Rat Cardiac Allograft Survival Research Articles

How Can Immuno suppression After Organ Transplantation be Made More Effective and Safer? – A Review on Liposomal Formulations with Consideration of Archaeal Tetraetherlipid

Immune-suppressive agents such as methylprednisolone and cyclosporine exert tremendous side effects, because of high dosage and long-term application required for immune suppression after organ transplantation. Major side effects of methylprednisolone include bleeding of the gastro-intestinal tract, hypertension, and osteoporosis, whereas cyclosporine is nephrotoxic. Liposomes are phospholipid particles that allow delivery of drugs preferentially to the reticuloendothelial system. They can be prepared from phospholipids such as lecithin from soybean or egg yolk, other specific or modified lipids or from membrane-spanning tetraether lipid (TEL), which can be extracted and purified from archaeal cell membranes. One advantage in the use of liposomal application is reduced toxicity of many drugs. We report on various liposomal preparations of cyclosporine, methylprednisolone (L-MPL) and its palmitate derivative (L-MPLP). It has been documented that liposomal cyclosporine A (L-CsA), 1.75 mg/kg/day for seven days has potential for use as an immune-suppressive agent in rats with increased efficacy and decreased nephrotoxicity compared to commercially available forms of intravenous CsA. Liposomal methylprednisolone (L-MPL) 2 mg/kg, intravenously (IV), twice a week shows significantly prolonged cardiac allograft survival in rats and tissue-selective sequestration of the drug in comparison with the same dosage regimen of methylprednisolone in solution, administered daily. We report on organ distribution of L-MPLP in rats after intraperitoneal (IP) administration. Conclusion: Liposomal preparations of immunosuppressants have significantly higher immune-suppressive potential and lower toxicity than non-liposomal preparations. Bipolar TEL can be extracted, fractionated and purified from archaea to form stable liposomes which are extremely resistant, even to gastric fluid. Hence, TEL liposomes allow (besides IV and IP) for oral administration of immuno suppressants after organ transplantation with pharmacological and toxicological advantages as common liposomal phospholipid bilayer preparations.

MicroRNA let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

Abstract Objective In this study, we investigated whether microRNA (MiR) let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently prolongs rat cardiac allograft survival. Methods QRT-PCR, ELISA, dual luciferase assay were performed to verify whether IL-10 was the target of let 7i. Tregs were assessed by flow cytometry and immunohistochemical study, and JAK1, STAT3 and pSTAT3 expressions were detected by western bolt. Lewis recipients of DA heart were transfused with PBS, LPS-mDCs, or let 7i inhibitor-mDCs. Allograft survival times were recorded, and histologic study were performed. Results Expression of IL-10 mRNA level and production of IL-10 were increased in let 7i inhibitor-mDCs as compared to LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let 7i mimic, but increased by let 7i inhibitor. MiR let 7i inhibitor suppressed DC maturation by depressing CD80 and CD86, however， pretreatment of IL-10 SiRNA attenuated the suppression. Expressions of JAK1, STAT3, and pSTAT3 in mDCs were suppressed by let 7i mimic and pretreatment of IL-10 SiRNA, however up regulated by let 7i inhibitor. Lewis recipients transfused with let 7i inhibitor-mDCs significantly prolonged DA cardiac allograft survival. The allografts transfused with let 7i inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let 7i increased Tregs and modulated cytokine profiles both in vivo and in vitro. Conclusion MiR let 7i regulated DCs maturation and function targeting the IL-10 through JAK1-STAT3 pathway. Moreover, transfusion of let 7i inhibitor-mDCs induced prolonged cardiac allograft survival and generated Tregs.

MicroRNA let-7i regulates dendritic cells maturation targeting interleukin-10 via the Janus kinase 1–signal transducer and activator of transcription 3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

