Eotaxin/CCL11 levels correlate with myocardial fibrosis and mast cell density in native and transplanted rat hearts.

INTRODUCTION: Myocardial fibrosis contributes to hemodynamic and cardiac functional alterations commonly observed posttransplantation. Cardiac mast cells (MC) have been linked to fibrosis in posttransplantation hearts. Eotaxin, which has been shown to be involved in fibrogenesis, has been demonstrated to be increased in production in cardiac macrophages. The aim of our study was to correlate myocardial fibrosis during heart transplant rejection in the rat with eotaxin/chemokine [c-c motif] ligand 11 (CCL11) expression, and with various subtypes of infiltrating cardiac MC, namely connective-type MC (CTMC) and mucosa-type MC (MMC). METHODS: We used tissues from 2 previous studies of ongoing acute rejection in allogeneic Brown-Norway to Lewis rat and an isogeneic Brown-Norway to Brown-Norway heterotopic heart transplantation models under cyclosporin/prednisolone immunosuppression. Collagen fibrils were stained with Masson's trichrome with myocardial fibrosis expressed as percent fibrotic area per total section area. Eotaxin/CCL11 previously measured in heart tissue using enzyme-linked immunosorbent assay (ELISA) was correlated with the extent of myocardial fibrosis. We compared values from native hearts (n = 4) as well as transplants on days 5, 16, and 28 (n = 4 in each group). RESULTS: The area of myocardial fibrosis was significantly increased in the allogeneic compared with the isogeneic group at day 16 (38% vs 21%) and at day 28 (49% vs 22%) after transplantation. Myocardial fibrosis correlated significantly with eotaxin/CCL11 concentrations and the density of MMC, but not with CTMC in heart tissue. CONCLUSIONS: Eotaxin-triggered MC infiltration of the heart may contribute to myocardial fibrosis after transplantation. Targeting eotaxin/CCL11 with monoclonal antibodies, such as bertilimumab, could reduce MC infiltration, possibly resulting in decreased myocardial fibrosis and improved contractile function after heart transplantation.

An experimental approach toward chronic pulmonary allograft rejection: Orthotopic lung versus heterotopic tracheal segment transplantation in rats.

BACKGROUND: Despite the impact of chronic rejection (CR) on long-term outcomes, clinically relevant experimental models are sparse, often including a design of subcutaneous implantation of tracheal segments. However, this latter site lacks anatomic correlation, adequate perfusion, and ventilatory function. In this study, we compared the spatial and sequential course of CR in models of orthotopic single lung transplantation (LT) versus heterotopically implanted tracheal segments in rats. METHODS: We performed 30 orthotopic left single LTs from Fisher 344 (F344) to Wistar Kyoto (WKY) rats for comparison with the outcomes of 3 tracheal segments implanted subcutaneously in every recipient. As a control group, 3 syngeneic tracheal segments were implanted into 12 WKY rats. For histopathologic examinations, tracheal segments and pulmonary allografts were harvested between days 1 and 112 and between weeks 4 and 18, respectively. RESULTS: Allogeneic tracheal segments showed rapid fragmentation of the respiratory epithelium, with complete luminal occlusion by week 4, whereas the lumen in isografts remained unobstructed. In contrast, bronchioles from orthotopically transplanted lungs did not show epithelial changes before week 14. However, marked lymphocytic sequestration into bronchioles occurred by week 8 with sequential destruction of all layers of the small airways, with loss of respiratory epithelium by week 16. CONCLUSIONS: Based on the different histomorphologic dynamics of CR, direct comparison between those 2 models is limited. When investigating CR in future studies, initial findings based on tracheal implantation experiments should be expanded in the site of orthotopic pulmonary transplantation.

Magnetic resonance imaging of transplanted mouse islets labeled with chitosan-coated superparamagnetic iron oxide nanoparticles.

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.

The changes of aquaporin 2 in the graft of acute rejection rat renal transplantation model.

