Rat Cardiac Transplant Model Research Articles

Cyclosporine does not enhance the development of chronic rejection. Experimental study in a rat cardiac transplant model

Background: Among a number of different factors Cyclosporine A (CsA) is linked with the development of chronic rejection after transplantation. The objective of this study was to investigate the influence of different CsA regimens on the development of chronic rejection after heart transplantation in rats.

New approach in the therapy of chronic rejection? ACE- and AT1-blocker reduce the development of chronic rejection after cardiac transplantation in a rat model

Background: Angiotensin II is one of the most potent mitogens of smooth muscle cell proliferation and plays a central role in the development of accelerated coronary artery disease (ACAD), which remains a serious consequence after heart transplantation and limits long-term survival. We investigated the effect of an angiotensin-II blocker, Losartan (angiotensin II Type 1 [AT 1]-blocker), and an angiotensin-converting enzyme (ACE) inhibitor, Enalapril, on experimental ACAD in a rat cardiac transplant model (Fisher to Lewis). Methods After grafting, recipients were treated with 10 mg/kg/day per os Losartan or 40 mg/kg/day per os Enalapril. Two groups of animals received additional pre-treatment with Losartan or Enalapril 7 days before transplantation. All study groups, including the control group, received immunosuppression with cyclosporine (3 mg/kg/day sub-cutaneously). We assessed the extent of ACAD of large and small arteries 80 days after grafting using digitizing morphometry. Results We observed significant reduction of neointimal proliferation in small arteries in Losartan pre- and post-treated and in Enalapril pre-treated recipients compared with the cyclosporine-treated group ( p < 0.05). In epicardial arteries, Enalapril pre- and post-treatment as well as Losartan post-treatment significantly reduced neointimal formation compared with the control group. Reduction of neointima by Enalapril post-treatment in small arteries and Losartan pre-treatment in large arteries trended toward but failed statistical significance. Conclusions Our results suggest the important role of the renin–angiotensin system in neointimal proliferation, which can be reduced equally with ACE inhibitors or the angiotensin-II blocker. Therefore AT 1 blockade with Losartan is a useful therapeutic strategy for inhibition of ACAD after cardiac transplantation.

Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts.

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.

Perillyl Alcohol Inhibits TCR-Mediated [Ca2+]i Signaling, Alters Cell Shape and Motility, and Induces Apoptosis in T Lymphocytes

Perillyl alcohol (POH) inhibits isoprenylation and has shown anticancer and chemopreventive properties in rodent models. The mechanism that underlies the anticancer activity of POH and other isoprenylation inhibitors is unknown but has been postulated to involve decreased levels of isoprenylated Ras and Ras-related proteins. Previously we demonstrated that POH effectively inhibits human T cell proliferation in vitro and can prevent acute and chronic rejection in a rat cardiac transplant model. In this report, we investigate the effects of POH on T lymphocytes at the single-cell level. POH disrupts the polarized shape and motility of antigen-specific murine 1E5 T cells. Using an optical trap to position anti-CD3-coated beads in contact with 1E5 T cells, we demonstrate that POH inhibits their TCR-mediated calcium response. Furthermore, we show that POH preferentially induces apoptosis in PHA-activated human T cells as well as in 1E5 T cells.

Sulfasalazine inhibits reperfusion injury and prolongs allograft survival in rat cardiac transplants

