Effect of anti-ICAM-1 antibodies on macromolecular leakage and leukocyte activation: a study of hindlimb allografts in the rat

We investigated the ability of anti-ICAM-1 monoclonal antibodies to reduce endothelial cell damage by assessing microvascular permeability and microcirculatory function during the acute phase of allograft rejection. The composite rat hindlimb-cremaster muscle transplantation model was employed in three experimental groups of 18 animals each. Isograft control transplantations were performed between genetically identical Lewis (LEW, RT11) rats. Allograft transplantations were performed across a major histocompatibility barrier between Lewis-Brown-Norway (LBN, RT-11+n), and Lewis (LEW, RT11) rats. In addition, a third group of animals receiving allografts was treated with 1 mg/kg/day of anti-ICAM-1 monoclonal antibody. After 24 hours, 72 hours, and 7 days, we measured microvascular permeability, leukocyte activation, functional capillary perfusion, red blood cell velocity, vessel diameters, and endothelial edema index in six animals per each follow-up period. Endothelial cell damage was assessed by measuring graft permeability to fluorescein isothiocyanate-labeled albumin (0.2 ml/100 g body weight) with computer-aided image analysis. Mean microvascular permeability was lower in the treated allograft group than in untreated controls at all follow-up times (p<0.001). In addition, anti-ICAM-1 treatment significantly reduced the activation of sticking leukocytes at 24 and 72 hours (p<0.001) and the activation of transmigrating leukocytes at 72 hours and 7 days (p<0.05). The allografts presented a characteristic microcirculatory pattern of acute rejection as early as 24 hours after transplantation. The dysfunction of the endothelial cell barrier at all time points was indicated by significant increases in the degree of allograft macromolecular permeability and in the number of activated sticking and transmigrating leukocytes. Treatment with anti-ICAM-1 antibodies significantly reduced the surge of leukocytes in the allograft transplants and protected the endothelial barrier from the acute effects of transplantation trauma.     

Role of ICAM-1 in chronic hepatic allograft rejection in the rat

The pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecule-1 (ICAM-1)-mediated cell recruitment in chronic hepatic rejection. Liver transplantation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg. kg(-1). day(-1) x 6 days ip) or a control preparation (either ICAM-1 missense oligonucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemistry, and survival rates were also examined. Leukocyte adhesion in terminal hepatic venules was significantly increased in the rejection group compared with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the antisense-treated groups compared with control-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 +/- 1.2 days) compared with the controls (25.2 +/- 2.7 days). These results showed that early leukocyte recruitment in the hepatic microvasculature of rejecting allografts is ICAM-1 dependent and suggest that impacting on early cell recruitment can significantly ameliorate chronic rejection.     

A new composite facial and scalp transplantation model in rats

There are limited sources of autogenous tissue available for reconstruction of severe facial and scalp deformities caused by extensive tumor ablation, burns, or trauma. Allografts from cadaveric sources may serve as a reconstructive alternative. However, technical and immunological aspects of harvesting and transplanting face and scalp flaps limit the routine use of such procedures. For evaluation of the feasibility of composite-tissue reconstruction, an experimental model of composite face/scalp flap transplantation in rats was designed. Technical aspects of the model, survival rates, and the complications encountered during development of the model are presented. A total of 64 animals, in three experimental groups, were studied. In group I, the anatomical study group (n = 6), the anatomical features of the face and scalp region in rats were explored. Groups II and III were the transplantation groups. Isograft transplantations were performed between identical Lewis rats (RT11 to RT11), and allografts were transplanted, across major histocompatibility complex barriers, between Lewis-Brown Norway rats (RT1l/n) and Lewis rats (RT11). In group II (the control group, n = 8), transplantation of nonvascularized composite face/scalp isografts and allografts was performed. In group III (the transplantation group, n = 50), vascularized face/scalp isografts (n = 36) and allografts (n = 14) were transplanted. Complications included partial or total flap necrosis, death attributable to food aspiration, and poor general condition. To prevent acute and chronic allograft rejection, cyclosporine A (16 mg/kg per day) therapy was initiated 24 hours after transplantation; the dose was tapered to 2 mg/kg per day within 4 weeks and was maintained at that level thereafter. Long-term survival (>170 days) was achieved, without any signs of rejection, with low-dose (2 mg/kg per day) cyclosporine A therapy. This is the first report documenting successful composite face/scalp flap transplantation in the rat model.     

