Untreated Allografts Research Articles

PTH Promotes Allograft Integration in a Calvarial Bone Defect

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

Profiling circulating microRNA expression in a mouse model of nerve allotransplantation

BackgroundThe lack of noninvasive biomarkers of rejection remains a challenge in the accurate monitoring of deeply buried nerve allografts and precludes optimization of therapeutic intervention. This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation with or without immunosuppression.ResultsBalb/c mice were randomized into 3 experimental groups, that is, (1) untreated isograft (Balb/c → Balb/c), (2) untreated allograft (C57BL/6 → Balb/c), and (3) allograft (C57BL/6 → Balb/c) with FK506 immunosuppression. A 1-cm Balb/c or C57BL/6 donor sciatic nerve graft was transplanted into sciatic nerve gaps created in recipient mice. At 1, 3, 7, 10, and 14 d after nerve transplantation, nerve grafts, whole blood, and sera were obtained for miRNA expression analysis with an miRNA array and subsequent validation with quantitative real-time PCR (qRT-PCR). Three circulating miRNAs (miR-320, miR-762, and miR-423-5p) were identified in the whole blood and serum of the mice receiving an allograft with FK506 immunosuppression, within 2 weeks after nerve allotransplantation. However, these 3 circulating miRNAs were not expressed in the nerve grafts. The expression of all these 3 upregulated circulating miRNAs significantly decreased at 2, 4, and 6 d after discontinuation of FK506 immunosuppression. In the nerve graft, miR-125-3b and miR-672 were significantly upregulated in the mice that received an allograft with FK506 only at 7 d after nerve allotransplantation.ConclusionsWe identified the circulating miR-320, miR-762, and miR-423-5p as potential biomarkers for monitoring the immunosuppression status of the nerve allograft. However, further research is required to investigate the mechanism behind the dysregulation of these markers and to evaluate their prognostic value in nerve allotransplantation.

The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing

Allografts remain the clinical “gold standard” for treatment of critical sized bone defects despite minimal engraftment and ∼60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a ‘tissue engineered periosteum’. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (∼2.4-fold), endochondral bone formation (∼2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification.

Nanoparticle PET-CT Detects Rejection and Immunomodulation in Cardiac Allografts

Macrophages predominate among the inflammatory cells in rejecting allografts. These innate immune cells, in addition to allospecific T cells, can damage cardiomyocytes directly. We explored whether sensitive positron emission tomography-computed tomography (PET-CT) imaging of macrophages-avid nanoparticles detects rejection of heart allografts in mice. In addition, we used the imaging method to follow the immunomodulatory impact of angiotensin-converting enzyme inhibitor therapy on myeloid cells in allografts. Dextran nanoparticles were derivatized with the PET isotope copper-64 and imaged 7 days after transplantation. C57BL/6 recipients of BALB/c allografts displayed robust positron emission tomography signal (standard uptake value allograft, 2.8±0.3; isograft control, 1.7±0.2; P<0.05). Autoradiography and scintillation counting confirmed the in vivo findings. We then imaged the effects of angiotensin-converting enzyme inhibitor (5 mg/kg enalapril). Angiotensin-converting enzyme inhibitor significantly decreased nanoparticle signal (P<0.05). Histology and flow cytometry showed a reduced number of myeloid cells in the graft, blood, and lymph nodes and diminished antigen presentation (P<0.05 versus untreated allografts). Angiotensin-converting enzyme inhibitor also significantly prolonged allograft survival (12 versus 7 days; P<0.0001). Nanoparticle macrophage PET-CT detects heart transplant rejection and predicts organ survival by reporting on myeloid cells.

The Potassium Channel KCa3.1 as New Therapeutic Target for the Prevention of Obliterative Airway Disease

The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD. Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28. Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes. Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.

No red cell alloimmunization or change of clinical outcome after using fresh frozen cancellous allograft bone for acetabular reconstruction in revision hip arthroplasty: a follow up study.