AIM: To investigate the significance and changes of Aquaporin 2 (AQP2) in the rat renal graft of acute rejection (AR). METHODS: Wistar recipients of Spraque-Dawley or Wistar renal grafts were treated with cyclosporine (CsA). Renal grafts were harvested at various times after transplantation for analysis of the levels of AQP2 mRNA and protein of by RT-PCR and immunohistochemistry. RESULTS: The expression of AQP2 mRNA and protein in the acutely rejecting were grafts significantly decreased (P < 0.05) compared with the control group. But the expression of AQP2 mRNA and protein in the syngeneic grafts (sTX) versus the immunosuppression group (aTX+CsA) showed no difference compared with a control group (P > 0.05). Furthermore, at day 5 and day 7 after transplantation the expressions of AQP2 expression in the allogeneic group (aTX) were decreased significantly compared with day 3 after transplantation (P < 0.05). In addition, there was no remarkable difference at day 5 or 7 after transplantation. CONCLUSION: AQP2 mRNA and protein expressions were down-regulated during renal transplant acute rejection, which had no relationship to the ischemia reperfusion injury and denervation damage. Furthermore, CsA administration after kidney transplantation blunted this down-regulation (P > 0.05).

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

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

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

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

Effect of remote ischemic preconditioning on rat estradiol serum levels and follicular development after ovarian transplantation.

PURPOSE: The purpose of this study was to evaluate estradiol serum levels and follicular development in rats subjected to ovarian autologous transplantation with or without remote ischemic preconditioning (R-IPC). METHODS: Seventy-two adult female Wistar EPM-1 rats were distributed into 3 groups: (1) controls; (2) ovarian transplantation; and (3) ovarian transplantation + R-IPC. The groups were divided into subgroups, according to the prefixed date for euthanasia: 24 hours, 48 hours, 72 hours, and 7th postoperative day (PO). R-IPC was performed by clamping the common iliac artery for a 15-minute period of ischemia followed by 15 minutes of reperfusion, before undergoing ovarian transplantation. The graft was fixed to the retroperitoneum with a simple 8-0 prolene thread. Blood samples were collected from the vena cava. For evaluation of follicular development, the ovarian follicles were classified as immature and mature follicles besides corpora lutea. Only the viable follicles and functioning corpora lutea were counted. RESULTS: At 72 hours, the R-IPC group showed higher estradiol values than the other groups, which were similar. After 24 hours the mean values were similar among all groups, and at 48 hours the R-IPC group was similar to the transplanted group without IPC. Animals undergoing R-IPC showed superior morphologic aspects, but 7 days after transplantation the morphology was worse in all groups. R-IPC enhanced the number of immature follicles at 48 hours (P > .05) and number of mature follicles from 24 hours to 48 hours after transplantation (P < .01). Functioning corpora lutea number was increased as well. CONCLUSION: R-IPC increased the estradiol levels in autologous ovarian transplants associated with better graft morphology and more mature follicles.

Perioperative glycine treatment attenuates ischemia/reperfusion injury and ameliorates smooth muscle dysfunction in intestinal transplantation.

BACKGROUND: Ischemia/reperfusion evokes a functionally relevant inflammatory response within the muscularis propria of small bowel grafts by activation of resident macrophages and leukocyte recruitment. We hypothesized that immunomodulatory perioperative treatment with glycine attenuates the proinflammatory cascade and improves smooth muscle dysfunction of small bowel grafts. METHODS: Orthotopic SBTx was performed in Lewis rats. Glycine (1 mg/g body weight) was infused (0.1 mL/g/hr) for 2 hr before harvest as preconditioning in the donor, and for 2 hr from the onset of reperfusion in the recipient. Transplanted vehicle (isotonic saline)-treated animals and naive animals served as controls. Rats were sacrificed after 3 hr and 24 hr. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by real-time-PCR. Jejunal circular smooth muscle contractility was assessed in a standard organ bath. RESULTS: Compared with vehicle controls, glycine-treated graft muscularis expressed a significant alleviation in mRNA peak expression for IL-6, IL-1beta, ICAM-1, MCP-1, TNFalpha, COX-2, and iNOS. Also glycine-treated grafts exhibited significantly less infiltration with ED-1-positive macrophages and MPO-positive neutrophils as well as reduced apoptosis. Concurrent to these results, vehicle controls showed an 80% decrease in smooth muscle contractility, whereas glycine-treated animals exhibited only a 40% decrease in contractile activity compared with controls. CONCLUSIONS: The data indicate that perioperative glycine treatment reduces the molecular and cellular inflammatory response within the grafts and improves smooth muscle dysfunction after transplantation. Therefore, the glycine-activated chloride channel on resident and infiltrating leukocytes could be a promising pharmacologic target to attenuate ischemia/reperfusion injury after ITx.