Introduction: Reperfusion injury is an inflammatory cell-mediated response that causes tissue damage immediately following transplantation, and has been implicated in the development of acute and chronic rejection. NF-κB is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1 following reperfusion. We hypothesized that treatment with sulfasalazine, a potent inhibitor of NF-κB, would decrease adhesion molecule expression, decrease reperfusion injury, and prolong allograft survival in rat cardiac transplants. Methods Heterotopic rat heart transplants were performed. Donor allografts were treated with saline, sulfasalazine (SSA), or lipopolysaccharide (LPS), a potent inducer of NF-κB activity. Reperfusion injury was assessed with cardiac edema (percent wet weight), neutrophil infiltration (MPO activity), and histologic damage (contraction band necrosis). Immunohistochemistry was performed to analyze protein expression. Acute rejection was determined by daily palpation. Results Treatment with a single 100 mg/kg intraperitoneal injection of sulfasalazine decreased reperfusion injury compared to saline controls (MPO activity, saline: 2.1 ± 0.3, SSA: 1.2 ± 0.31, P < 0.005; % wet weight, saline 77.6 ± 1.1%; SSA 75.8 ± 1.0%, P < 0.005; contraction band necrosis, saline: 13.1 ± 2.5%, SSA: 6.1 ± 3.4%, P < 0.001). LPS administration increased all parameters of reperfusion injury. Treatment with sulfasalazine prior to LPS also decreased reperfusion injury compared to LPS and saline groups. Sulfasalazine treatment decreased ICAM-1 and VCAM-1 protein expression. Administration of 500 mg/kg sulfasalazine increased graft survival to 15.4 ± 1.8 days compared to saline (6.8 ± 1.4 days, P < 0.005). Conclusion Treatment with sulfasalazine is an effective method to decrease reperfusion injury and prolong allograft survival in a rat cardiac transplantation model.

Induction of T-independent xenotolerance in a semi-discordant hamster-to-presensitized, nude rat model.

We have previously demonstrated that in a concordant hamster-to-nude rat cardiac transplant model, T-independent specific B-lymphocyte and natural killer (NK)-cell tolerance could be induced, leading to long-term xenograft (Xg) survival. Here, we investigated whether the same could be achieved in a clinically more relevant semi-discordant model involving hamster hearts transplanted into pre-sensitized, nude rats. Sensitized, nude rats with high titers of anti-hamster immunoglobulin (Ig)M xenoantibodies (XAbs) were prepared by transplanting a first hamster heart without treatment. One week after rejection, a complete tolerizing regimen was given, including the following: a) an i.v. injection of hamster heart antigens; b) a 4-week administration of malononitriloamide; and c) a single injection of an anti-NK antiserum. Two weeks later, a second hamster heart was grafted. The isotype and level of XAb were examined by fluorescence-activated cell sorting. NK cytotoxicity was evaluated by a standard 4-hr 51Cr release assay. Hamster heart Xgs were examined by conventional histologic and immunohistochemical analysis. Untreated, presensitized, nude rats developing high titers of IgM XAb underwent hyperacute rejection within 4 hr (n=4) after transplantation of the second hamster heart. Immunohistochemical analysis showed intensive staining for IgM and C3 along the vascular endothelia in the rejected Xgs. In contrast, presensitized, nude rats receiving the complete tolerizing regimen had a rapid decrease in anti-hamster IgM XAb. The second hamster hearts were not rejected and showed long-term survival even after withdrawal of malononitriloamide (n=6). Moreover, tolerant rats showed specific B-lymphocyte tolerance and a specific continuous absence of anti-hamster NK-cell reactivity. T-independent B-lymphocyte and NK-cell xenotolerance can also be achieved in recipients with pre-existing IgM XAb.

The effects of leflunomide and cyclosporin A on rejection of cardiac allografts in the rat.

Leflunomide is a new low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate-dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and cyclosporin A (CsA) are different, we postulated a synergistic effect of the two drugs and tested graft survival following leflunomide administration alone or in combination with CsA in a rat cardiac transplantation model. Low- and high-responder rat strain combinations were used in parallel and the experiments were performed both with and without challenge with Linomide, an immunomodulator which promotes graft rejection in this model. In the low-responder rat strain combination (Piebald Virol Glaxo graft to Dark Agouti recipient; PVG to DA), graft survival appeared to be a dichotomous variable, being characterized by tolerance or early rejection. Leflunomide (10 or 5 mg/kg) given for 10 days induced tolerance and CsA did likewise; the addition of Linomide abolished the immunosuppressive effect of leflunomide but not that of CsA. In the high-responder combination (DA to PVG), no tolerance was seen and graft survival was moderately prolonged both after leflunomide and after CsA treatment; the addition of Linomide to CsA or to leflunomide (5 mg/kg) abolished the immunosuppressive effect of the drugs. However, when CsA-Linomide or leflunomide-Linomide were supplemented with the second immunosuppressive drug, leflunomide or CsA respectively, graft survival was significantly prolonged (P < 0.001 in both cases). This suggests leflunomide and CsA have additive potential.