Cyclosporine and experimental skin allografts: long-term survival in rats treated with low maintenance doses

Although cyclosporine (CsA) is a powerful immunosuppressive agent in organ transplantation, its efficacy in skin transplantation has not been examined completely. We have tested it as primary immunosuppression in a rat skin allograft model. Histoincompatible Brown-Norway skin grafts are rejected in untreated Lewis hosts within 9 +/- 1 days but survive for 22 +/- 3, 34 +/- 2, or 41 +/- 8 days after 7, 14, or 21 days of CsA treatment (15 mg/kg per day subcutaneously), respectively (p less than 0.001). Animals treated daily for 4 weeks died from drug toxicity; however, an initial 2-week course followed by a low maintenance dose (15 mg/kg every fourth day) produced indefinite (greater than 150 days) graft acceptance without side effects. The long-surviving grafts were supple, grew long hair, and showed normal histology. When the drug was stopped at any time during this maintenance period, early signs of rejection (hair loss, epidermal breakdown, and localized ulceration) occurred, which could be reversed completely by a short CsA "pulse" (15 mg/kg per day for 7 days). These experimental data support the potential application of CsA immunosuppression in human skin allotransplantation.     

Characterization of acute renal allograft rejection by proteomic analysis of renal tissue in rat

Rapid and reliable biomarkers of renal allograft rejection have not been available. This study aimed to investigate biomarkers in renal allograft tissue using proteomic analysis. Orthotopic kidney transplantations were performed using Fisher (F344) or Lewis rats as donors and Lewis rats as recipients. Syngenic control group (Group I) constituted F344-to-F344 orthotopic kidney allo-transplantations (n = 8); and allogenic group (Group II) consisted of F344-to-Lewis orthotopic kidney allo-transplantations (n = 8). Renal tissues were harvested 7 days after transplantation. Samples were analyzed using 2-D electrophoresis and matrix assisted laser desorption ionization-time of flight mass spectrometry. 6 differentially expressed proteins were identified between allogenic group and syngenic control group. A rat model of acute renal allograft rejection was successfully set up. Differentially expressed proteins in renal allograft tissue of rat were detected using proteomic analysis and might serve as novel diagnostic and therapeutic targets in human. Quantitative proteomics, using MALDL-TOF-MS methodology has the potential to provide a profiling and a deeper understanding of acute renal rejection.     

Prevention of neutrophil migration ameliorates rat lung allograft rejection

Chemokines activate and recruit specific leukocyte subpopulations. We sought to determine whether neutrophil migration, which can contribute to the development of ischemia-reperfusion injury, correlates with lung allograft rejection. Orthotopic left lung allotransplantation was performed from Brown Norway (donor) to Fisher 344 (recipient) rats. Because the role of activated neutrophils in the development of allograft rejection is believed to be biphasic, we used specific CXC receptor inhibition with antileukinate in 2 dosing regimens. Recipients were allocated into 4 groups; A (early administration) received 2 doses of antileukinate (10.0 mg/kg) intramuscularly 24 h before and immediately after transplantation; B (continuous administration) continuously received antileukinate intraperitoneally (10.0 mg/kg/day) for 7 days after surgery. Groups A or B were compared with individual controls that received PBS alone. The progression of rejection was assessed radiographically. Histologic evaluation of allograft rejection based on pathologic rejection grade, performed on day 7, demonstrated significantly lower histologic rejection in group B compared with the control group (2.1+/-1.0 vs. 3.3+/-0.5; P=0.018), whereas there was no significant difference in group A compared with the control group. There were no significant differences between the aeration scores of groups A or B compared with their control groups. Our data suggest that neutrophils may play a promoting role in the development of allograft rejection, and blockage of neutrophil migration may suppress acute lung allograft rejection.     