BackgroundPossible immunization to blood group or other antigens and subsequent inhibition of remodeling or incorporation after use of untreated human bone allograft was described previously. This study presents the immunological, clinical and radiological results of 30 patients with acetabular revisions using fresh frozen non-irradiated bone allograft.MethodsAB0-incompatible (donor-recipient) bone transplantation was performed in 22 cases, Rh(D) incompatible transplantation in 6 cases. The mean follow up of 23 months included measuring Harris hip score and radiological examination with evaluation of remodeling of the bone graft, implant migration and heterotopic ossification. In addition, all patients were screened for alloimmunization to Rh blood group antigens.ResultsCompared to the whole study group, there were no differences in clinical or radiological measurements for the groups with AB0- or Rh(D)-incompatible bone transplantation. The mean Harris Hip Score was 80.6. X-rays confirmed total remodeling of all allografts with no acetabular loosening. At follow up, blood tests revealed no alloimmunization to Rh blood group donor antigens.ConclusionsThe use of fresh frozen non-irradiated bone allograft in acetabular revision is a reliable supplement to reconstruction. The risk of alloimmunization to donor-blood group antigens after AB0- or Rh-incompatible allograft transplantation with a negative long-term influence on bone-remodeling or the clinical outcome is negligible.

Surface Treatment of Flexor Tendon Autograft and Allograft Decreases Adhesion Without an Effect of Graft Cellularity: A Pilot Study

Flexor tendon grafting is often required to reconstruct a failed tendon repair. Previous reports have demonstrated flexor grafts coated with lubricants such as carbodiimide derivatized hyaluronic acid (cd-HA) decrease adhesion formation and improve digit function. However, whether this surface modification would affect graft adhesion and cellularity is unknown. Adhesion score and the cellularity of the graft of untreated and cd-HA surface-modified autograft and allograft tendons were studied using a canine forepaw in vivo model. The peroneus longus tendons (n = 6) and flexor digitorum profundus tendons (n = 8) were used as extrasynovial autograft and intrasynovial allograft, respectively. The flexor digitorum profundus (FDP) tendons in the second and fifth digits in each dog were reconstructed with one digit treated with cd-HA and the other treated with saline as a control. Six weeks after surgery, the grafted tendons were harvested for histological evaluation with hematoxylin and eosin staining. During dissection, the adhesions were observed and scored. The adhesion score was greatest in the extrasynovial autograft without surface modification and the least in the intrasynovial allograft with surface modification. Autograft tendons had a higher cell density than the allografts regardless of surface treatment. Cd-HA graft treatment did not affect cellularity when compared with controls. Our observations suggest surface modification of a tendon graft with cd-HA decreased the adhesion formation without altering the cellularity in either autologous or allograft tendon. We therefore presume this surface modification would not adversely affect graft healing.

Autologous Mesenchymal Stem Cells Prevent Transplant Arteriosclerosis by Enhancing Local Expression of Interleukin-10, Interferon-γ, and Indoleamine 2,3-dioxygenase

Transplant arteriosclerosis (TA) remains the major limitation of long-term graft survival in heart transplantation despite the advances in immunosuppressants. Mesenchymal stem cells (MSCs) have been demonstrated to suppress allogeneic immune responses by numerous in vitro studies. However, the immunomodulatory effects of MSCs in vivo are controversial and the underlying molecular mechanisms are not conclusive. In this study, we investigated the therapeutic potential of autologous bone marrow-derived MSCs on TA in a porcine model of femoral artery transplantation. MSCs or saline were injected into the soft tissue surrounding the arterial grafts immediately postanastomosis. Four weeks after transplantation, neointimal formation increased significantly in untreated allografts compared with the MSC-treated grafts as assessed by intravascular ultrasound (maximum luminal area stenosis: 40 ± 12% vs. 18 ± 6%, p < 0.001). Grafts harvested at 4 weeks showed dense perivascular lymphocyte infiltration accompanied by significant intimal hyperplasia in the untreated but not in the MSC-treated allografts. Serial angiographic examination showed that all of the untreated allografts became occluded at the 8th week whereas the majority of the MSC-treated grafts remained patent at the 12th week posttransplantation (n = 12 each group, p < 0.001). Quantitative PCR analysis revealed that Foxp3 expression was comparable between the untreated and the MSC-treated groups. However, expression of interleukin-10 (IL-10), interferon-γ (IFN-γ), and indoleamine 2,3-dioxygenase (IDO) was increased significantly in the MSC-treated allografts compared with that in the allograft controls (p = 0.021 for IL-10, p = 0.003 for IFN-γ, and p = 0.008 for IDO). In conclusion, local delivery of autologous MSCs alleviates TA by inducing allograft tolerance via enhanced expression of IL-10, IFN-γ, and IDO but not Foxp3-positive cells in the vessel wall. These results suggest that MSCs induce immune tolerance by activating the type 1 regulatory T-like cells.

