Model Of Allograft Rejection Research Articles

Cytomegalovirus infection enhances connective tissue growth factor mRNA expression in a rat model of chronic renal allograft rejection

Cytomegalovirus infection enhances connective tissue growth factor mRNA expression in a rat model of chronic renal allograft rejection

MRI and ultrasonographic detection of morphologic and hemodynamic changes in chronic renal allograft rejection in the rat.

The purpose of this study is to describe the sonographic, MRI, and histopathologic findings in a rat model of chronic renal allograft rejection. Allogeneic renal grafts (male DA kidney into male Lewis rat with unilateral nephrectomy, N = 27) and syngeneic renal grafts (male Lewis kidney into male Lewis rat, N = 19) were examined serially with ultrasound, MRI, and histology. Nonparametric Spearman rank correlation showed significance between the histologic score and the following parameters: the MRI score (r(s) = 0.91, P < 0.01, N = 46), the ultrasound score (r(s) = 0.9, P < 0.01, N = 46), the power Doppler score (r(s) = 0.86, P < 0.01, N = 46), and the MRI perfusion (r(s) = -0.80, P < 0.01, N = 45). Positive correlations were also found between the MRI volume estimations (graft r(s) = 0.49, P < 0.01, N = 46; native r(s) = 0.59, P < 0.01, N = 46), and the ultrasound volume estimations (graft r(s) = 0.39, P < 0.01, N = 45; native r(s) = 0.64, P < 0.01, N = 46) as well as with actual graft weight. This study shows that both MRI and ultrasound can provide complementary, accurate information compared to histology in regard to the alterations in anatomy and hemodynamic changes associated with chronic allograft nephropathy.

Induction of Vascular Adhesion Protein-1 during Liver Allograft Rejection and Concomitant Cytomegalovirus Infection in Rats

Vascular adhesion protein-1 (VAP-1) is an adhesion molecule controlling lymphocyte recirculation through high endothelial venules of the lymph nodes. It has also been shown to be induced and to mediate lymphocyte adhesion at sites of inflammation. We studied the expression of VAP-1 and two other inducible adhesion molecules ICAM-1 and VCAM-1 in our experimental model of rat liver allograft rejection and, in addition, the effect of concomitant rat cytomegalovirus (RCMV) infection on this expression. Expression of VAP-1, ICAM-1, and VCAM-1 was studied in rat liver allografts with or without RCMV infection, isografts, and normal rat liver. Immunoperoxidase technique and monoclonal antibodies including a novel anti-VAP-1 reagent were used. VAP-1 expression was induced by acute rejection in sinusoids, hepatocytes, and also in bile ducts, when compared to the isografts or normal liver, where only blood vessels were consistently positive. Sinusoidal and hepatocyte expression of VAP-1 was prolonged by the presence of RCMV. ICAM-1 and VCAM-1 expression was also induced by acute rejection. However, RCMV increased sinusoidal VCAM-1 expression compared to uninfected grafts. The present experimental study shows that VAP-1 is up-regulated in acute rejection of liver allografts, and that this up-regulation is prolonged by RCMV infection.

Anti-TCR Vbeta-specific DNA vaccination prolongs heart allograft survival in adult rats.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

Anti-TCR Vβ-specific DNA vaccination prolongs heart allograft survival in adult rats

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-β chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vβ13 and Vβ2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vβ, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vβ13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vβ families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vβ13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways

Background: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. MethodsLewis RT11 (LEW) rats sensitized by transplantation with Wistar-Furth RT1u (WF) skin grafts received WF cardiac allografts 7 days later—a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. ResultsControl animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6 ± 0.6 days (p < 0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1n cells was ineffective in this regard. Intrathymic injection of γ-irradiated donor cells marginally extended cardiac allograft survival to 3.0 ± 0.9 days (p < 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. ConclusionThe superiority of non-modified donor spleen cells over γ-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.