In this study, we investig1ated whether microRNA let-7i regulates dendric cell maturation targeting interleukin-10 (IL-10) via the Janus kinase 1-signal transducer and activator of transcription 3 (JAK1-STAT3) signal pathway subsequently prolongs rat cardiac allograft survival. Quantitative real-time reverse transcriptase polymerase chain reaction, enzyme linked immunosorbent assay, and dual-luciferase assay were performed to verify whether IL-10 was the target of let-7i, and regulatory T cells were assessed by flow cytometry and immunohistochemical study. Western blot was performed to detect JAK1, STAT3, and phosphorylated STAT3 expression. Lewis recipients of Dark Agouti hearts were transfused with phosphate-buffered saline, lipopolysaccharide (LPS)-mature dendritic cells (mDCs), or let-7i-inhibitor-mDCs. Allograft survival times were recorded, and histologic studies were performed. Expression of IL-10 messenger RNA level and production of IL-10 were increased in let-7i-inhibitor-mDCs compared with LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let-7i mimic but increased by let-7i-inhibitor. MicroRNA let-7i inhibitor suppressed DC maturation; however, pretreatment of IL-10 small interfering RNA attenuated the suppression. Expression of JAK1, STAT3, and phosphorylated STAT3 in mDCs were suppressed by let-7i mimic, and pre-treatment of IL-10 small interfering RNA, however, were upregulated by let-7i inhibitor. Lewis recipients transfused with let-7i-inhibitor-mDCs significantly prolonged Dark Agouti cardiac allograft survival. The allografts transfused with let-7i-inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let-7i increased CD4(+)CD25(+)forkhead box P3(+) regulatory T cells and modulated cytokine profiles in vivo and in vitro. MicroRNA let-7i regulated DC maturation and function targeting IL-10 through the JAK1-STAT3 pathway. Moreover, transfusion of LPS-induced mDCs transfected with let-7i inhibitor induced prolonged cardiac allograft survival and generated regulatory T cells.

IL-34 is a Treg-specific cytokine and mediates transplant tolerance

Cytokines and metabolic pathway-controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8+CD45RClo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ and CD4+ Tregs. IL-34 was involved in the suppressive function of both CD8+ and CD4+ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8+ and CD4+ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8+ and CD4+ FOXP3+ Tregs, with a superior suppressive potential of antidonor immune responses compared with non-IL-34-expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg-specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.

Injection of Cardiac Stem Cells Prolongs the Survival of Cardiac Allograft Rats

Cardiac c-kit+cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. The present study was undertaken to determine the isolation and culture of c-kit+cardiac stem cells (CSCs), and the roles of myocardial injection of CSCs on the survival of rat cardiac allograft. Recipient Sprague–Dawley rats were transplanted with hearts from Wistar rats. in the in vitro experiment, c-kit+cells were isolated from mouse heart fragment culture by magnetic cell sorting. CSCs expressed of cardiomyocyte specific protein cardiac troponin I, α smooth muscle actin and von Willebrand factor in conditioned culture. CsC injection increased graft survival of cardiac allograft rats. The effects of CSCs on increase in graft survival of cardiac allograft rats were blocked by stromal-derived factor-1 (SDF-1) knockdown. The expression of SDF-1 was increased after CSC injection into the cardiac of cardiac allograft rats. These results indicate that CSC injection into the cardiac prolongs graft survival of cardiac allograft rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.

Immunomodulation Induced By a Novel Immunosuppressant, NK026680 Plus Donor Specific Transfusion Permits a Long-Term Cardiac Allograft Survival in Mice.

Background: We have recently demonstrated that a novel triazolopylimidine derivative, NK026680 (NK), inhibits T cell proliferation and prolongs rat cardiac allograft survival. In this study, we examined the immunomodulatory effect of NK in combination with donor specific transfusion (DST) in mice. Methods: A fully MHC incompatible BALB/c-to-C57BL/6 mouse heart transplantation was performed on day 0. DST, 20x106 donor-splenocytes, was given intravenously on day -7. NK (40 mg/kg/day) was orally administered from day -7 to day +6. Recipient-splenocytes were cocultured with irradiated donor-splenocytes, and T cell proliferation (3H-thymidine incorporation), IFN-g production (ELISPOT) and cell phenotypes (FACS) were assessed. Results: Untreated mice promptly rejected cardiac allograft at a graft median survival time (gMST) of 6.5 days. DST or NK monotherapy prolonged gMST to 24.5 and 25.5 days, respectively. In a sharp contrast, NK plus DST treatment (NK+DST) markedly prolonged gMST to 75.5 days. (A). In the NK+DST group, both proliferation and IFN-g production against donorantigens transiently increased on day 0, while they were both significantly suppressed on day 7 (p<0.05, vs. Untreated). Following in vivo antigen-challenge by infusing 20x106 donor-splenocytes on day 0, the splenic Foxp3+CD4+ T cells increased[Figure 1](B); however, CD69+ effector T cells were abolished.Figure: No Caption available.(C and D) in the NK+DST group. Conclusion:NK together with DST induces a potent immunomodulatory effect by shifting immune-balance towards a regulatory milieu, and markedly prolongs cardiac allograft survival in mice. DISCLOSURES:Saiga, K.: Employee, salary.