Indoleamine 2,3-Dioxygenase and Metabolites Protect Murine Lung Allografts and Impair the Calcium Mobilization of T Cells

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.

The role of speckle tracking imaging in the noninvasive detection of acute rejection after heterotopic cardiac transplantation in rats

Objective Acute cardiac allograft rejection continues to be the cause of graft loss and contributes to the morbidity and mortality after cardiac transplantation. Repetitive endomyocardial biopsies are necessary to monitor the eff ects of immunosuppressants after cardiac transplantation. In this study, we investigate whether speckle tracking imaging (STI) is a valuable method in assessing acute cardiac rejection.Methods and resultsHearts from Brown Norway rats or Lewis rats were transplanted into other Brown Norway rats. Isografts and groups of allografts, either untreated or treated with cyclosporine A (CsA) at a low dose (3 mg •kg-1• d-1) or a high dose (10 mg •kg-1• d-1), were compared 7 days after transplantation. Echocardiography-derived left ventricular post wall thickness was increased only in untreated allografts. The left ventricular ejection fraction was signifi cantly lower in the allografts compared with the isografts, but allografts treated without or with low-dose CsA showed similar results .The radial velocity and systolic radial strain rate showed a lower value in untreated allografts than other grafts, but there is no signifi cant diff erence between allografts treated with high- or low-dose CsA and isografts. The circumferential strain and circumferential strain rate were comparable among the 4 groups. However, the radial strain exhibited a clear gradient in these groups (2.8 ± 1.3 in untreated allografts, 5.2 ± 0.9 in allografts treated with lowdose CsA, 6.3 ± 1.8 in allografts treated with high-dose CsA, and 12.7 ± 7.9 in isografts, P < 0.001).Conclusions STI is able to off er a noninvasive method for detecting transplant allograft rejection.

Evaluating acute rejection after heterotopic cardiac transplantation in rats by speckle tracking imaging

Objective To examine whether speckle tracking imaging(STI) could provide for the assessment of acute cardiac rejection. Methods Hearts from Brown Norway rats or Lewis rats were transplanted into other Brown Norway rats. Isografts and groups of allografts either untreated or treated with cyclosporin A (CsA) at a low dose (3 mg ·kg-1 ·d-1) or high dose (10mg · kg-1 ·d-1) from 1 day before transplantation were compared at posttransplantation day 7. Results Echocardiography-derived left ventricular post wall thickness was increased only in untreated allografts. The left ventricular eject fraction was significant lower in the allografts compared with isograft, but allografts treated without or with low-dose CsA showed similar results. The radial systolic radial strain rate showed a lower value in untreated allografts than other grafts,but there was no significant differences between allograft treated with high- or low-dose CsA and isografts. The circumferential strain and circumferential strain rate was comparable among the 4 groups. However the radial strain exhibited a clear gradient in these groups [(2. 8 ± 1.3)％ in untreated allografts, (5.2 ± 0.9)％ in allografts treated with low-dose CsA, (6.3 ± 1.8 )％ in allografts treated with high-dose CsA,and (12.7 ± 7.9) in isografts, P＜0.001]. The radial strain exhibited a clear correlation with the severity of rejection ( r =-0.812, P＜ 0.0000). Conclusions The radial strain decreased as the severity of rejection worsen. STI offers promise as a noninvasive method for detecting transplant allograft rejection. Key words: Echocardiography; Heart transplantation; Graft rejection; Speckle tracking imaging