Transforming growth factor–β1 gene transfer ameliorates acute lung allograft rejection

Background: The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor–β1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. Methods: The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor–β1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. Results: On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor–β1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor–β1 sense construct compared with antisense controls (arterial oxygen tension = 411 ± 198 vs 103 ± 85 mm Hg, respectively; P = .002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 ± 0.5 vs 2.8 ± 0.6; P = .04; airway rejection scores: 1.3 ± 0.7 vs 2.3 ± 0.8, respectively; P = .02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. Conclusions: The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor–β1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology. (J Thorac Cardiovasc Surg 2000;119:913-20)

Effect of angiotensin-converting enzyme inhibition on growth factor mRNA in chronic renal allograft rejection in the rat

Despite considerable progress in immunosuppression, the incidence of chronic renal allograft rejection has not decreased. Recent studies have revealed that angiotensin-converting enzyme (ACE) inhibition ameliorates graft arteriosclerosis, glomerulosclerosis, and tubular atrophy. Moreover, it decreases systemic and glomerular capillary hydrostatic pressure in a rat kidney allograft model. We evaluated the effects of the ACE inhibitor enalapril on cytokine and growth factor expression in chronically rejecting rat kidney allografts. Kidneys of Fisher (F344) rats were orthotopically transplanted into Lewis (Lew) rats. To prevent acute rejection, cyclosporine A (1.5 mg/kg/day) was given to all recipients during the first 10 days after transplantation. Enalapril (60 mg/L) or vehicle was added to the drinking water 10 days after transplantation. Animals were harvested 20 weeks after transplantation for histologic and immunohistologic studies, as well as for evaluation of cytokine and growth factor mRNA by semiquantitative polymerase chain reaction. Controls developed severe signs of chronic rejection, such as glomerular and vascular lesions, associated with a large number of infiltrating leukocytes. Enalapril-treated animals developed less proteinuria and other signs of chronic rejection. The mRNA levels of transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor A and B chain (PDGF A and B), insulin-like growth factor-I (IGF-I), interleukin-1 (IL-1), and monocyte chemoattractant protein-1 (MCP-1) were significantly reduced in the enalapril group and were most pronounced for IL-1 and PDGF A. In addition, we found an increased level of renal angiotensinogen mRNA after treatment with enalapril. Treatment with enalapril attenuated the development of proteinuria, ameliorated morphological damage, decreased leukocyte infiltration, and prevented a rise in renal mRNA levels of growth factors and cytokines in kidney grafts in a rat model of chronic renal allograft rejection.

SDZ-RAD prevents manifestation of chronic rejection in rat renal allografts.

Chronic rejection remains the most frequent cause of renal graft loss over the long term. However, effective treatment of this process is not yet available. SDZ-RAD (40-O-[2-hydroxyethyl]-rapamycin) is a new, orally active rapamycin derivative with potent immunosuppressive activity. We have examined the effects of SDZ-RAD in a well-established model of chronic renal allograft rejection in rats. Kidneys of Fisher (F334) rats were orthotopically transplanted into bilaterally nephrectomized Lewis recipients. To suppress an initial episode of acute rejection, rats were briefly treated with low doses of cyclosporine for the first 10 days. Thereafter they received either SDZ-RAD (0.5 mg/kg(day) or vehicle. At 24 weeks, functional evaluations were performed, kidneys were harvested, and histological, immunohistological, and reverse transcription-polymerase chain reaction evaluations were performed. Animals treated with SDZ-RAD developed lower proteinuria and less glomerulosclerosis as compared with controls. Additionally SDZ-RAD reduced the infiltration of macrophages and lymphocytes and the expression of intercellular adhesion molecule-1, laminin, and fibronectin. Furthermore, we observed a reduced expression of growth factor mRNA (transforming growth factor-beta and platelet-derived growth factor-AA) in these animals. Our results demonstrated that SDZ-RAD effectively ameliorates chronic renal allograft rejection in rats, probably mediated by suppression of growth factors.

Antiviral and immunomodulatory effects of desferrioxamine in cytomegalovirus-infected rat liver allografts with rejection.