Combined intrathymic and intravenous injection of mesenchymal stem cells can prolong the survival of rat cardiac allograft associated with decrease in miR-155 expression

BackgroundMesenchymal stem cells (MSCs) have the potential to improve graft outcomes and promote allograft tolerance. In this study, we examined the effects and mechanism of combined intrathymic (IT) and intravenous (IV) injection of MSCs on the survival of transplanted hearts in a rat allograft model. MethodsRecipient Sprague-Dawley rats were transplanted with hearts from Wistar rats. Wistar rat MSCs were infused via IT or IV or combined IT and IV (IT/IV) injection at designated intervals. In vitro mixed lymphocyte reaction assays were performed to assess the immunosuppressive capacity of MSCs. Mesenchymal stem cell surface markers and CD4+, CD25+, and Foxp3+ T-cells in the peripheral blood were detected using flow cytometry analysis. The expression of microRNAs and cytokines in graft infiltrating lymphocytes was analyzed by real-time polymerase chain reaction. ResultsThe MSCs cultured in vitro had multipotential differentiation capacity. Mixed lymphocyte reaction assays showed that donor-derived MSCs could not stimulate a proliferative response of recipient lymphocytes and could markedly suppress T-cell responses. Survival of the allografts was significantly prolonged by administration of IT/IV injection of MSCs compared with controls, with a mean survival of 32.2 versus 6.5 d, respectively. Compared with the syngeneic groups posttransplant, miR-155 expression was significantly increased in the allogeneic group, and could be restored by injection of MSCs, especially IT/IV injection of MSCs. Moreover, IT/IV injection of MSCs decreased the level of interleukin (IL)-2 and interferon-gamma, but increased the levels of IL-4 and IL-10 in the allogeneic group. More important, IT/IV injection of MSCs was the best way to increase the percentage of CD4+, CD25+, and Foxp3+ T-cell peripheral blood. ConclusionsOur results indicated that IT/IV injection of MSCs can prolong the survival of rat cardiac allograft, which may be associated with down-regulating miR-155 expression, a shift in the Th1/Th2 balance, and up-regulation of Treg cells expression.

A New Derivative of a Natural Flavonoid Icariin Extracted from Chinese Herb Epimedii Prolongs Cardiac Allograft Survival in Rats through Inhibition of Lymphocyte Proliferation and Activation

Background: Novel immunosuppressive agents, which could avoid deleterious adverse effects of current immunosuppressants such as nephrotoxic calcineurin inhibitors (CNIs), are required to prolong long-term survival of organ transplant patients and allografts. A new derivative of a natural flavonoid icariin extracted from Chinese herb Epimedii (also named YinYangHuo) was chemically synthesized, and had shown immunosuppressive effects both in vitro and in vivo in this present study. Materials and methods: The new derivative cycloicaritin was synthesized by hydrolysis of a flavonoid icariin. The allogeneic heterotopic heart transplantation model was established with Brown Norway (BN) and Lewis rats. The cycloicaritin was orally administered at daily dosage of 12 mg/kg and 24 mg/kg to prevent acute rejection. Human peripheral blood mononuclear cells (PBMCs) were stimulated by ConA, anti-CD3/anti-CD28, PMA/inonmycin or inactivated donor PBMCs in the presence or absence of indicated concentrations of cycloicaritin. The PBMCs proliferation was examined by chemiluminescence analysis with CCK-8 kit or by CFSE proliferation analysis with flow cytometer. Membrane expression of CD25 and CD69 on CD4+ and CD8+ T cells was examined by flow cytometry. Soluble IL-2 and INF-γ was examined by ELISA analysis. Results: The new cycloicaritin significantly prolonged cardiac allograft survival from 6.8 ± 0.7 days to 18 ± 4.2 days (12 mg/kg/day) and 30 ± 4.9 days (24 mg/kg/day) (p< 0.05). The cycloicaritin significantly inhibited PBMC proliferation in concentration dependent manner (10-11 M˜10-3 M). The highest inhibition rate was up to 89%, and concentration required to inhibit proliferation by 50% (IC50) was 1.8×10-6 M. Expression level of CD25 and CD69 was respectively decreased by 59.5% and 71.5% on CD4+ T cells, and was decreased by 55.8% and 73.8% on CD8+ T cells when the cycloicaritin was administered (p< 0.05). The cycloicaritin (10-8 M˜10-4 M) decreased soluble IFN-γ level of human PBMCs by 18%˜70% (p< 0.05). In mixed lymphocyte reaction (MLR) model, the cycloicaritin (10-6 M˜10-4 M) reduced soluble IFN-γ secretion by 33%˜75% when compared to control group (p< 0.05). Conclusion: The new structurally modified derivative (cycloicaritin) of the natural extract flavonoid icariin is able to suppress inhibition and activation of human peripheral lymphocytes, and thus prevent acute allograft rejection in rats.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