大鼠肝移植急性排斥反应模型的建立与评价

目的：建立大鼠肝脏移植急性排斥反应模型,并对所建模型进行评价。方法：采用改良Kamada“二袖套”法,以近交系大鼠Dark Agouti（DA）为供体、Lewis（LEW）为受体（A组）建立大鼠原位肝移植急性排斥反应模型,通过观察手术情况、生存情况及肝功能和病理学检查对此模型进行评价,同时以LEW→LEW作为对照组（B组）。结果：手术成功率为91.3%;手术时间为（90.70±5.68）min;无肝期时间（9.96±1.19）min;平均存活时间A组为（12.44±3.43）d,B组超过100d;A组血清谷丙转氨酶、谷草转氨酶及血清总胆红素术后不断升高,在第10～14d最为明显,血清白蛋白在术后第3d开始逐渐降低,在相同时相点,与B组相比差异显著;A组移植肝脏病理检查有明显的排斥反应,而B组没有。结论：DA→LEW为稳定、强烈的大鼠肝移植急排模型,是研究肝移植排斥及免疫耐受的理想动物模型。     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Long-term dietary L-arginine supplementation attenuates proteinuria and focal glomerulosclerosis in experimental chronic renal transplant failure.

Glomerular endothelial nitric oxide synthase expression is decreased in humans during acute rejection and chronic renal transplant failure (CRTF). This may contribute to vascular damage through changes in the renal hemodynamics and enhanced endothelial adhesion of leukocytes and platelets. Dietary supplementation of L-arginine may increase endothelial NO production, thereby protecting the vascular wall and improving renal hemodynamics. We tested the hypothesis that long-term L-arginine supplementation attenuates the development of CRTF in an experimental model for renal transplantation. In the Fisher 344 to Lewis rat model for renal transplantation, renal function and histology of untreated rats was compared with rats receiving L-arginine in the drinking water (10g/L), starting 2 days before transplantation. Every 4 weeks systolic blood pressure was measured and serum and urine were collected for measurement of nitrite and nitrate (NO(x)), creatinine, and proteinuria. At 34 weeks the histological renal damage was assessed by scoring focal glomerulosclerosis and measurement of alpha-smooth muscle actin (alpha-SMA) expression. Urinary NO(x) was significantly increased in treated animals. Proteinuria was significantly lower in L-arginine-treated animals from week 24 onward (p<0.05). Plasma creatinine and creatinine clearance did not differ between the groups. The focal and segmental glomerulosclerosis (FGS) score (max 400) at week 34 was also significantly lower in treated rats arbitrary U (20+/-21 vs 61+/-67 arbitrary U; p<0.05). The expression of alpha-SMA was lower in L-arginine-treated rats than in untreated rats (1.93+/-0.8% area surface vs 3.64+/-2.5% area surface). In conclusion, in this experimental model for CRTF, L-arginine administration significantly reduced FGS and proteinuria, without affecting renal function. Our data suggest that dietary L-arginine supplementation attenuates progression of CRTF and may therefore be an additional therapeutic option in human renal allograft recipients.     

Electroporation-mediated interleukin-10 overexpression in skeletal muscle reduces acute rejection in rat cardiac allografts

OBJECTIVES: Human interleukin 10 (hIL-10) may reduce acute rejection after organ transplantation. Our previous data shows that electroporation-mediated transfer of plasmid DNA to peripheral muscle enhances gene transduction dramatically. This study was designed to investigate the effect of electroporation-mediated overexpression of hIL-10 on acute rejection of cardiac allografts in the rat. METHODS: The study was designed to evaluate the effect of hIL-10 gene transfer on (a) early rejection pattern and (b) graft survival. Gene transfer was achieved by intramuscular (i.m.) injection into the tibialis anterior muscle of Fischer (F344) male recipients followed by electroporation 24 h prior to transplantation. Heterotopic cardiac transplantation was performed from male Brown Norway rat to F344. Four groups were studied (n = 6). Treated animals in groups B1 and B2 received 2.5 microg of pCIK hIL-10 and control animals in groups A1 and A2 distilled water. Graft function was assessed by daily palpation. Animals from group A1 were sacrificed at the cessation of the heart beat of the graft and those in group B1 were sacrificed at day 7; blood was taken for ELISA measurement of hIL-10 and tissue for myeloperoxidase (MPO) measurement and histological assessment. To evaluate graft survival, groups A2 and B2 were sacrificed at cessation of the heart beat of the graft. RESULTS: Histological examination revealed severe rejection (IIIB-IV) in group A1 in contrast to low to moderate rejection (IA-IIIA) in group B1 (p = 0.02). MPO activity was significantly lower in group B1 compared to group A1 (18 +/- 7 vs. 32 +/- 14 mU/mg protein, p = 0.05). Serum hIL-10 levels were 46 +/- 13 pg/ml in group B1 vs. 0 pg/ml in group A1. At day 7 all heart allografts in the treated groups B1 and B2 were beating, whereas they stopped beating at 5 +/- 2 days in groups A1 and A2 vs. 14 +/- 2 days in group B2 (p = 0.0012). CONCLUSIONS: Electroporation-mediated intramuscular overexpression of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in rats. This study demonstrates that peripheral overexpression of specific genes in skeletal muscle may reduce acute rejection after whole organ transplantation.     