Acute rejection and the muscularis propria after intestinal transplantation: the alloresponse, inflammation, and smooth muscle function.

BACKGROUND: It has been shown that in transplantation the intestinal muscularis may act as an immunologically active layer via the activation of resident macrophages and the recruitment of leukocytes. Thus we hypothesized that inflammation within the intestinal muscularis is involved in the promotion of acute rejection in intestinal allografts and that this causes smooth muscle dysfunction. METHODS: Orthotopic allogenic and small bowel transplantation (Brown-Norway rats-Lewis rats) was performed without immunosuppression. Animals were sacrificed 1, 4, and 7 days after small bowel transplantation. Isogenic transplanted grafts (Brown-Norway rats-Brown-Norway rats) as well as nontransplanted bowel served as controls. Mediator mRNA expression was determined by real-time reverse-transcriptase polymerase chain reaction. Leukocyte infiltration was evaluated in muscularis whole mounts by immunohistochemistry. Apoptosis was evaluated by TdT-mediated dUTP-X nick end labeling assay. Contractility was assessed in a standard organ bath under bethanechol stimulation. Statistical analysis was performed using a Student's t test and one-way analysis of variance. RESULTS: Transplanted animals showed a significant early inflammatory response within the graft muscularis because of reperfusion injury. Only allogenic transplanted animals exhibited a significant second molecular inflammatory peak in the muscularis during rejection (mRNA induction for interleukin (IL)-6, intercellular adhesion molecule-1, monocyte chemoattractant protein (MCP)-1, interferon-gamma, IL-2, tumor necrosis factor-alpha, IL-10, inducible nitric oxide synthase). These findings were associated with significant leukocyte infiltration within the muscularis, increasing apoptotic cells and massive impairment of smooth muscle contractile activity by 78%. CONCLUSIONS: The data shows that transplantation results in an early and temporary inflammatory response within the intestinal graft muscularis, that is reactivated and intensified during acute allograft rejection. The immunoreaction within the intestinal muscularis leads to intestinal allograft smooth muscle dysfunction.

Maximal inflammatory response benefits syngeneic skin graft acceptance.

OBJECTIVE AND DESIGN: We investigated the influence of acute inflammation in skin isograft acceptance. METHODS: Two mouse lines selected for maximal (AIRMAX) or minimal inflammatory response (AIRMIN) were transplanted with syngeneic skin. Cellular infiltrates and cytokine production were measured 1, 3, 7 or 14 days post-transplantation. The percentage of CD4+CD25+Foxp3+ cells in the lymph nodes was also evaluated. RESULTS: Grafts were totally accepted in 100% of AIRMAX and in 26% of AIRMIN mice. In the latter, partial acceptance was observed in 74% of the animals. Emigrated cells were basically PMN and were enhanced in AIRMAX transplants. IL-10 production by graft infiltrating cells showed no interline differences. IFN-gamma was increased in AIRMIN grafts at day 14 and lower percentages of CD4+CD25+Foxp3+ cells in the lymph nodes were observed in these mice. CONCLUSIONS: Our data suggest that differences in graft acceptance might be due to a lack of appropriate regulation of the inflammatory response in AIRMIN mice compromising the self/non-self recognition.

Two-layer method (UW solution/perfluorochemical plus O2) for lung preservation in rat lung transplantation.