BackgroundThe CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.ResultsIn this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.ConclusionsSCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment. SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Eicosapentenoic Acid Attenuates Allograft Rejection in an HLA-B27/EGFP Transgenic Rat Cardiac Transplantation Model.

The development of an animal model bearing definite antigens is important to facilitate the evaluation and modulation of specific allo-antigen responses after transplantation. In the present study, heterotopic cardiac transplantation was performed from F344/EGFPTg and F344/HLA-B27Tg rats to F344 rats. The F344 recipients accepted the F344/EGFPTg transplants, whereas they rejected the cardiac tissue from the F344/HLA-B27Tg rats by 39.4 ± 6.5 days, due to high production of anti-HLA-B27 IgM- and IgG-specific antibodies. In addition, immunization of F344 rats with skin grafts from F344/HLA-B27Tg rats resulted in robust production of anti- HLA-B27 IgM and IgG antibodies and accelerated the rejection of a secondary cardiac allograft (7.4 ± 1.9 days). Of interest, the F344 recipients rejected cardiac grafts from double transgenic F344/HLA-B27&EGFPTg rats within 9.0 ± 3.2 days, and this was associated with a significant increase in the infiltration of lymphocytes by day 7, suggesting a role for cellular immune rejection. Eicosapentenoic acid (EPA), one of the ω-3 polyunsaturated fatty acids in fish oil, could attenuate the production of anti-HLA IgG antibodies and B-cell proliferation, significantly prolonging double transgenic F344HLA-B27&EGFPTg to F344 rat cardiac allograft survival (36.1 ± 13.6 days). Moreover, the mRNA expression in the grafts was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealing an increase in the expression of the HO-1, IL-10, TGF-β, IDO, and Foxp3 genes in the EPA-treated group. Hence, our data indicate that HLA-B27 and/or GFP transgenic proteins are useful for establishing a unique animal transplantation model to clarify the mechanism underlying the allogeneic cellular and humoral immune response, in which the transplant antigens are specifically presented. Furthermore, we also demonstrated that EPA was effective in the treatment of rat cardiac allograft rejection and may allow the development of novel immunomodulatory strategies for organ transplantation.

NK026680 inhibits T-cell function in an IL-2-dependent manner and prolongs cardiac allograft survival in rats

NK026680 is a triazolopyrimidine derivative that has been shown to inhibit dendritic cell maturation and activation. Here, we examined the immunosuppressive properties of NK026680 on T-cell function and assessed its immunosuppressive efficacy in an ACI (RT1 av1 haplotype) to Lewis (RT1 l) rat heart transplantation model. The effects of NK026680 on T-cell proliferation, activation, and cytokine production were investigated in vitro. Heart transplant recipient rats were administered NK026680 daily for 14 days post-transplantation. In addition to graft survival time, alloimmune responses and graft histology at 4–10 days post-transplantation were assessed. NK026680 was found to inhibit proliferation, CD25 upregulation, IL-2 production, and cell cycle progression in αCD3/αCD28-stimulated murine T cells. These effects were likely due to suppression of the p38 mitogen-activated protein kinase pathway and the subsequent inhibition of p65, c-Fos, and to a lesser extent, c-Jun. Daily NK026680 treatment suppressed alloimmune responses, prevented cellular infiltration into allografts, and prolonged graft survival. The anti-rejection effects of NK026680 were enhanced by tacrolimus. In conclusion, NK026680 inhibits the activation of T cells and prolongs cardiac allograft survival in rats. These features make it a potential candidate immunosuppressant for the treatment of organ transplant patients in the future.