The Effects of Pharmacologic Plasminogen Activator Inhibitor-1 Inhibition in Acute and Chronic Rejection in Murine Cardiac Allografts

Acute rejection and graft arterial disease (GAD) in cardiac transplantation limit the long-term survival of recipients; these processes are enhanced by inflammation and thrombus formation. Plasminogen activator inhibitor (PAI)-1 is critical in the inflammation and thrombus formation. However, little is known about the effect of PAI-1 in heart transplantation. Thus, the objective was to clarify the role of PAI-1 in the progression of cardiac rejection. Murine hearts were heterotopically transplanted using major mismatch combinations for evaluation of acute rejection and class II mismatch combinations for the GAD. We administered the specific PAI-1 inhibitor (IMD-1622) into murine recipients after cardiac allografts. Nontreated allografts of the major mismatch group were acutely rejected, whereas the PAI-1 inhibitor prolonged their survival. Although severe cell infiltration and intimal thickening with enhancement of inflammatory factors were observed in untreated allografts of class II mismatch group on day 60, the PAI-1 inhibitor attenuated these changes. The PAI-1 inhibitor is potent for the suppression of both acute rejection and GAD.

Steroid-free maintenance of islet allografts using mycophenolate mofetil and cyclosporine in the non-human primate

Islet transplantation continues to be a promising treatment for type 1 diabetes, however, numerous limitations still prevent its widespread use. Many immunosuppressive medications used for islet transplantation are known to be diabetogenic. The goal of this study is to evaluate the short-term follow-up (90 day) of a steroid-free maintenance immunosuppression protocol of mycophenolate mofetil (MMF) and cyclosporine (CSA) in non-human primate islet allotransplantation. Diabetes was induced in the primate Macaca fascicularis via total pancreatectomy. Freshly isolated islets were autotransplanted (n=5) or allotransplanted (n=5) into the portal vein. Immunosuppression consisted of induction with rabbit anti-thymocyte globulin (ATG) and prednisone. CSA (25 mg/daily) and MMF (250 mg daily) were used for maintenance and given orally once daily throughout the 90 day study period. Fasting blood glucose measurements were used to monitor graft function. Intravenous glucose tolerance tests (IVGTT) were performed prior to pancreatectomy and at the study endpoint. Average fasting blood glucose levels were elevated in diabetic controls (313.6 mg/dl ±51.8) and untreated allograft recipients (257 mg/dl ±61.0) post-transplant. Auto-transplanted animals and allograft recipients treated with immunosuppression, on the other hand, maintained normoglycemia (74.5 mg/dl ±20.9, 62.3 mg/dl ±4.40) throughout the follow-up period. Additionally, beta cell function and first phase insulin secretion did not differ significantly between auto-graft and immunosuppressed allo-graft recipients post-transplant. We conclude that this steroid-free maintenance immunosuppression regimen is effective in maintaining islet allograft survival in the non-human primate and offers comparable graft function to that of autografts for up to 3 months post-transplant.

Glutaraldehyde treatment of allograft tissue decreases allosensitization after the Norwood procedure

Cryopreserved allograft tissue used in the Norwood procedure for infants with hypoplastic left heart syndrome causes a marked immunologic sensitization that may complicate future heart transplantation. Treatment of the allograft tissue before implantation may prevent this sensitization. The purpose of this study was to assess the anti-human leukocyte antigen antibody response to glutaraldehyde-treated allograft tissue used in the repair of hypoplastic left heart syndrome. Since June 2005, the University of Alberta has subjected allograft vascular tissue used in the Norwood procedure to glutaraldehyde treatment. An observational study was designed to assess whether glutaraldehyde treatment of the allograft tissue affected subsequent panel reactive antibody after patch implantation. Panel reactive antibodies for class I (human leukocyte antigen-A, B, C) and class II (human leukocyte antigen-DR, DQ) antibodies were measured 4 months postoperatively using flow cytometry. Fourteen patients underwent a Norwood procedure using glutaraldehyde-treated allograft tissue. Historical controls consisted of 12 patients who underwent a Norwood procedure using untreated allograft tissue. At 4 months, infants who had received glutaraldehyde-treated allograft tissue had lower class I panel reactive antibody (7.3% +/- 17.4% [median, 0%] vs 61.9% [median, 73%] +/- 39.9%; P = .0005) and class II panel reactive antibody (6.1% [median, 0%] +/- 22.7% vs 49.3% [median, 63%] +/- 41.9%, P = .001) compared with the historical controls. Intraoperative glutaraldehyde treatment of allograft tissue used in hypoplastic left heart syndrome repair prevents the profound immunologic sensitization that occurs in the majority of infants undergoing surgical palliation. In patients requiring subsequent heart transplantation, this decreases the risk of antibody-mediated rejection and increases the likelihood of finding a suitable donor, thus improving access to transplantation.

