (10 mg/kg) or tacrolimus (1 mg/kg) administered at 24 hr or 7 days before being subjected to 2 hr of cold ischemia and then transplanted. Pharmacologic preconditioning significantly improved renal function, as assessed by serum creatinine and inulin clearance, and histologic score versus vehicle-treated rats. There were no differences between cyclosporine and tacrolimus in the measured outcomes. This renoprotective effect, although not complete, was seen with only one dose of calcineurin inhibitor, and the effect was sustained for at least 7 days before IRI. This approach may represent a viable pharmacologic intervention to decrease IRI at the time of organ transplantation.

Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Relationship between TH1/TH2 cytokines and immune tolerance in liver transplantation in rats.

OBJECTIVE: To explore the relationship between Th1/Th2 cytokine expressions and immune tolerance in rat liver transplantation. METHODS: Recipients were divided into three groups (each group = 12 rats). The allograft group underwent orthotopic liver transplantation from male Wistar-to-Sprague-Dawley (SD) rats. The isograft group of SD-to-SD liver transplantation was complemented with a control group of normal male SD rats that had sham operations. We evaluated the 2-week survival rates histological changes, as well as serum and mRNA expression levels of Th1/Th2 cytokines: interferon-gamma (IFN-gamma) and interleukin-10 (IL-10). RESULTS: The 1-week survival in the isograft and allograft groups were 100% and 41.67%, respectively. The 2-week survival rates were 75% and 8.33% (P < .05). Light microscopic inspection revealed severe acute rejection in the allograft but not the isograft group in the first week postoperation. This observation was verified by the cellular morphology under transmission electron microscopy. Serum IFN-gamma (Th1 cytokine) levels (pg/mL) determined by enzyme-linked immunosorbent assay in the allograft, isograft, and control groups were 386.67 +/- 14.36, 159.83 +/- 16.53, and 87.83 +/- 8.67, respectively; for IL-10 (Th2 cytokine), they were 126.33 +/- 13.10, 288.33 +/- 17.10, and 70.50 +/- 7.23, respectively (P < .05, allograft vs isograft). The semiquantitative reverse-transcriptase polymerase chain reaction assay showed that expression of IFN-gamma and IL-10 mRNA was similar to that of serum levels. IFN-gamma mRNA was more highly expressed in the allograft group and IL-10 mRNA, in the isograft group (P < .05). CONCLUSIONS: The dynamic equilibrium of Th1/Th2 cytokines is critically involved in immune tolerance. The deviation of Th1 to Th2 may be one mechanism of immune tolerance.