Targeting Lymphatic Vessel Activation and CCL21 Production by Vascular Endothelial Growth Factor Receptor-3 Inhibition Has Novel Immunomodulatory and Antiarteriosclerotic Effects in Cardiac Allografts

Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C(+) inflammatory cell and hyaluronan receptor-1 (LYVE-1)(+) lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3(+), contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein-positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8(+) effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62(+) dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4(+) T cells in chronically rejecting mouse cardiac allografts. These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel-targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

The role of recipient mast cells in acute and chronic cardiac allograft rejection in C57BL/6-KitW-sh/W-sh mice

Mast cells are hypothesized to promote rejection and adverse remodeling in cardiac allografts. In contrast, it has been reported that mast cells may enhance cardiac allograft survival in rats. We used C57BL/6-Kit(W-sh/W-sh) mast cell-deficient and corresponding wild-type mice to investigate possible contributions of recipient mast cells to acute or chronic cardiac allograft rejection. FVB (H-2(q); acute rejection), or C-H-2(bm12)KhEg (H-2(bm12); chronic rejection) donor hearts were heterotopically transplanted into C57BL/6-Kit(W-sh/W-sh) (H-2(b)) or C57BL/6-Kit(+/+) (H-2(b)) mice. The degree of acute rejection was assessed at 5 days and chronic rejection, at 52 days. In the acute rejection model, donor heart vascular cell adhesion molecule-1 (VCAM-1) expression was significantly lower in C57BL/6-Kit(W-sh/W-sh) than in wild-type recipients; however, acute rejection scores, graft survival, inflammatory cells, or cytokine expression did not differ significantly. In the chronic rejection model, the number of mast cells/mm(2) of allograft tissue was significantly increased 52 days after transplantation in allografts transplanted into C57BL/6-Kit(+/+) but not C57BL/6-Kit(W-sh/W-sh) mice; however, no substantial differences were noted in graft coronary artery disease, graft inflammatory cells, or levels of graft tissue expression of cytokines or adhesion molecules. Cardiac allografts undergoing chronic rejection in wild-type C57BL/6-Kit(+/+) mice exhibit increased numbers of mast cells, but acute or chronic cardiac allograft rejection can develop in C57BL/6-Kit(W-sh/W-sh) mice even though these recipients virtually lack mast cells. These findings indicate that recipient mast cells are not required for acute or chronic cardiac allograft rejection in the models examined.

Preconditioning With Ozone Abrogates Acute Rejection and Prolongs Cardiac Allograft Survival in Rats

Acute cardiac allograft rejection remains a major cause of morbidity and mortality after heart transplantation and predisposes for the development of graft vasculopathy. The aim of this study was to investigate the immunomodulatory effect of preconditioning of the donor and recipient with medical ozone (O 3/O 2) on acute allograft rejection. Minimizing the initial ischemia-reperfusion injury may result in a reduction of graft vasculopathy and ameliorate long-term outcomes after cardiac transplantation. Lewis rats were challenged with Wistar-Furth cardiac allograft. In donor and recipient animals a medical ozone (O 3/O 2)-pneumoperitoneum was induced by single (1×) or repetitive (5×) insufflation (concentration: 50 μg/mL, 80 mL/kg body weight) of medical ozone intraperitoneally. Without immunomodulatory therapy (n = 11) cardiac allograft survival was 5.9 ± 0.9 days. Preconditioning with medical ozone alone (single bolus as well as repetitive administration, n = 7) of the donor and recipient animals prolonged cardiac allograft survival significantly to 7.6 ± 1.4 days ( P < .05), without any adjunctive immunosuppressive therapy. In this pilot study, the intraperitoneal administration of ozone in donor and recipient animals protected from ischemia-reperfusion injury, reduced the immunogenicity of the graft, and prolonged cardiac allograft survival. Further studies are warranted to elucidate the underlying mechanisms and—more important—to investigate the effect on the development of graft vasculopathy, the major obstacle to long-term graft and patient survivals.

Hydrodynamics‐based delivery of plasmid DNA encoding CTLA4‐Ig prolonged cardiac allograft survival in rats

Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.