Cytomegalovirus (CMV) infection is associated with acute and chronic allograft rejection. We have recently shown that rat CMV increases portal inflammation and bile duct destruction in a model of rat liver allograft rejection. Desferrioxamine (DFO), an iron chelator and antioxidant, has recently been demonstrated to have antiviral as well as immunomodulatory effects in vitro. We therefore investigated whether DFO inhibits (a) CMV infection and (b) graft destruction in our rat model. One day after liver transplantation, PVG (RT1c) into BN(RT1n), the rats were infected with rat CMV (RCMV, Maastricht strain; 10(5) plaque-forming units i.p.). The effects of 100 mg/kg body weight and 200 mg/kg body weight DFO were examined. In the untreated group, the grafts were uniformly RCMV culture-positive. In the group receiving 200 mg/kg DFO, RCMV replication was effectively inhibited. Inflammatory response in the graft, and especially the number of macrophages, was significantly reduced by DFO. Portal inflammation and bile duct destruction were also significantly reduced. In the untreated group, the bile duct epithelial cells were found to be strongly positive for tumor necrosis factor-alpha and this expression was clearly decreased by DFO. In addition, DFO significantly inhibited vascular cell adhesion molecule-1 expression on sinusoidal endothelial cells. Our in vivo transplant study strongly supports the inhibitory effects of metal chelators on CMV infection and their possible usefulness in the treatment of CMV-induced pathogenic changes.

Feasibility of in vivo intravascular ultrasound tissue characterization in the detection of early vascular transplant rejection.

Unprocessed ultrasound radiofrequency (RF) signal analysis has been shown to distinguish different tissue structures more reliably than gray-scale interpretation of conventional ultrasound images. The objective of this study was to test the feasibility of in vivo intravascular ultrasound (IVUS) RF signal analysis in an animal model of allograft rejection. Six cynomolgus monkeys underwent transplantation of 3-cm aortic allograft segments distal to the renal arteries from immunologically mismatched donors. IVUS imaging with a 30-MHz system was performed 84 to 105 days after the operation. RF signals were acquired from cross sections of the recipient and the allograft aortas in real time with a digitizer at 500 MHz with 8-bit resolution. Sixty-five cross sections and 68 regions of interest (31 in host aorta and 37 in allograft) were analyzed in the adventitial layer with a total number of 8568 vectors processed. For each region of interest, a weighted-average attenuation was calculated on the basis of the attenuation and length for each individual vector. Histological examination was performed at every cross section imaged by IVUS. When the gray-scale images of conventional IVUS scored by an independent observer were compared, no distinction between adventitia of the native aorta and allograft was possible. Analysis of the average RF backscatter power also showed no significant difference (70.32+/-3.55 versus 70.72+/-3.38 dB). However, the average attenuation of allografts was significantly lower than that of the host aortas (2.64+/-1.38 versus 4.02+/-1.16 dB/mm, P<0.001). Histology demonstrated a marked adventitial inflammatory response in all allografts, with no inflammation observed in the host aortas. In vivo IVUS tissue characterization can be performed during routine imaging. In this model of transplant vasculopathy, RF attenuation measurements were more sensitive than visual or quantitative gray-scale analysis.

Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture.

The counter receptors intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are lymphocyte cell surface adhesion proteins the interaction of which can provide signals for T cell activation. This binding event is important in T cell function, migration, and general immune system regulation. The ability to inhibit this interaction with monoclonal antibodies has proved to be therapeutically useful for several allograft rejection and autoimmune disease models. Short peptides representing counter-receptor contact domains of LFA-1 and ICAM-1 were examined for their ability to inhibit T cell adhesion and T cell function. Peptides encompassing amino acids Q1-C21 and D26-K50 of ICAM-1, I237-I261 and G441-G466 of the LFA-1 alpha-subunit, and D134-Q159 of the LFA-1 beta-subunit inhibited LFA-1/ICAM-1-dependent adhesion in a phorbol-12,13-dibutyrate-induced model of tonsil T cell homotypic adhesion. This inhibition was specific to the peptide sequence and occurred without stimulation of T cell proliferation. The peptides also were effective in preventing T cell function using a one-way mixed lymphocyte reaction model for bone marrow transplantation. Our data suggest that these peptides or their derivatives may be useful as therapeutic modulators of LFA-1/ICAM-1 interaction during organ transplants.

Disruption of CD154:CD40 Blocks Generation of Allograft Immunity Without Affecting APC Activation

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-gamma, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154-/- mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.