A pilot study of fat allograft transplantation in immunocompetent rabbits for potential neurosurgical applications

The authors investigated the feasibility of using fat allografts (chemically treated to reduce the host immune response) for neurosurgical applications. Subcutaneous fat specimens collected from New Zealand White rabbits were treated with DNAse I and sodium deoxycholate to reduce immunogenicity before subcutaneous, midscapular implantation in immunocompetent recipient rabbits. Allograft incorporation and the host-allograft response were examined at 1, 6, and 11 weeks by histopathological analysis. Control specimens of autograft and untreated fat allograft implants were examined for comparison. The host immune response was markedly reduced in the region around the chemically treated fat allografts when compared with untreated allografts, and was similar to the tolerant host response to autografts. Based on their results, the authors suggest that fat allografts processed for reduced immunogenicity may be a convenient, viable alternative for neurosurgical applications.

Syngeneic Bone Marrow Cell Therapy Prevents Intimal Proliferation in Allogeneic Vascular Transplantation

Transplant arteriosclerosis is characterized by intraluminal obstructive proliferation occurring in response to immune-mediated arterial wall injury. Cell therapy with vascular progenitor cells have been suggested to repair intimal lesions following endothelial injury. The aim of the current study was to assess the effects of autologous bone marrow cell direct transfer and of Fucan-mobilization bone marrow-derived progenitor cells on intimal thickening in vascular grafts. Aortic allografts were performed in Brown Norway (BN) and Lewis (LEW) rats. Cell therapy was performed by injection of two doses of 10 million LEW bone marrow mononuclear cells to recipient LEW following aortic grafting. Fucan, a low molecular weight sulfated polysaccharide (LMWF) was used to mobilize bone marrow-derived progenitor cells. Five groups of 10 rats included: untreated isografts (BN to BN), untreated allografts, and three allografted groups, respectively, treated by fucan therapy, cell therapy, or cell and fucan therapy. Aorta were studied by morphometric analysis at 30 d. In the absence of treatment, intimal thickening was greater in allograft than in isograft groups (299±50 versus 3.5±1.7 μm, P<0.001). Cell therapy alone, fucan therapy alone, and the combined treatment were shown to prevent intimal thickening in allografts (5.1±1.7, 6.1±2.3, 4.1±2.5, versus 299±50 μm respectively, P<0.001). In the three treated groups, the intimal lining was a single layer of endothelial cells expressing CD34 positive, endothelial nitric oxide synthase (eNOS) positive, and Ox3 specific-recipient monoclonal antibody positive. These results provide the proof of concept of recipient syngenic bone-marrow cell therapy for the prevention of chronic vasculopathy.

Abstract 5671: The Effects of Pharmacological PAI-1 Inhibition in Acute and Chronic Rejection in Murine Cardiac Allografts