Effects of pravastatin on chronic rejection of rat cardiac allografts.

Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, inhibits coronary transplant vasculopathy in the clinical setting. To further delineate the immune modulatory effect of this agent, it was tested in a rat cardiac transplant model of chronic rejection. Rat heterotopic abdominal cardiac transplants were performed using a Lewis to Fischer 344 combination. Fischer 344 recipients received a brief course of cyclosporine to decrease the incidence of acute rejection. Experimental groups were treated with either high-dose (10 mg/kg) or low-dose (5 mg/kg) pravastatin for 120 days, while a control group did not receive pravastatin. The effect of pravastatin on chronic rejection of cardiac allografts was analyzed by histology, and the expression of laminin, fibronectin, macrophages, and T cells was assessed by immunohistochemistry. Coronary transplant vasculopathy was inhibited in both groups of pravastatin-treated animals, as compared with controls. Immunohistochemistry revealed that control animals had degraded laminin and fibronectin which paralleled the degree of tissue necrosis. In contrast, pravastatin-treated animals had modest amounts of extracellular matrix proteins retained within intermyocytes and endothelium, a pattern seen in native cardiac tissue. The pravastatin-treated groups also had fewer graft-infiltrating macrophages, specifically within the arterial intima and perivascular areas. Progressive chronic vascular rejection, a leading cause of allograft failure, can be inhibited by pravastatin in a well-defined rat cardiac transplant model. Pravastatin appears to inhibit the synthesis and subsequent degradation of extracellular matrix proteins and block the infiltration of macrophages to the graft, which emphasizes that this inflammatory cell plays a major role in the pathogenesis of transplant chronic rejection.

Mycophenolate mofetil, azathioprine and cyclophosphamide enhanced efficacy combined with cyclosporine in rat cardiac transplantation.

Anti-proliferative drugs have been used for immunosuppression since the introduction of clinical transplantation. Most transplant centres include azathioprine (Aza) and cyclosporine (CyA) in their standard regimens, despite several controlled studies having failed to confirm the benefit of this combination. Aza is still the most commonly used anti-proliferative drug, although no major differences in immunosuppressive or toxic effects have been shown between Aza and cyclophosphamide (Cph). Cph as an adjunct to CyA has never been tested in a randomized study. Recently, mycophenolate mofetil (MMF) has been developed as the most selective inhibitor of T- and B-cell proliferation and promoted as an adjunct to CyA treatment. In the present study, the additive or synergistic effects of these three anti-proliferative agents in combined treatment with CyA have been investigated using a rat cardiac transplantation model in which the immunomodulator linomide (Lin) was included as a potentiator of rejection. As single drug treatment, CyA, Cph and MMF, but not Aza, exerted a beneficial effect on graft survival. This prolongation of graft survival was abrogated when any one drug was administered together with Lin. The addition of MMF, Aza or Cph to CyA plus Lin treatment improved the graft survival significantly, thus demonstrating each of the anti-proliferative drugs to exert additive or synergistic effects in conjunction with cyclosporine. MMF seemed to be the most effective and least toxic of the drugs tested.

Effects of vesnarinone, a novel orally active inotropic agent with an immunosuppressive action, on experimental cardiac transplantation in rats.

