Rat Allograft Model Research Articles

The effects of local administration of mesenchymal stem cells on rat corneal allograft rejection

BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-γ expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8 ± 1.2 days). Injection twice of MSCs (MST 12.6 ± 1.4 days) was more effective than a single injection (MST 10.8 ± 1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4 + T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-γ expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection.

BackgroundThe association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts.MethodsWe prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course.ResultsqRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.ConclusionsWe conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.

Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.

Protein tyrosine phosphatase receptor-type Z (PTPRZ) is aberrantly over-expressed in glioblastoma and a causative factor for its malignancy. However, small molecules that selectively inhibit the catalytic activity of PTPRZ have not been discovered. We herein performed an in vitro screening of a chemical library, and identified SCB4380 as the first potent inhibitor for PTPRZ. The stoichiometric binding of SCB4380 to the catalytic pocket was demonstrated by biochemical and mass spectrometric analyses. We determined the crystal structure of the catalytic domain of PTPRZ, and the structural basis of the binding of SCB4380 elucidated by a molecular docking method was validated by site-directed mutagenesis studies. The intracellular delivery of SCB4380 by liposome carriers inhibited PTPRZ activity in C6 glioblastoma cells, and thereby suppressed their migration and proliferation in vitro and tumor growth in a rat allograft model. Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.

Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: A potential new drug for treatment of glioblastoma multiforme

Dioscin, a natural product obtained from medicinal plants shows lipid-lowering, anti-cancer and hepatoprotective effects. However, the effect of it on glioblastoma is unclear. In this study, dioscin significantly inhibited proliferation of C6 glioma cells and caused reactive oxygen species (ROS) generation and Ca²⁺ release. ROS accumulation affected levels of malondialdehyde, nitric oxide, glutathione disulfide and glutathione, and caused cell apoptosis. In addition, ROS generation caused mitochondrial damage including structural changes, increased mitochondrial permeability transition and decreased mitochondria membrane potential, which led to the release of cytochrome C, nuclear translation of programmed cell death-5 and increased activities of caspase-3,9. Simultaneously, dioscin down-regulated protein expression of Bcl-2, Bcl-xl, up-regulated expression of Bak, Bax, Bid and cleaved poly (ADP-ribose) polymerase. Also, oxygen stress induced S-phase arrest of cancer cells by way of regulating expression of DNA Topo I, p53, CDK2 and Cyclin A and caused DNA damage. In a rat allograft model, dioscin significantly inhibited tumor size and extended the life cycle of the rats. In conclusion, dioscin shows noteworthy anti-cancer activity on glioblastoma cells by promoting ROS accumulation, inducing DNA damage and activating mitochondrial signal pathways. Ultimately, we believe dioscin has promise as a new therapy for the treatment of glioblastoma.

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.

Inflammatory lymphangiogenesis in a rat transplant model of interstitial fibrosis and tubular atrophy.

We have previously reported de novo lymphangiogenesis in human renal allograft nephrectomy specimens that exhibited interstitial fibrosis and tubular atrophy (IFTA). This study examined whether a similar pathology developed in an experimental model of renal transplantation in the rat. Renal transplants were carried out in rats comprising both isografts (Lewis kidneys → Lewis rats) and allografts (Fisher kidneys → Lewis rats). Animals were immunosuppressed in the immediate postoperative period and sacrificed at 12 months. Experimental readouts included lymphatic vessel number and location, inflammatory cell infiltration, interstitial fibrosis, renal function, blood pressure and proteinuria. Rat allografts demonstrated the characteristic features of IFTA with increased macrophage and T cell infiltration and scattered B cells aggregates. Rat allografts exhibited impaired renal function and proteinuria. Although there was no difference in the number of perivascular lymphatic vessels, there was a striking 18-fold increase in the number of interstitial lymphatic vessels in renal allografts. Furthermore, the lymphatic vessel number correlated with the extent of interstitial fibrosis. This rat allograft model of IFTA demonstrates a marked increase in the number of interstitial lymphatic vessels and mirrors previous work in failing human renal allografts.

RNA interference of hypoxia-inducible factor-1 alpha improves the effects of transcatheter arterial embolization in rat liver tumors

Transcatheter arterial embolization (TAE) and transcatheter arterial chemoembolization are widely used palliative treatments for patients with nonsurgical hepatocellular carcinoma. TAE has been shown to increase levels of hypoxia-inducible factor-1 alpha (HIF-1α) in tumors. In this study, we investigated whether RNA interference (RNAi) of HIF-1α could improve the efficacy of TAE to treat HCC. Four treatment groups were assessed using a rat allograft model of HCC: RNAi of HIF-1α combined with TAE, RNAi of HIF-1α only, TAE only, and a control group. Rats were treated by TAE by retrograde placement of a microcatheter into the gastroduodenal artery. The results demonstrated that the RNAi of HIF-1α visibly reduced the expression of HIF-1α and vascular endothelial growth factor, suppressed tumor angiogenesis, and attenuated metastasis, which were all enhanced by TAE. The RNAi of HIF-1α synergized with TAE to significantly inhibit tumor growth. In conclusion, RNAi of HIF-1α augmented the therapeutic effects of TAE and diminished its undesirable effects.