Immunologic and ultrastructural changes during early rejection of vascularized bone allografts

This investigation evaluated ultrastructural changes during the earliest phase of immunologic rejection of vascularized bone allografts in a genetically defined rat model. These results were correlated with the cell-mediated and humoral immunologic responses during this time period. Employing a model for heterotopic allograft transplantation, 33 rats divided into four categories were evaluated. Group I consisted of ungrafted (naive) Lewis and Brown Norway rats; group II consisted of Lewis-to-Lewis vascularized bone isografts; group III consisted of Lewis-to-Brown Norway vascularized bone allografts; and group IV consisted of Lewis-to-Brown Norway vascularized bone allografts in rats receiving cyclosporine (10 mg/kg/day). Experimental animals were sacrificed at 3, 5, and 7 days. Immunologic analysis was performed using a cell-mediated lymphocytotoxicity assay and a complement-dependent cytotoxic antibody assay. The results of this study show that rejection of vascularized bone allografts appears as early as 3 days postoperatively, with osteocytes and vascular endothelium being the first elements affected. This early rejection is probably a manifestation of the humoral response. All changes secondary to rejection were arrested by cyclosporine.     

Atrial natriuretic factor (ANF) levels are elevated in rats bearing rejecting heart-lung allografts

Intra-abdominal heart-lung grafts were transplanted into 8 rats across a major histocompatibility barrier. Four of the 8 rats were treated with Cyclosporin A (CsA) to prevent rejection. Atrial natriuretic factor (ANF) levels measured 6 days after transplantation revealed a significantly (p less than .005) higher mean ANF concentration in rats bearing a rejecting heart-lung allograft (642 +/- 148.0 pg/ml) compared to rats bearing a heart-lung allograft not undergoing rejection (200.8 +/- 13.07 pg/ml). ANF might be a useful noninvasive marker in the diagnosis of rejection in heart and heart-lung transplants.     

Reduction of skeletal muscle injury in composite tissue allotransplantation by heat stress preconditioning

Ischemia-reperfusion injury is a dominant factor limiting tissue survival in any microsurgical tissue transplantation, a fact that also applies to allogeneic hand transplantation. The clinical experience of the 12 human hand transplantations indicates that shorter ischemia times result in reduced tissue damage and, ultimately, in better hand function. Heat stress preconditioning and the accompanying up-regulation of the heat shock protein 72 have been shown to reduce the ischemia-reperfusion injury following ischemia of various organs, including organ transplantation. The aim of this study was to reduce the ischemia-reperfusion injury in a model of composite tissue allotransplantation. Allogeneic hind limb transplantations were performed from Lewis (donor) to Brown-Norway rats. Donor rats in group A (n = 10) received a prior heat shock whereas rats in group B (n = 10) did not receive any prior heat shock. Group C served as a control group without transplantation. The transplantations were performed 24 hours after the heat shock, at which time the heat shock protein 72 was shown to be up-regulated. The outcome was evaluated 24 hours after transplantation by nitroblue tetrazolium staining and wet-to-dry weight ratio of muscle slices (anterior tibial muscle). The nitroblue tetrazolium staining showed a significant reduction of necrotic muscle in group A (prior heat shock) (p = 0.005). The wet-to-dry ratio was significantly reduced in group A (prior heat shock), indicating less muscle edema and less tissue damage (p = 0.05). Heat shock preconditioning 24 hours before an ischemic event leads to an up-regulation of heat shock protein 72 in muscle and to a tissue protection reducing ischemia-reperfusion injury in composite tissue transplantation.     