Vesnarinone (VES) has been used for treatment of patients with congestive heart failure. In addition to inotropic effects, it seems to have immunosuppressive action. We tested the hypothesis that VES suppresses graft rejection, inotropic dysfunction caused by early rejection, and chronic coronary obstruction in a heterotopic rat cardiac transplantation model. (1) To study acute rejection, hearts from Lewis-Brown Norway (LBN) rats were transplanted into Lewis rats, which were treated with or without VES (50 or 100 mg/kg/day orally). (2) In a functional study, LBN hearts with or without VES (100 mg/kg/ day) were isolated and perfused on day 3 after transplantation to assess inotropic response to isoproterenol (3 x 10(-8) M). (3) To study chronic rejection, Lewis hearts were transplanted into Fisher 344 rats, which were treated with or without VES (50 mg/kg/day) for 90 days. Coronary obstructive disease was assessed by morphometric analysis. There were five to six animals in each group. (1) VES (100 mg/kg/day) prolonged LBN heart survival (11.7 +/- 0.7 vs. 9.6 +/- 0.7 days in control; P < 0.05). (2) Left ventricular developed pressure was depressed in transplanted hearts regardless of VES treatment (84 +/- 12, 90 +/- 8 vs. 144 +/- 16 mmHg in untransplanted hearts; P < 0.01). The developed pressure after administration of isoproterenol in VES-treated hearts (184 +/- 20 mmHg) was higher than transplanted hearts without VES (118 +/- 16 mmHg; P < 0.05), and similar to untransplanted hearts (203 +/- 27 mmHg; P = NS). (3) Transplanted hearts treated with or without VES showed similar grades of rejection (2.0 +/- 0.3 vs. 2.6 +/- 0.2; P = NS), intimal area (6,996 +/- 3,186 vs. 13,441 +/- 5,165 microns2; NS), and coronary luminal obstruction (45 +/- 16% vs. 67 +/- 14%; NS). VES produces mild prolongation in survival of rat heart grafts, but has no significant effect on chronic graft atherosclerosis. VES preserves the positive inotropic effects of isoproterenol that are otherwise deteriorated by early acute rejection.

An examination of tissue chimerism in the ACI to Lewis rat cardiac transplant model

While the existence of chimeric cells in host tissue following organ transplantation is well documented, its distribution, temporal evolution and relationship to allograft survival is less clear. To explore this phenomenon, Lewis recipients of ACI cardiac allografts representing a wide range of immunosuppressive protocols and graft survival times were examined for the presence of chimerism using a sensitive polymerase chain reaction assay. Four groups of animals were examined: untransplanted animals receiving donor specific transfusion (DST)/cyclosporine A (CsA); allograft recipients with no treatment; recipients treated with DST/CsA/supplementary immunosuppression with rejection at 21–183 days; and recipients sacrificed with functioning allografts, treated with DST/CsA/supplementary immunosuppression and surviving >200 days. To elucidate variations in the tissue distribution of chimeric cells, bone marrow, skin, liver, spleen, and thymus were examined in each animal. Untransplanted animals receiving DST/CsA displayed no evidence of chimerism. In animals receiving a cardiac allograft but no treatment, there was extensive evidence of chimerism in four of five animals. Chimerism was also detected in seven of nine animals with intermediate graft survival at the time of rejection. In animals with long-term graft survival, only four of eight displayed chimerism. These results suggest that, without immunosuppression, early chimerism does not lead to prolonged graft survival and that, even when graft survival is moderately prolonged, these cells are not sufficient to prevent rejection. In conclusion, chimerism appears to be a common phenomenon following transplantation, is not a result of DST, and may not be necessary for maintenance of long-term graft survival.

Thalidomide prolonged graft survival in a rat cardiac transplant model but had no inhibitory effect on lymphocyte function in vitro