A new preservation method using perfluorochemicals (PFC) with oxygen administered continuously was developed for lung preservation and compared with traditional cold preservation methods for rat lung transplantation. Male Sprague-Dawley rats underwent orthotopic left lung transplantations of grafts preserved in lactiated Ringers solution (LR), University of Wisconsin solution (UW), Celsior solution, or a two-layer (PFC plus O2) solution for 6 hours. One hour after reperfusion, the right pulmonary artery and bronchus were clamped and 5 minutes later we recorded peak airway pressure and PaO2 level. The isograft was excised for measurement of myeloperoxidase activity, wet-to-dry ratio, and histologic examination to evaluate isograft function. The mean peak airway pressure was 29.80+/-6.72 mm H2O in the LR group, 28.80+/-5.76 mm H2O in the UW group, 33.60+/-5.17 mm H2O in the Celsior group, and 32.40+/-2.60 in the two-layer group. The mean PaO2 level was 99.78+/-76.09 mm Hg in the LR group, 87.84+/-33.58 mm Hg in the UW group, 104.50+/-72.93 mm Hg in the Celsior group, and 62.08+/-31.34 mm Hg in PFC and UW solution plus O2 group (two layers). The mean net myeloperoxidase activity OD level was 0.110+/-0.104 in the LR group, 0.392+/-0.328 in the UW group, 0.351+/-0.620 in the Celsior group, and 0.532+/-0.616 in the two-layer group. The mean wet-to-dry ratio was 7.47+/-1.60 in the LR group, 6.56+/-0.62 in the UW group, 7.54+/-2.19 in the Celsior group, and 5.32+/-2.20 in the two-layer group. The differences between groups in these parameters were not significant. Upon histologic examination, more inflammatory cell aggregates were seen in the two-layer group, less in the LR and the Celsior groups. The function of the lung graft after 6 hours of storage was not better using this two-layer method for preservation than traditional preservation methods in rat lung transplantation. Histologic examination revealed more inflammatory cell aggregates in the lung graft preserved using a two-layer method.

New anti-CD30 human pancreatic ribonuclease-based immunotoxin reveals strong and specific cytotoxicity in vivo.

Although the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma has been considerably improved during the last decades, high therapeutic toxicity, relapses, secondary tumors, and primary treatment failure(s) occur. Both malignancies are well suited for CD30-targeted immunotherapy because of their strong CD30 expression. We constructed an immunotoxin composed of a single chain variable fragment of a CD30 antibody fused to the human pancreatic ribonuclease, showing CD30-specific binding and ribonucleolytic activity resistant to the inhibitor RNasin. This immunotoxin revealed CD30-specific anti-tumor activity in BALB/c mice that were challenged with CD30-positive or CD30-negative syngeneic tumor cells.

A mouse model of orthotopic vascularized aerated lung transplantation.

Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient-type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.

Vascular endothelial growth factor (VEGF) ligand and receptor induction in rat renal allograft rejection.

BACKGROUND: Acute rejection is the single most important risk factor for the subsequent development of chronic allograft nephropathy (CAN), which is still the primary reason for late allograft loss in kidney transplantation. Vascular endothelial growth factor (VEGF) is a proangiogenic factor that has an important role in the development and maintenance of physiological endothelium. While its role has been characterized in the pathology of diabetic nephropathy and preeclampsia, its role in the development of acute and chronic allograft rejection remains unclear. METHODS: Kidney transplantations were performed from DA to WF rats and syngeneic control transplantations were performed between DA rats. Normal kidneys were used as controls to evaluate physiological VEGF and VEGFR-1 expression. Allografted rats were immunosuppressed with cyclosporine (CsA) (1.5 mg/kg/d subcutaneously); and no immunosuppression was given to syngeneic grafts. Grafts were harvested at 5 and 90 days after transplantation for histology and immunohistochemistry (VEGF, VEGFR-1). RESULTS: In normal kidneys VEGF ligand and receptor expression was almost nonexistent. Only mild glomerular, arterial, and tubular VEGF expression was seen. In syngeneic grafts, no histological signs of acute or chronic rejection were seen, whereas characteristics of both acute and chronic rejection were seen in CsA-treated allografts. Altough VEGF expression was increased in syngenic grafts when compared to controls it still remained mild in both the early and the late posttransplant period. In CsA-treated allografts moderate VEGF expression was seen already 5 days after transplantation; the expression increased at 90 days after transplantation. The same pattern was also discovered for VEGFR-1 expression although the difference was not as remarkable after 5 days. CONCLUSIONS: Our results demonstrated that VEGF ligand and receptor expression was increased in both acute and chronic rejection. Our data suggested that VEGF may have an important role in the pathology of chronic rejection. Based on our findings VEGF inhibition could be a potential intervention to prevent CAN in clinical kidney transplantation.

Effect of FTY720 in rat small bowel transplantation: apoptosis of crypt cells and lymphocytes in gut-associated lymphoid tissues.