Blockade of platelet endothelial cell adhesion molecule-1 (PECAM-1) protects against ischemia-reperfusion injury in muscle flaps at microcirculatory level

Several lines of evidence show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a component of endothelial cell junctions, is required for leukocyte transmigration through endothelial cell monolayers. Polymorphonuclear leukocytes play an important role in ischemia-reperfusion injury. We sought to determine whether administering an anti-PECAM-1 antibody would prevent or attenuate ischemia-reperfusion injury in a rat cremaster muscle flap injury model. Eighteen male Sprague-Dawley rats were divided into three groups. Group I (control): Cremaster muscle island flaps were dissected for baseline measurements of eight indicators: numbers of rolling, sticking, and transmigrating neutrophils, numbers of rolling and sticking lymphocytes, number of perfused capillaries, endothelial edema, and vessel permeability. Group II: The prepared cremaster flap was subjected to 4 hours of ischemia and 24 hours of reperfusion. Group III: The muscle flap was subjected to ischemia and reperfusion as in group II, and anti-PECAM-1 antibodies (1 mg/kg) were injected subcutaneously 15 minutes before reperfusion. Blood vessels were observed in vivo under an intravital microscopy system. Microvascular permeability was made visible with injected fluorescein isothiocyanate-labeled albumin and evaluated with Kontron Elektronik computer software. The ischemia-reperfusion-alone group (group II) presented a 225-percent increase in the activation of sticking leukocytes (2.4 +/- 0.4 to 7.8 +/- 0.8, p < 0.05) (p < 0.01). This leukocyte activation was reduced by 83 percent following anti-PECAM-1 monoclonal antibody treatment (1.3 +/- 0.5 per 100 microm) (p < 0.01). At 24 hours, endothelial injury in group II was confirmed by a 4-fold increase in the number of transmigrating leukocytes into the interstitial space (7.6 +/- 1.2 per field versus 1.9 +/- 0.4 per field in controls). This phenomenon was reduced by 85 percent following anti-PECAM-1 monoclonal antibody treatment (1.1 +/- 0.2 per field) (p < 0.01). Analysis showed that the number of flowing capillaries was 67 percent lower in group II (6.8 +/- 0.3 to 2.2 +/- 0.7, p < 0.01). Anti-PECAM-1 antibody treatment caused a 2.5-fold increase in this number (5.6 +/- 0.5, p < 0.01). Microcirculatory permeability index showed a 180-percent increase in group II (p < 0.05) when compared with baseline values. This increased albumin leakage was effectively attenuated by antibody treatment (p < 0.05). Blocking the action of PECAM-1 in vivo by administering monoclonal antibodies significantly attenuated ischemia-reperfusion injury, presumably by inhibiting transendothelial migration of neutrophils and by increasing capillary perfusion at a muscle flap microcirculatory level.     

Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells

Composite-tissue (e.g., hand allograft) allotransplantation is currently limited by the need for immunosuppression to prevent graft rejection. Inducing a state of tolerance in the recipient could potentially eliminate the need for immunosuppression but requires reprogramming of the immunological repertoire of the recipient. Skin is the most antigenic tissue in the body and is consistently refractory to tolerance induction regimens using bone marrow transplantation alone. It was hypothesized that tolerance to skin allografts could be induced in rats by injecting epidermal cells with bone marrow cells during the first 24 hours of life of the recipients. Brown Norway rats (RT1n) served as donors for the epidermal cells, bone marrow cells, and skin grafts. Epidermal cells were injected intraperitoneally and bone marrow cells were injected intravenously into Lewis (RT1l) newborn recipient rats. In control groups, recipients received saline solution with no cells (group I, n = 12), bone marrow cells only (group II, n = 15), or epidermal cells only (group III, n = 15). In the experimental group (group IV, n = 18), recipients received epidermal and bone marrow cells simultaneously. Skin grafts were transplanted from Brown Norway (RT1n) rats to the Lewis (RT1l) rats 8 weeks after cell injections. Skin grafts survived an average of 8.5 days in group I (10 grafts), 9.2 days in group II (12 grafts), and 12 days in group III (14 grafts). Grafts survived 15.5 days (8 to 26 days) in group IV (15 grafts). The difference was statistically significant (p < 0.05). Hair growth was observed in some accepted grafts in group IV but never in the control groups. This is the first report of prolonged survival of skin allografts in a rat model after epidermal and bone marrow cell injections. Survival prolongation was achieved across a major immunological barrier, without irradiation, myeloablation, or immunosuppression. It is concluded that the presentation of skin-specific antigens generated a temporary state of tolerance to the skin in the recipients that could have delayed the rejection of skin allografts.     