The effects of thalidomide on in vitro interleukin 2 (IL-2) production and thymidine uptake by human peripheral blood lymphocytes or rat splenocytes were investigated. Phytohaemagglutinin-stimulated human lymphocytes were incubated in the presence of thalidomide added at culture initiation. No immunosuppressive effect of thalidomide was observed in these experiments. Primary human mixed lymphocyte cultures treated with thalidomide for 6 days were also unaffected. A microsomal rabbit liver homogenate was prepared for metabolizing thalidomide. Stimulated lymphocytes secreted significantly more IL-2 in the presence of microsomal-treated thalidomide than did controls. The effect of thalidomide was then studied either as single therapy or in combination with cyclosporin A (CyA) in a rat allograft cardiac transplantation model. In addition, T cell subsets were analysed by flow cytometry in untransplanted rats treated with thalidomide. Treatment was given as induction therapy from the day of transplantation until day 9. Graft survival in rats treated with thalidomide was significantly prolonged compared to the untreated group. No difference in graft survival was detected between rats treated with thalidomide or CyA only. Graft survival was found to be slightly prolonged in rats given thalidomide and CyA in combination compared to rats treated with CyA alone. In untransplanted rats given thalidomide a decrease of CD4 positive T cells was detected on days 3 and 5. The T helper/cytotoxic-suppressor cell ratio was significantly diminished but, after 1 week of treatment, values for T cell subsets had almost returned to baseline levels. No inhibitory effect was obtained when phytohaemagglutinin-stimulated rat splenocytes were cultured with metabolized thalidomide. In summary, the ability of thalidomide to improve allograft survival in a solid organ transplant model was verified. The occurrence of thalidomide-induced changes in T cell subset ratios was demonstrated. In in vitro studies, however, there was no decrease but an increase in IL-2 production, and no change in thymidine uptake. The mechanism responsible for the immunosuppressive effect of thalidomide remains to be elucidated.

Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection.

NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.

Corticosteroids inhibit expression of inducible nitric oxide synthase during acute cardiac allograft rejection.

We have recently demonstrated that (1) nitric oxide (NO) is produced during experimental cardiac allograft rejection by the expression of inducible nitric oxide synthase (iNOS) in the rejecting organ and that (2) inhibition of NO production by iNOS attenuated acute rejection. The present study examined the interaction of corticosteroids, iNOS gene expression, and iNOS enzyme activity in a rat cardiac transplant model. Increased NO production in rejecting allografts was demonstrated by elevated serum nitrite/nitrate levels (67 +/- 5 versus 18 +/- 2 microM for isografts; P < 0.001) that were significantly reduced by pulse therapy with dexamethasone for 2 days prior to animal sacrifice or continuous dexamethasone treatment (34 +/- 2 and 19 +/- 4 microM, respectively; P < 0.001 versus untreated allografts). Increased iNOS enzyme activity was demonstrated in the allograft heart (5.11 +/- 1.00 versus 0.3 +/- 0.05 pmol L-citrulline.mg protein-1.min-1 for isografts) and was significantly reduced with dexamethasone treatment (1.13 +/- 0.47 for 2-day pulse-treated allografts and 0.02 +/- 0.01 for continuously treated allografts). Increased allograft iNOS enzyme activity resulted from induction of iNOS mRNA expression, which was more than 99% inhibited by dexamethasone treatment. Dexamethasone pulse therapy reduced but did not eliminate the histological changes of acute rejection. Thus corticosteroid treatment results in inhibition of iNOS expression during allograft rejection. These results further demonstrate that iNOS expression during acute rejection is immune-mediated and suggest that the immunosuppressive actions of corticosteroids in the treatment of acute rejection may include inhibition of iNOS expression.

NUTRITIONAL IMMUNOMODULATION ENHANCES CARDIAC ALLOGRAFT SURVIVAL IN RATS TREATED WITH DONOR-SPECIFIC TRANSFUSION AND CYCLOSPORINE

The aim of this study was to assess the efficacy of an enteral diet fortified with arginine, RNA, and fish oil (Impact), alone and in combination with cyclosporine (CsA) and donor-specific transfusion (DST) on allograft survival in the ACI:Lewis rat cardiac transplant model. Animals were fed ad libitum with either standard rat chow or Impact diet. Six groups were studied; these consisted of untreated recipients fed either standard diet or Impact diet; recipients treated with CsA 10 mg/kg on the day prior to engraftment (day-1) followed by 2.5 mg/kg/d, day 0-->day+13 and fed with either standard diet or Impact; and animals given a DST (1 ml) on day-1, CsA as described previously and fed either standard diet or Impact. Untreated animals standard diet (group 1, n = 8) rejected their allografts at 7.0 +/- 0.0 days, while those fed Impact (group 2, n = 9) had graft survival of 12.8 +/- 2.1 days, (P = .01 versus group 1). Animals treated with CsA alone and standard diet (group 3, n = 9) rejected at 30.3 +/- 4.8 days, while the combination of CsA and Impact diet (group 4, n = 8) rejected at 33.0 +/- 9.5 days--minimally improved survival compared with group 3. Animals treated with DST/CsA and standard diet (group 5, n = 7) rejected at 72.1 +/- 6.8 days, while the substitution of Impact for standard diet (group 6, n = 8) led to a significant graft prolongation to 275 +/- 53 days, n = 8 (P < .015 vs. groups 1-5). These data suggest that Impact diet alone can have potent immunomodulatory properties but may require the addition of DST/CsA to realize its potential. These findings underscore the potential of dietary immunomodulatory therapy to prevent rejection and promote tolerance to solid organ allografts.