Abstract B247: The MEK inhibitor BAY 869766 inhibits tumor growth and metastatic spread, prolongs the survival and acts synergistically with standard of care drugs in models of hepatocellular carcinoma and pancreatic cancer.

Abstract Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B247.

Treatment with Immunosuppressants FTY720 and Tacrolimus Promotes Functional Recovery after Spinal Cord Injury in Rats

Spinal cord injury (SCI) occurs frequently and is a leading cause of permanent disability in young adults. Many immune inhibitors including tacrolimus (FK506) are shown to be helpful in the regeneration of neural tissue following spinal cord injury. FTY720 belongs to a new class of immunosuppressants. The combination of FTY720 and tacrolimus has been reported to elicit synergistic immunosuppresive effects in rat allograft models without causing critical adverse effects. This study was to determine whether the combination of FTY720 and tacrolimus is superior to FTY720 or tacrolimus alone in the treatment of SCI. Forty-eight rats were subjected to a weight-drop contusion at the tenth thoracic level (a 10-g rod dropped from a height of 25 mm). At 30 min after the operation, they were randomly divided into four groups and received treatment with either FTY720 (0.5 mg/kg), tacrolimus (0.5 mg/kg), FTY720 + tacrolimus (0.5 mg/kg and 0.5 mg/kg respectively) or saline via gavage. Functional recovery was evaluated during 42 days after SCI via open-field test, inclined plane test, footprint analysis, somatosensory evoked potentials (SSEPs), and electron microscopic analysis. Rats from three treatment groups showed significantly better locomotor functional outcomes, higher SSEP amplitude, shorter SSEP latency, and milder pathological changes compared with those of control group. Moreover, rats treated with a combination of FTY720 and tacrolimus demonstrated significantly greater functional recovery by day 14 after SCI than those treated with either FTY720 or tacrolimus alone. These results suggest that the combination of FTY720 and tacrolimus could be a potentially effective therapeutic strategy to treat SCI.

Trophic factor supplemented UW solution reduces intimal hyperplasia in the rat aortic transplant model

Background Chronic allograft nephropathy (CAN) is associated with delayed graft function, cold ischemic injury, and is an important cause of premature graft loss. A characteristic vascular lesion of CAN is intimal hyperplasia (IH). The goal of this study was to evaluate the effect of cold storage in University of Wisconsin solution supplemented with trophic factors (UW-TF) on IH in rat aortic isograft (IG) and allograft (AG) models. Methods F344 → F344 and Lewis → F344 orthotopic abdominal aortic transplants were performed after 48 h of cold storage in either UW or UW-TF solution with and without immunosuppression. Results Significant reduction in IH was observed when IG were stored in UW-TF solution compared to UW solution. A significant reduction in intimal inflammation was observed in UW-TF stored, nonimmunosuppressed AG. In immunosuppressed recipients, AG stored in UW-TF solution evidenced significantly less IH compared to those stored in UW alone. Conclusions UW-TF solution decreased IH in both alloindependent and dependent models.

Administration of Donor-Derived Mesenchymal Stem Cells Can Prolong the Survival of Rat Cardiac Allograft

Background Mesenchymal stem cells (MSCs) are multipotent adult elements that have recently been shown to have profound immunomodulatory effects both in vitro and in vivo. Herein we have examined the impact of intravenous infusion of donor MSCs on the survival of transplanted hearts in a rat allograft model. Methods Recipient Fisher344 rats were transplanted with hearts from inbred Wistar rats. Wistar rat MSCs were infused via the tail vein at designated intervals. In vitro mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays were performed to assess whether MSCs downregulated T-cell responses in vivo. Real-time polymerase chain reaction (PCR) was used to analyze the Th1/Th2 balance in MSC-treated and control groups. Results The MSCs cultured in vitro exhibited multipotential for differentiation. Survival of the allografts was markedly prolonged by administration of MSCs compared with the controls, namely mean survivals of 12.4 vs 6.4 days, respectively. Real-time PCR showed a shift in the Th1/Th2 balance toward Th2. By MLR and CML assays, untreated control rats showed greater alloreactivity than did MSC-treated rats. Conclusion Our results indicated that MSCs suppressed allogeneic T-cell responses both in vitro and in vivo. Intravenous administration of MSCs prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance through changing the Th1/Th2 balance.