Calcium-supplemented University of Wisconsin solution in long-term myocardial preservation

The purpose of this study was to assess the effects of the addition of calcium to University of Wisconsin solution in long-term myocardial perfusion. In a heterotopic heart transplantation model, performed in pigs, the donor heart was preserved for 24 hours by means of continuous perfusion in this solution, without (24hUW group) or with calcium, 2.4 mmol/L (24hUW+Ca). During this period, the oxygenation and pH of the solution were measured, as were the calcium and lactate concentrations and enzyme release. After two hours of reperfusion, samples were collected from both ventricles for the morphological study. In the control group, there were no signs that reperfusion had triggered the calcium paradox. The addition of this cation to the preservation solution improved the intercellular junction integrity but, at the same time, favored intracellular calcium overload. This is manifested by increased enzyme release during preservation (LDH: 242+/-95 vs 140+/-25; CK: 668+/-371 vs 299+/-83 (U/L). p<0.01 in both cases) and signs of ventricular contracture: hardness and stiffness were significantly more prominent than in the group without calcium supplementation. Moreover, in comparison with the control group, the structural morphology of 24hUW+Ca is characterized by the more prominent and extensive presence of contraction bands and disorganized actin structure. Thus, under the experimental conditions employed in this study, we consider the addition of calcium to Wisconsin solution to be unadvisable.     

Combined CXCR3/CCR5 blockade attenuates acute and chronic rejection

Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 +/- 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4(+)CD25(+) T cells expressing the Foxp3 gene. Depletion of CD25(+) cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 +/- 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25(+) regulatory T cell recruitment and control of T lymphocyte proliferation.     

Chronobiological serial sections for core temperature monitoring after transplantation of the murine pancreas

In order to study any early sign of rejection of pancreas transplantation, rhythmometry was carried out on female adult inbred Lewis rats. Animals previously kept in continuous light, more or less synchronized in frequency by cyclic human activities, were transferred to a regimen of light (L) and darkness (D) alternating at 12-h intervals in single cages at the room temperature of 24 +/- 1 degrees C, with food and water ad libitum. At this time under ether anesthesia, temperature transensors were implanted in healthy rats and rats rendered diabetic by the administration of streptozotocin. Some of the diabetic rats were left untreated; casual blood sampling showed gross hyperglycemia. Other rats were treated by pancreatic grafts from ethionine-prepared donors, either by isografts (in rats of the Lewis strain) or by allografts (of the pancreas from inbred Fischer rats transplanted to Lewis rats). Intraperitoneal temperature was telemetered at 10-min intervals for 3 weeks following transplantation. Urine volumes were determined from rats housed in metabolic cages. Data were analyzed rhythmometrically. Chronobiological serial sections and single cosinors served this purpose. Following sensor implantation and transfer to an LD 12:12 regimen, the adjustment of the thermal acrophase consistently near the middle of the daily dark span occurred within approximately 7 days in healthy rats and in streptozotocin-diabetic rats cured by isograft. Thermal acrophase adjustment was slower for animals rendered diabetic by streptozotocin and left untreated or for animals thus rendered diabetic which had rejected the pancreatic allograft (as documented by hyperglycemia in casually sampled blood). The eventual synchronization of the circadian temperature rhythm of allografted rats differed from one rat to the other and, for some allografted animals, from the consistent synchronization of the circadian rhythm in telemetered intraperitoneal temperature of diabetic and non-diabetic Lewis rats. The acrophase of the circadian rhythm in urine volume of healthy rats or of a rat with a pancreatic isograft (which cured a prior streptozotocin-induced diabetes) differed with statistical significance from those of rats with untreated diabetes, some in this state after the rejection of a pancreatic allograft. Both urine volume and core temperature are ready marker rhythms, not only for rats but also for human beings. Both variables can be self-monitored by the cooperation of instructed but not necessarily extensively educated patients. Temperature, in particular, can also be monitored with automatic devices and alterations of certain of its rhythm characteristics may signal changes preceding fever. The use of such admittedly unspecific yet eminently practical and possibly informative marker rhythmometry awaits clinical testing.     

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.