Upregulation and Modulation of Inducible Nitric Oxide Synthase in Rat Cardiac Allografts With Chronic Rejection and Transplant Arteriosclerosis

The Lewis-F344 rat cardiac transplantation model produces cardiac allografts with chronic rejection characterized by arteriosclerotic lesions composed of macrophages and smooth muscle cells. Modulation of the inflammatory response with a diet deficient in essential fatty acids protects against the development of intimal thickening. Little is known about the components of the inflammatory response mediating this process. The cytokine-inducible isoform of nitric oxide synthase (iNOS) regulates the high-output nitric oxide pathway that confers activation properties to macrophages and regulates vasomotion, monocyte adherence, and smooth muscle cell proliferation in the vasculature. The purpose of the present study was to determine whether the iNOS pathway was upregulated during the course of chronic cardiac rejection. We studied iNOS mRNA and protein expression patterns in a series of Lewis-F344 cardiac allografts with early and late chronic rejection and after modulation of the inflammatory response (in an effort to attenuate arteriosclerosis). Relative gene transcript levels were measured with a 32P-dCTP reverse-transcriptase polymerase chain reaction assay designed to amplify iNOS mRNA. The distribution of the iNOS gene product was examined by immunocytochemistry with a polyclonal antibody against iNOS. NOS transcript levels increased significantly in cardiac allografts (days 7, 14, 28, and 75) compared with paired host hearts (exposed to the same circulation) and syngrafts (P < .003). Immunostaining localized the iNOS antigen within subpopulations of mononuclear inflammatory cells in cardiac allografts--presumably, activated macrophages. The number of iNOS-positive mononuclear cells was 25-fold higher in cardiac allografts compared with paired host hearts and syngrafts (P < .009). In cardiac allografts of 75 days or older, there also was striking iNOS staining within some medial and intimal smooth muscle cells in various vessels. Modulation of the inflammatory response (with a diet deficient in essential fatty acids) produced significant decreases in the intimal thickening score and in the percentage of diseased vessels in 28-day cardiac allografts compared with allografts from rats fed a control diet. There was a correlate decrease in iNOS transcript levels and in the number of iNOS-positive mononuclear cells in the 28-day cardiac allografts from rats fed the essential fatty acid-deficient diet. The early and persistent upregulation of iNOS in chronic cardiac rejection and the coincident reduction in arteriosclerosis and downregulation of iNOS suggest that this inducible regulator may contribute to the inflammatory response mediating transplant arteriosclerosis.

Enhancement of the immunosuppressive effect of cyclosporin A by ciprofloxacin in a rat cardiac allograft transplantation model