Anti-Macrophage Migration Inhibitory Factor Antibody Suppresses Chronic Rejection of Heterotopically Transplanted Trachea in Rats

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine essential for delayed hypersensitivity in vivo, and is involved in bronchiolitis obliterans and late rejection in lung transplantation. We tested here whether neutralization of MIF using anti-MIF antibody prevents such a response. We examined the MIF mRNA expression level and changes in allograft tracheal epithelium and intraluminal obstruction in a rat allograft model. Lewis rat (RT1l) underwent heterotopic tracheal transplantation from Brown Norway rats (RT1n) in the omentum. Anti-MIF antibody was injected in the peritoneum. Rats were divided into three groups (non-treated allograft, allograft treated with normal rabbit IgG and allograft treated with anti-MIF antibody). Implants were harvested on days 7 or 21 for histological analysis. MIF mRNA expression was higher in the allograft at days 7 and 21 than in the isograft. The epithelium in non-treated allograft was almost absent at day 7. The epithelial height in the anti-MIF-treated graft was higher than that in normal IgG-treated grafts. The intraluminal space was mostly replaced by granulation tissue at day 21 in the untreated group. The proportion of obliterans was lowest in the anti-MIF group, the second lowest in the untreated group and the third lowest in the normal IgG-treated grafts, and the difference was significant (p<0.001) between the first two groups. Our results indicate that anti-MIF antibody suppresses allogenic tracheal rejection.

Inhibition of hepatic tumor cell proliferationin vitroand tumor growthin vivoby taltobulin, a synthetic analogue of the tripeptide hemiasterlin

To investigate the inhibitory effects of taltobulin (HTI-286), a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L +/- 1 nmol/L) in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allograft model). HTI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

Captopril suppresses Post-transplantation angiogenic activity in rat allograft coronary vessels

Background The development of transplant coronary artery disease is associated with neovascularization in the thickened neointima. We previously reported that captopril inhibits neointimal proliferation in a rat allograft model. We postulated that angiogenic inducers are upregulated post-transplantation and captopril ameliorates transplant coronary artery disease by suppressing the angiogenic activity of coronaries. Methods Animals received no treatment or captopril (50 mg/kg/day). Allograft hearts were analyzed at post-transplantation Days 0, 14, and 21 and angiogenic inducer, plasma platelet-activating factor, determined. The conditioned media from coronaries and myocardium were tested for vascular endothelial growth factor, thrombospondin-1 and angiogenic activity using an endothelial migration assay and rat corneal neovascularization assay. Results The captopril-treated group had reduced plasma platelet-activating factor and coronary media revealed earlier upregulation of thrombospondin-1 secretion, diminished vascular endothelial growth factor and no angiogenic activity. At Day 0, the coronary and myocardial conditioned medium had inhibitory activity due to thrombospondin-1, and circulating levels of platelet-activating factor were negligible. By 21 days post-transplantation, plasma platelet-activating factor was elevated and the conditioned medium from untreated coronaries had significantly higher angiogenic activity due to increased vascular endothelial growth factor whereas the myocardium remained non-angiogenic. Conclusions After transplantation, coronary vessels switch to an angiogenic phenotype and vascular endothelial growth factor contributes to the high angiogenic activity, possibly exacerbated by high circulating levels of platelet-activating factor. The ability of captopril to modulate angiogenic mediators and maintain the allograft coronary to its normal anti-angiogenic phenotype may be one mechanism by which it suppresses transplant coronary artery disease.

Reversal of diabetes-induced rat graft transplant coronary artery disease by metformin

Background Induction of diabetes in rat heterotopic heart transplantation models leads to an accelerated form of severe transplant coronary artery disease (TxCAD). We undertook this study to determine whether treatment of diabetes with metformin would favorably affect TxCAD. Methods Heterotopic abdominal heart transplantation was performed in rat isograft and allograft models. After transplantation, diabetes was induced with streptozotocin. Fifty percent of the animals received metformin at 500 mg/kg twice daily. We quantitatively assessed TxCAD using histologic sections of harvested hearts at 30 and 60 days with computer-assisted morphometry. We compared vessels in the first tertile of the area distribution with vessels in the last tertile. Results Fasting glucose levels in metformin-treated animals were 161 ± 45 mg/dl compared with 400 ± 120 mg/dl ( p < 0.05) in untreated rats. Treatment with metformin led to decreased diabetes-induced TxCAD in the larger vessels. This effect was sustained during the study course in the isografts but not in the allografts. Treatment with metformin did not prevent progression of TxCAD in the smaller vessels at 60 days. Conclusions Metformin reduced luminal occlusion and severe TxCAD in the larger vessels but did not alter the course of TxCAD in the smaller vessels. These results may have therapeutic implications for patients.