Rosiglitazone Suppresses Cyclosporin-Induced Chronic Transplant Dysfunction and Prolongs Survival of Rat Cardiac Allografts

The lack of effective treatment for chronic transplant dysfunction restricts the long-term survival of solid organ allografts. Peroxisome proliferator-activated receptor ligands can suppress vascular inflammation. The aim of this study was to analyze the effects of rosiglitazone on chronic transplant dysfunction in a rat cardiac transplant model. Inbred male Fisher 344 (F344, RT1lvl) and Lewis (LEW, RT1(1)) rats were subjected to heterotopic abdominal heart transplantation according to standard procedures. Cyclosporine A was administered intraperitoneally to cover acute rejection, and rosiglitazone was administered orally by gavage daily from 3 days before the operation to the end of experiments. Rosiglitazone significantly prolonged the survival of cardiac allografts in rats (F344 to LEW) that had received a 10-day course of cyclosporin A compared to treatment with immunosuppressant alone. Analysis of allografts at 120 days posttransplantation showed that rosiglitazone reduced the inflammatory cell infiltrate in both the vessels and graft parenchyma as were neointimal formation, vascular occlusion, and fibrosis. Expression of transforming growth factor-beta and related proteins was less abundant after cyclosporin A/rosiglitazone treatment. The findings reported here demonstrate that rosiglitazone given under the cover of short-term treatment with cyclosporin A prolongs cardiac allograft survival and reduces the severity of chronic transplant dysfunction. This may be mediated in part through the downregulation of transforming growth factor-beta and related proteins. The combined effects of rosiglitazone and immunosuppressive drugs are potentially beneficial to patients receiving organ transplants.

Gene transfer of interleukin-18-binding protein attenuates cardiac allograft rejection

Interleukin (IL) 18 is a potent pro-inflammatory Th1 cytokine that exerts pleiotropic effector functions in both innate and acquired immune responses. Increased IL-18 production during acute rejection has been reported in experimental heart transplantation models and in kidney transplant recipients. IL-18-binding protein (IL-18BP) binds IL-18 with high affinity and neutralizes its biologic activity. We have analyzed the efficacy of an adenoviral vector expressing an IL-18BP-Ig fusion protein in a rat model of heart transplantation. IL-18BP-Ig gene transfer into Fisher (F344) rat donor hearts resulted in prolonged graft survival in Lewis recipients (15.8 +/- 1.4 days vs. 10.3 +/- 2.5 and 10.1 +/- 2.1 days with control virus and buffer solution alone, respectively; P < 0.001). Immunohistochemical analysis revealed decreased intra-graft infiltrates of monocytes/macrophages, CD4(+), CD8alpha(+) and T-cell receptor alphabeta(+) cells after IL-18BP-Ig versus mock gene transfer (P < 0.05). Real-time reverse transcriptase polymerase chain reaction analysis showed decreased cytokine transcripts for the RANTES chemokine and transforming growth factor-beta after IL-18BP-Ig gene transfer (P < 0.05). IL-18BP-Ig gene transfer attenuates inflammatory cell infiltrates and prolongs cardiac allograft survival in rats. These results suggest a contributory role for IL-18 in acute rejection. Further studies aiming at defining the therapeutic potential of IL-18BP are warranted.

Administration of the stress protein gp96 prolongs rat cardiac allograft survival, modifies rejection-associated inflammatory events, and induces a state of peripheral T-cell hyporesponsiveness

High-dose gp96 has been shown to inhibit experimental autoimmune disease by a mechanism that appears to involve immunoregulatory CD4+ T cells. This study tested the hypothesis that high-dose gp96 administration modifies allograft rejection and associated inflammatory events. Wistar cardiac allografts were transplanted into Lewis recipient rats and graft function was monitored daily by palpation. Intradermal administration of gp96 purified from Wistar rat livers (100 microg) at the time of transplantation and 3 days later significantly prolonged allograft survival (14 vs 8 days in phosphate-buffered saline [PBS]-treated recipients; P = 0.009). Rejected allografts from gp96-treated animals were significantly less enlarged than allografts from their PBS-treated counterparts (2.8 vs 4.3 g; P < 0.004). Gp96 was also effective when administered on days 1 and 8 (13 vs 7 days), but not if it was derived from recipient (Lewis) liver tissue or administered on days 0, 3, and 6. In parallel studies, CD3+ T cells from gp96-treated untransplanted animals secreted less interleukin (IL)-4, IL-10, and interferon (IFN)-gamma after in vitro polyclonal stimulation than CD3+ T cells from PBS-treated animals. Gp96 administration might therefore influence the induction of immunity to coencountered antigenic challenges and inflammatory events by inducing what appears to be a state of peripheral T-cell hyporesponsiveness.