Role of circulating lipid abnormalities in chronic renal allograft rejection

The evidence that circulating lipid abnormalities may play a role in the pathogenesis of chronic renal allograft rejection is tantalizing but circumstantial. In animal models of cardiac and aorta allograft rejection, lipogenic diets accelerate vascular injury, and treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors reduce vascular injury. Histological studies have demonstrated foam cells and apolipoprotein deposits in the intima of arteries from chronically rejecting human kidneys. Observational studies have found correlations between plasma lipid levels and both acute and chronic rejection. The association between lipid levels and acute rejection in cyclosporine A (CsA)-treated renal transplant recipients suggests the possibility that plasma lipids may influence the immunosuppressive effects of CsA by modulating the lipoprotein-free levels of CsA. If true, such an effect could also explain the result of recent controlled trials showing that HMG-CoA reductase inhibitors reduced graft coronary artery disease and that they prolonged survival in heart transplant recipients. In any case, the hypothesis that circulating lipid abnormalities contribute to chronic renal allograft rejection deserves further testing in well-designed, clinical trials.

Inhibition of endothelin-converting enzyme attenuates transplant vasculopathy and rejection in rat cardiac allografts.

Transplant vasculopathy in kidney and heart allografts is associated with marked elevation of endothelin-1 (ET-1), but a role for ET-1 in the pathogenesis of transplant vasculopathy and chronic rejection has not been established. We, therefore, tested whether inhibition of ET-1-converting enzyme by phosphoramidon (PA) would attenuate rejection in a rat model of chronic cardiac allograft rejection (Lewis [LEW] to F344). Donor LEW rats were pretreated 24 hr before transplantation with a bolus injection of vehicle (water) or PA. Twenty- four hour after transplantation, water or PA was continuously administered through an osmotic mini-pump. Plasma ET-1 levels in Fisher 344 (F344) recipients were 0.8+/-0.1 pg/ml in water-treated rats and 0.2+/-0.2 pg/ml (P<0.01) in PA-treated rats, demonstrating that the PA treatment protocol effectively lowered ET-1 biosynthesis. LEW cardiac allografts treated with water survived (i.e., palpable heart beat) for 16.0+/-0.5 days (n=6). Inhibition of ET-1 secretion by PA improved allograft survival to 28.8+/-3.3 days (P<0.01, n=8). An analysis of cardiac arteries demonstrated that PA treatment attenuated transplant vasculopathy. A morphometric scale of neointima formation (0-5) was 1.4+/-0.2 and 3.6+/-0.2 in PA- or water-treated rats, respectively (P<0.01). The percent of luminal occlusion, as measured by microscopic image analysis, was 19+/-6% in PA-treated animals and 38+/-6% (P<0.01) in animals treated with water. PA treatment also reduced infiltration of ED-1-positive monocytes/macrophages into the vascular neointima. We conclude that, even in the absence of concomitant immunosuppression, inhibition of ET-1 biosynthesis significantly attenuates transplant vasculopathy and improves survival of LEW to F344 cardiac allografts.

Immunomodulatory functions of low-molecular weight hyaluronate in an acute rat renal allograft rejection model.

Low molecular weight hyaluronate (LMW-HA) blocks interactions between T lymphocyte CD44 and hyaluronate (HA), a heteropolysaccharide that is expressed on the surface of endothelial cells and ubiquitously in the extracellular matrix. This study was undertaken to assess the ability of LMW-HA to modify the course of experimental acute renal allograft rejection. Lewis (LEW) rats were bilaterally nephrectomized and received an orthotopic, fully MHC-mismatched, Wistar-Furth (WF) kidney transplant. Animals received either no treatment, low doses of cyclosporin A (CsA) on days 0 to 5, LMW-HA on days 0 to 5, or CsA plus LMW-HA on days 0 to 5 after transplantation. With no treatment, CsA monotherapy, or HA monotherapy, animals rejected their allografts at a median of 15, 13, and 7.5 d, respectively (P = NS). In contrast, combined CsA plus LMW-HA therapy prevented acute rejection and significantly prolonged graft survival (P = 0.008) to a median of 49.0 d. CsA/LMW-HA-treated grafts also demonstrated better preservation of renal function at day 30 (serum creatinine level, 1.38+/-0.8 mg/dl), compared with surviving animals treated with CsA alone (2.9+/-0.55 mg/dl, P<0.05). Histologic graft analysis of CsA/LMW-HA-treated animals at day 7 after transplantation showed minimal rejection and leukocyte infiltration, compared with all other groups. Intragraft gene expression analysis, using semiquantitative reverse transcription-PCR, at the same time point showed reductions of CD4, CD8, and interferon-gamma transcript levels in the combined-treatment group. This is the first study demonstrating the immunomodulatory functions of LMW-HA in vivo in the setting of organ transplantation. Defining the exact mechanisms that underlie this immunomodulation may provide the rationale to develop novel strategies for use in clinical transplantation.