Backgrounds: Acute rejection and graft arterial disease (GAD) in cardiac transplantation are enhanced by inflammation and thrombus formation. However, little is known about the effect of plasminogen activator inhibitor-1 (PAI-1) in heart transplantation. Thus, the objective was to clarify the role of PAI-1 in the progression of rejection. Methods and Results: Murine hearts were heterotopically transplanted using major mismatch combinations for evaluation of acute rejection and class II mismatch combinations for the GAD. We performed administration of the specific PAI-1 inhibitor (IMD-1622) into murine recipients of cardiac allografts. Nontreated allografts of the major mismatch group were acutely rejected (n=6; 7.3±0.2 days), while the PAI-1 inhibitor prolonged their survival (n=6; 13.7±2.4 days, P &lt; 0.05). Pathologically, severe myocardial cell infiltration (40.8±3.3 %) and fibrosis (44.5±2.8 %) were observed in untreated allografts, the PAI-1 inhibitor attenuated infiltration (25.8±1.9 %, p&lt;0.05) and fibrosis (25.8±3.1 % p&lt;0.05) in the class II mismatch group on day 60. Although severe GAD (luminal occlusion 28.6±6.4 %) was observed in untreated allografts, the PAI-1 inhibitor suppressed the development of neointimal formation (8.1±3.5 %, p&lt;0.05). Immunohistochemically, CD8 and ICAM-1 were enhanced in nontreated allografts, while the PAI-1 inhibitor markedly attenuated expression of these factors. Zymography showed that increased gelatinase activity was observed in the non-treated allograft group, however, the PAI-1 inhibitor decreased the activity. RNAse protection assay revealed that PAI-1 inhibitor suppressed IFN-gamma compared with that of non-treated group. Conclusion: The PAI-1 inhibitor is potent in the suppression of both allograft rejection and arterial disease because they are critically involved in the development of rejection through the suppression of inflammation.

Donor Apoptotic Lymphocyte Transfusion–Induced Liver Allograft Tolerance by Up-Regulation of CD4 +CD25 + Regulatory T Cells in Peripheral Blood

Uptake of apoptotic cells by antigen-presenting cells (APC) may be involved in tolerance maintenance with an immunoregulatory role. The aim of this study was to evaluate the consequences of preoperative transfusion of donor apoptotic lymphocytes on survival of orthotopic liver transplantations (OLT). OLT was performed between Lewis (donor) and Brown Norway (BN recipient) inbred rats using a double-cuff technique. Apoptotic splenic lymphocytes induced by ultraviolet-C (UVC) irradiation were infused intravenously at 7 days before OLT. Changes in regulatory T cells in blood were determined using flow cytometry. UVC irradiated lymphocytes were sensitive and effective, as evidenced by increased peripheral blood CD4 +CD25 + T cells compared with recipients that received infusion of untreated donor lymphocytes or a control. Apoptotic lymphocyte transfusion prolonged hepatic allograft survival, with significantly lower histological stages of inflammation and cellular infiltration than in untreated allografts. Our results demonstrated that donor apoptotic cells promoted allograft acceptance and up-regulated CD4 +CD25 + regulatory T cells (Treg) in blood.

Nicotine Attenuates Macrophage Infiltration in Rat Lung Allografts

Monocytes and macrophages play an important role in acute pulmonary allograft rejection. Acetylcholine has been shown to exert anti-inflammatory effects on these cells via nicotinic acetylcholine receptors. The aim of this study was to test for the hypothesis that a global nicotinic stimulation of pulmonary allograft recipients attenuates acute rejection. Orthotopic left lung transplantation was performed in the Fischer 344-Wistar Kyoto rat strain combination. Graft recipients treated with nicotine added to the drinking water were compared with untreated allograft recipients. Graft histopathology, leukocytic infiltration, expression of inducible nitric oxide (NO) synthase and cytokine expression were analyzed during the process of acute rejection on Day 7 post-transplantation using quantitative reverse transcript-polymerase chain reaction (RT-PCR), enzyme-linked immunoassay (ELISA) and immunohistochemistry. The right native lung of the experimental animals was included as an internal control. Nicotine treatment resulted in a marked reduction in lung allograft infiltration by CD68-like antigen(+) alveolar and tissue macrophages, whereas resident mature macrophages (CD163(+)) and T cells remained unchanged. Concomitantly, inducible NO synthase expression, which was predominantly localized in alveolar macrophages of control allografts, decreased in response to nicotine. In contrast, cytokine mRNA and peptide levels were only marginally affected by nicotine. Stimulation of nicotinic acetylcholine receptors results in a marked attenuation of important hallmarks of pulmonary allograft rejection, indicating that cholinergic therapies may be beneficial for lung allograft recipients.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.