Effect of tranilast on chronic graft vascular disease and tracheal proliferation in a rat allograft model

Effect of tranilast on chronic graft vascular disease and tracheal proliferation in a rat allograft model

Late multiglycosidorum tripterygium treatment ameliorates established graft coronary arteriosclerosis after heart transplantation in the rat

GRAFT coronary arteriaosclerosis (GCA) is the major determinant of long-term survival following heart transplantation. GCA consists of concentric intimal arterial lesions that are diffusely distributed within epicardial and intramural coronary arteries of engrafted hearts and may result in myocardial ischemia, infarction, and sudden death. The clinical diagnosis of GCA is often delayed until late progress of GCA because patients with denervated engrafted hearts do not experience anginal symptoms, and the diffuse and concentric nature of occlusive lesions may obscure the early angiographic findings. In addition, therapeutic interventions for GCA such as angioplasty and coronary bypass grafting are limited because of the diffuse nature of the process. Therapeutic options are, therefore, limited, and retransplantation is often the last choice. Recent reports have suggested GCA is a type of chronic vascular rejection. It remains controversial whether established GCA can be improved by modification in immunosuppressive therapy. Previously our study indicated that multiglycosidorum tripterygium (MT) has effective immunosuppressive effect and could prevent the development of GCA following transplantation. The purpose of this study was to examine whether the therapy of MT initiated after GCA was already established could halt or reverse the progress of GCA in a rat allograft model.

FTY720, a novel immunosuppressant, shows a synergistic effect in combination with FK 506 in rat allograft models

FTY720, a novel immunosuppressant, shows a synergistic effect in combination with FK 506 in rat allograft models

The relationship of blood transfusion and immunosuppression to the Th1/Th2 paradigm

A lthough current drug protocols used to prevent organ rejection in transplant recipients have been highly successful, recipients must take anti-rejection drugs for the rest of their lives and endure risk of opportunistic infections and lymphoproliferative disorders. An alternative strategy would be to induce donor-specific tolerance using a short term protocol. We have previously demonstrated that a single donor-specific blood transfusion (DST) combined with a short course of cyclosporine A (CsA) significantly prolongs the survival of cardiac allografts in the high responding AC1 to Lewis rat model.’ The addition of a third agent, such as rapamycin, IL-4 or nutritional therapy to this protocol produces long-term tolerance in a significant number of recipients.2-4 Overly aggressive immunosuppression in this model does not improve allograft survival and in some cases decreases it.5 The mechanisms responsible for these observations are not completely understood. Recent studies of cytokine profiles associated with allograft rejection and tolerance induction suggest the ThUTh2, Tcl/Tc2 paradigm as a mechanism of tolerance with a Th2 phenotype being associated with tolerance and a Thl phenotype closely linked to rejection.‘j In order to elucidate the possible role played by these cell types and cytokines, we monitored the production of IL-4 and INF-y from CD3+CD4’ and CD3’CD8+ lymphocytes in an AC1 to Lewis rat allograft model.

Synergism of calcium-ethanehydroxybisphosphonate (CaEHBP) and FeC1 3: controlled release polymers for preventing calcification of bioprosthetic aortic wall

Controlled release delivery implants based on ethylene-vinyl acetate (EVA) copolymer were studied for prevention of calcification of aortic wall in an intracirculatory rat allograft model. The calcium salt of ethanehydroxybisphosphonate (CaEHBP) and ferric chloride (FeCl 3) were used as anti-calcification drugs either in combination or separately in solvent-cast EVA films. These matrices were characterized in vitro for their drug release at 37°C at pH 7.4 (0.05 M HEPES buffer). Inulin was included in the single drug loaded systems as an inert filler to obtain comparable loadings. The films released the drugs in vitro continuously over 60 days without any rapid burst phase. For rat allograft studies controlled release matrices or non-drug EVA films were sutured periadventitially to the aortic wall allografts to study the anticalcification efficacy for 30 days. The calcium and phosphorous levels of the explanted allografts were quantified. Controlled release films releasing both the drugs (CaEHBP and FeCl 3) together synergistically inhibited calcification of the aortic walls. CaEHBP alone releasing from EVA polymer was partially effective, and EVA films releasing only FeCl 3 did not inhibit calcification at all. Overall, no adverse effects on somatic growth or recipient bone morphology were noted following controlled release drug administration.