In vivo migration of lymphocytes in chronically rejecting rat kidney allografts.

Histological analyses have identified lymphocytes and macrophages as the predominant leukocyte populations that infiltrate organs undergoing chronic rejection. In order to define the time frame of this infiltration, we investigated the in vivo migration pattern of lymphocytes in a well-established rat model of chronic kidney allograft rejection. F344 kidneys were orthotopically transplanted into bilaterally nephrectomized Lewis rats. Recipients were treated with cyclosporin A (1.5 mg/kg/per day) for the first 10 days. After anti-CD18 or vehicle pretreatment, peripheral blood lymphocytes obtained from naive Lewis rats and labeled with 3H-uridine were injected into transplanted rats 12 and 16 weeks after transplantation. Organs were harvested 4, 8, and 12 h thereafter. After 12 weeks, proteinuria developed, accompanied by all signs of chronic rejection including glomerular sclerosis. Labeled lymphocytes rapidly infiltrated grafted kidneys 4 h after injection. Even more lymphocytes had accumulated in the grafts 12 h after injection. After 16 weeks, few lymphocytes had emigrated into the graft at 4 h, while infiltration was most pronounced by 12 h. Pretreatment with anti-CD18 inhibited the influx of lymphocytes. There was no difference between the patterns of lymphocytes derived from naive and transplanted rats. Our results emphasize the importance of endothelial cells in chronically rejecting kidneys for the control of leukocyte influx. Beta2-integrins may play a central role in determining the transendothelial migration during this process.

TNP-470, AN ANGIOGENESIS INHIBITOR, INTERRUPTS THE DEVELOPMENT OF GRAFT ARTERIOSCLEROSIS IN A RAT CARDIAC MODEL OF CHRONIC ALLOGRAFT REJECTION

884 We have previously found neovascularization within the aortic intimal lesions of graft arteriosclerosis. However the function of angiogenesis in lesion formation is not known. We speculate that angiogenesis facilitates the development of intimal proliferation by facilitating mononuclear cell recruitment and provision of growth factors In this study we assessed the effects of a well established angiogenesis inhibitor (TNP-470) on graft survival and development of graft arteriosclerosis in the Lew into F344 rat heterotopic cardiac transplant model. Control F344 recipients of Lew donor cardiac grafts are treated with low dose cyclosporin A for 30 days. Allografts surviving beyond 100 days exhibit significant graft arteriosclerosis. The following groups were established: Group 1 (n=6): CsA (5mg/kg/d s.c.) for 30 days post transplant (control group); Group 2 (n=10): CsA (5mg/kg/d s.c.) and TNP-470 (20mg/Kg s.c 3x/wk) for 30 days post transplant ("prevention" protocol); and Group 3 (n=6): CsA (5mg/kg/d s.c.) for 30 days post transplant followed by TNP-470 (20mg/kg s.c 3x/wk) until cardiac allografts were harvested ("interruption" protocol). Allografts were harvested following cessation of palpable heart beat or at day 120. Allografts were fixed in formalin, sectioned and stained with Verhoeffs elastin stain for arteriosclerosis scoring. Arteriosclerosis scoring was performed using a standard grading system (score 0 to 5). We find that prolonged administration of TNP-470 (group 3) significantly reduced graft arteriosclerosis compared to the control group 1 (mean score 0.96±0.14 vs 2.16±0.58; p<0.05). In contrast, an early post-operative 30 day course of TNP-470 (group 2) did not inhibit graft arteriosclerosis (mean score 2.3±0.33). We further find that there was a trend to improved graft survival in TNP-470 treated animals. We propose that the ability of TNP-470 to interrupt the development of graft arteriosclerosis in this model is a direct consequence of inhibiting neovascularization within the vessel intima or alternatively TNP-470 could have a direct immunosuppressive effect. Indeed, we find that TNP-470 is a potent inhibitor of the rat mixed lymphocyte reaction (IC50 0.1-1ug/ml). Furthermore, in vivo we find that TNP-470 prolongs graft survival in the WF to LEW model of acute cardiac allograft rejection. Animals in the TNP-470 treatment group (20mg/kg s.c 3x/wk) had a mean graft survival of 14 days vs 7 days in controls (p<0.01). When Grafts were routinely harvested on day 5 control grafts showed significantly worse tissue necrosis, hemmorrhage and infiltrates than TNP-470 treated allografts. Furthermore, splenocytes isolated from TNP-470 treated animals exhibit significantly reduced cytotoxic activity than splenocytes from controls. TNP-470 attenuates graft arteriosclerosis in a rat model of chronic cadriac allograft rejection. We speculate that TNP-470 functions through combined anti-angiogeneic and immunosuppressive properties.