AIM: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 during rejection. METHODS: Orthotopic small bowel transplantations (SBTs) were performed from BN to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, and 7 after operation: isograft, untreated allograft, allograft with FTY720. FTY720 was orally administered by gavage (1 mg/kg/d) to allograft recipients on 7 consecutive days. Cryostat sections were prepared from grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/Fas-L. RESULTS: Graft survival was prolonged in the FTY720-treated group. The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7 compared with the isograft group. In the FTY720-treated group, the number of ISEL-positive enterocytes in the allografts was down-regulated significantly on days 3, 5, and 7 compared with untreated allograft group. In the PPs, the number of ISEL-positive mononuclear cells increased significantly in the allografts compared with the isograft group. In the FTY720-treated groups, the number of ISEL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. The number of Fas/FasL-positive enterocytes were increased significantly in allografts compared with isograft group. In FTY720-treated groups, the number of Fas/FasL-positive enterocytes were down-regulated significantly on day 7 compared with the untreated allograft group. In the PPs, Fas/FasL-positive mononuclear cells also increased significantly on day 7 in the allografts compared with isografts. In the FTY720-treated groups, Fas/FasL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. CONCLUSIONS: The number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes increased during small bowel graft rejection. FTY720 prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes while also prolonging small bowel allograft survival.

Acute rejection in allogeneic rodent small bowel transplantation causes smooth muscle dysfunction via an inflammatory response within the intestinal muscularis.

INTRODUCTION: Isogeneic intestinal transplantation elicits an inflammatory response within the intestinal muscularis that is associated with dysmotility. Usually the inflammation and the postoperative motor dysfunction resolve within a few days after small bowel transplantation (SBTx). However, the onset of acute rejection in allogeneic SBTx is again associated with dysmotility. We hypothesized that dysmotility during acute rejection is based on coexpression of kinetically active mediators by the alloreactive leucocyte infiltrate. MATERIALS AND METHODS: Rat SBTx (BN to Lew and BN to BN) was performed without immunosuppression. Animals were sacrificed at 1, 4, and 7 days after SBTx. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by reverse transcriptase polymerase chain reaction. Muscle contractility was assessed in a standard organ bath. RESULTS: Transplanted animals showed a significant inflammatory response within the muscularis at day 1 after SBTx. However, allogeneic transplanted animals exhibited a significant second inflammatory peak at day 7 (mRNA induction: iNOS 150-fold; tumor necrosis factor-alpha 18-fold; interferon-gamma 397-fold), parallel to the onset of rejection. This change was associated with a significant leukocyte infiltration. Compared to controls, allogeneic transplanted animals showed a 29% decrease in smooth muscle contractility on days 1 and 4 and a 71% decrease of contractility on postoperative day 7. CONCLUSIONS: The motor dysfunction of transplanted small bowel during acute rejection is associated with an inflammatory reaction in the intestinal muscularis. The initial unspecific inflammation process immediately after transplantation is reactivated and intensified during acute rejection.

Composite vascularized skin/bone transplantation models for bone marrow-based tolerance studies.

There is an ongoing need to understand the mechanisms of bone marrow-based allograft tolerance. This is important in clarifying the diverse variables influencing the ultimate outcome of the solid organ and composite tissue transplants. To establish bone marrow transplantation as a routine clinical application, further experimental studies should be conducted to overcome the obstacles related to the bone marrow transplantation. These obstacles include graft versus host disease, immunocompetence, and toxicity of the conditioning regimens. For these purposes, novel experimental models are needed. In an attempt to provide a reliable research tool for bone marrow-based tolerance induction studies, we introduced different experimental models of modified vascularized skin/bone marrow (VSBM) transplantation technique for tolerance induction, monitoring, and maintenance studies. In this skin/bone transplantation model, the technical feasibility of concurrent or consecutive transplantation of the combination of bilateral vascularized skin, vascularized bone marrow, or vascularized skin/bone marrow transplants was investigated. Isograft transplantations were performed between genetically identical Lewis (LEW, RT1) rats. Five different experimental designs in 5 groups of 5 animals each were studied. Group I: Bilateral vascularized skin (VS) transplantation; group II: bilateral vascularized skin/bone transplantation; group III: vascularized skin transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group IV: vascularized bone transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group V: vascularized bone transplantation on one side and vascularized skin transplantation on the contralateral side. Successful transplantations were performed in all groups. The survival of the isograft transplants was evaluated clinically and histologically. All skin flaps remained pink and pliable and grew new hair. The viability of the compact bone, bone marrow and skin at 100 days posttransplant was confirmed by histologic evaluation, and bone marrow revealed active hematopoiesis. Bilateral skin/bone transplantation model may serve as an experimental tool to study new strategies in tolerance induction by altering the amount of the immunogenic load in the form of skin transplant and bone marrow delivery in the vascularized form, allowing for expedited engraftment of stem and progenitor cells.