LONG-TERM SURVIVAL OF CARDIAC ALLOGRAFTS INDUCED BY COSTIMULATORY BLOCKADE MAY REQUIRE INDIRECT RECOGNITION

45 Previous studies have indicated that both the direct and indirect pathways of allorecognition can contribute to allograft rejection in the absence of immunosuppression. The present studies examined the effectiveness of T cell costimulatory blockade when only the direct or indirect pathway was operative in a mouse model of acute vascularized cardiac allograft rejection. To study the pathways in vivo, we used genetically altered mouse strains in the following manner: 1. B6 MHC class II-deficient mice (II-) were used as donors of cardiac grafts to normal B10.D2 recipients to eliminate the direct CD4+ T cell response. 2. II-4+ mice, which are MHC class II-deficient mice expressing a MHC class II transgene only on thymic epithelium, were used as recipients of normal B10.D2 cardiac grafts. These mice have normal numbers of peripheral CD4+ T cells but lack expression of MHC class II on their APCs and, therefore, cannot mount an indirect CD4+ T cell response. The following costimulatory blockade regimens were used: Murine CTLA4Ig (250μg ×1 i.p.) on day 2 or MR1 anti-CD40L antibody (250μg ×1 i.p.) on day 0 posttransplant. (Table)TableThese results support previous findings with skin transplants that either pathway of allorecognition can initiate rejection in the absence of immunosuppression. They also indicate that costimulatory blockade prolongs allograft survival more effectively in recipients that can mount only an indirect response, and in recipients with both pathways, than in recipients that can mount only a direct response. Thus, stimulation through the indirect pathway seems to be important for costimulatory blockade to be effective. (NIH-AI-38397 and 34965)

Immunomodulatory functions of hyaluronate in the LEW-to-F344 model of chronic cardiac allograft rejection.

CD44 is an important leukocyte cell surface glycoprotein with diverse functions including cell adhesion, homing, migration, and activation. Because administration of the principal ligand of CD44, hyaluronate (HA), in soluble form, can inhibit CD44-HA interaction, we tested the effects of HA in vivo in an established model of chronic allograft rejection. Control F344 recipients of LEW hearts received either no treatment or low-dose cyclosporine (CsA) for 30 days from the day of transplantation. Experimental animals received 30 days of CsA in combination with 30 or 90 days of low molecular weight HA (LMW-HA). CsA therapy alone resulted in approximately 40% long-term (>100 days) graft survival, whereas CsA + LMW-HA (30-day and 90-day protocols) significantly increased long-term graft survival to 60% and 92%, respectively. Light microscopy and immunohistology of CsA-treated and CsA + LMW-HA-treated grafts harvested at day 30 after transplantation demonstrated that LMW-HA + CsA therapy decreased mononuclear cell infiltration and afforded better preservation of myocardial architecture. In addition, LMW-HA + CsA-treated grafts exhibited decreased expression of interferon-gamma and the growth factors transforming growth factor-beta, platelet-derived growth factor, and fibrogenic growth factor-beta. Long-term surviving grafts were assessed for arteriosclerosis, the sine qua non of chronic rejection in this model. Using a standardized scoring system, significantly less arteriosclerosis was seen in grafts from LMW-HA + CsA-treated animals at 120 days after transplantation compared with CsA alone-treated grafts. This difference was not significant, however, in grafts harvested at >150 days. This is the first report indicating that CD44-HA interactions play an important role in chronic allograft rejection.