Integrity of airway epithelium is essential against obliterative airway disease in transplanted rat tracheas.

BACKGROUND: The pathogenesis of obliterative bronchiolitis after lung transplantation requires further elucidation. In this study we used rat trachea transplantation to examine the role of epithelium in the progression of obliterative airway disease. METHODS: Normal and denuded (i.e., epithelium removed) trachea grafts from Lewis (LEW) and Brown Norway (BN) rats were transplanted sub-cutaneously into LEW rats. Viable trachea epithelial cells (to recover epithelium) were seeded into the lumen of some of the denuded tracheas. Grafts were removed at different time-points between 2 days and 8 weeks after transplantation. Histologic analysis was performed to evaluate cellular infiltration of inflammatory cells, loss of epithelium, and obliteration of trachea lumen. RESULTS: Obliteration was found to occur in trachea transplants after loss of epithelium, caused by rejection in allografts or by enzymatic denudation in isografts. In these situations, fibroblasts started to proliferate and migrate into the lumen in the second week after transplantation. Obliteration could be prevented when epithelial integrity was restored by seeding epithelial cells; no obliteration occurred when denuded trachea isografts were seeded with epithelial cells, whereas non-seeded denuded tracheas were obliterated at Day 6 after transplantation. CONCLUSIONS: We conclude that integrity of airway epithelium is essential for rat trachea transplants to be safeguarded from obliterative airway disease. For clinical lung transplantation the results of our study suggest that protection of the integrity of airway epithelium may be important in preventing the development of obliterative bronchiolitis.

Effect of FTY720 in rat small bowel transplantation: expression of mucosal addressin cell adhesion molecule-1.

AIM: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated tissues (GALT). We performed a semiquantitative analysis of MAdCAM-1 expression during small bowel graft rejection in rat treated with FTY720. METHODS: Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, 7 after operations (Isograft, untreated allograft, allograft with FTY720). FTY720 was orally administered by gavage (1 mg/kg/d) to allograft models on 7 consecutive days. Cryostat sections were prepared from grafts, including Peyer's patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propia from 1 (faint), to 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villi tip). RESULTS: The graft survival was prolonged in the FTY720-treated group. MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection. In the FTY720-treated groups, MAdCAM-1 expression on HEVs in PPs was up-regulated and its expression on endothelial cells of vessels in the lamina propria was down-regulated compared with untreated allograft group. CONCLUSIONS: Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intestine immune reactivity during the early phase of small bowel allograft rejection. FTY720 was found to prevent the down-regulation of MAdCAM-1 expression on HEVs in PPs and the up-regulation of its expression on endothelial cells of vessels in the lamina propria while also prolonging small bowel allograft survival.

The effect of allo-injury in an in vitro model of allograft microvasculature.

Endothelial cells are critical to the integrity of allograft vasculature and can be damaged by alloreactive T cells or soluble mediators of alloreactivity. The biochemical effects of T cell-mediated damage to the endothelial cells have been studied, but not the structural and morphological effects of allo-injury on endothelial cells in the allograft. We utilized an assay that reproduces microvasculature in vitro to study the effect of alloreactivity on endothelial cells. In this assay, endothelial cells are induced into capillary-like networks that simulate microvascular capillaries. We studied the effect of allogeneic T cells and of soluble mediators from both mixed lymphocyte cultures (MLCs) and rejected heart allograft tissue on the in vitro capillaries. We found that both allogeneic T cells and soluble mediators inhibit the formation of the in vitro endothelial capillaries, suggesting that they cause a mild-to-moderate dysfunction of the endothelial cells. The inhibitory effect of the soluble mediators seems to be mediated, at least partly, by IFN-gamma, since this effect was prevented by antibody to IFN-gamma. Furthermore, pre-incubation of the in vitro capillaries with IFN-gamma appeared to magnify the effect of allogeneic T cells, as shown by a complete breakdown of well-formed in vitro capillary networks. Our experiments suggest that the in vitro capillary-tube model reflects structural injury to allograft vasculature by alloreactive T cells and their soluble mediators.