Abstract Ciprofloxacin hyperinduces interleukin-2 production in stimulated human and mouse lymphocytes. In this study, an enhanced and prolonged interleukin-2 response was also detected in polyclonally stimulated rat splenocytes in the presence of ciprofloxacin (5–80 μ/ml) compared to control cells without any antibiotic. Ciprofloxacin was able to counteract the immunosuppressive effect of 10 ng/ml cyclosporin A (CyA) but did not interfere with higher CyA concentrations. In parallel, ciprofloxacin did not influence thymidine uptake in mixed lymphocyte reactions in the presence of CyA. To obtain an in vivo application of these findings, graft survival was studied by performing rat cardiac allograft transplantations in the presence or absence of CyA. Brown Norway rats served as donors and Wistar Furth rats as recipients. Ciprofloxacin was injected intraperitoneally either at a high-dose regimen (240 mg/kg per 24 h) into rats every 8th h starting 1 day before transplantation until day 21 or graft loss, or it was injected at a low and clinically relevant dose regimen (45 mg/kg per 24 h) until day 9. CyA was administered orally (10 mg/kg per 24 h) from day 1 through day 9. Ciprofloxacin given alone at a high-dose regimen resulted in a median graft survival of 14.8 days, which was significantly longer than graft survival in rats without treatment (median 8.0 days). A low-dose regimen of ciprofloxacin alone did not affect graft survival. Ciprofloxacin at a highdose regimen combined with CyA prolonged graft survival to a median of 24.0 days compared to 20.5 days with CyA alone. Ciprofloxacin administered in the drinking water (200 mg/kg per 24 h) until day 9 in addition to CyA did not affect graft survival. However, when the same dose regimen was used in experiments with PVG rats as donors and Wistar/Kyoto as recipients, graft survival was significantly prolonged to a median of 45 days. Ciprofloxacin, given orally without the addition of CyA, did not influence graft survival in either of the two strain combinations. Thus, our data show that ciprofloxacin has no negative impact on heart graft survival rats. It remains to be clarified whether ciprofloxacin influences graft survival in humans.

Enhancement of the immunosuppressive effect of cyclosporin A by ciprofloxacin in a rat cardiac allograft transplantation model.

Ciprofloxacin hyperinduces interleukin-2 production in stimulated human and mouse lymphocytes. In this study, an enhanced and prolonged interleukin-2 response was also detected in polyclonally stimulated rat splenocytes in the presence of ciprofloxacin (5-80 micrograms/ml) compared to control cells without any antibiotic. Ciprofloxacin was able to counteract the immunosuppressive effect of 10 ng/ml cyclosporin A (CyA) but did not interfere with higher CyA concentrations. In parallel, ciprofloxacin did not influence thymidine uptake in mixed lymphocyte reactions in the presence of CyA. To obtain an in vivo application of these findings, graft survival was studied by performing rat cardiac allograft transplantations in the presence or absence of CyA. Brown Norway rats served as donors and Wistar Furth rats as recipients. Ciprofloxacin was injected intraperitoneally either at a high-dose regimen (240 mg/kg per 24 h) into rats every 8th h starting 1 day before transplantation until day 21 or graft loss, or it was injected at a low and clinically relevant dose regimen (45 mg/kg per 24 h) until day 9. CyA was administered orally (10 mg/kg per 24 h) from day 1 through day 9. Ciprofloxacin given alone at a high-dose regimen resulted in a median graft survival of 14.8 days, which was significantly longer than graft survival in rats without treatment (median 8.0 days). A low-dose regimen of ciprofloxacin alone did not affect graft survival.(ABSTRACT TRUNCATED AT 250 WORDS)

CARDIAC ALLOGRAFT ATHEROSCLEROSIS IN THE RAT

Cardiac transplant atherosclerosis is thought to result from immune-mediated vessel wall injury. The present experiments were designed to test whether CsA alone or in combination with the ACE-inhibitor cilazapril has any effect on graft atherosclerosis in a rat cardiac transplant model. Cardiac grafts were transplanted heterotopically into either syngeneic or allogeneic recipients and followed by daily palpation; long-surviving grafts were removed after 100 days and the extent and degree of atherosclerosis was assessed using computerized morphometry. Atherosclerosis was more extensive in grafts removed from untreated allogeneic recipients compared with syngeneic recipients; CsA treatment increased the extent of atherosclerosis in syngeneic transplants. The extent and degree of vascular occlusion in allogeneic grafts from recipients treated with 15 mg/kg of CsA every other day was not different from that in grafts removed from recipients that received initially higher CsA doses. Cilazapril had no effect on the extent of graft atherosclerosis but decreased the degree of luminal narrowing in grafts from CsA-treated recipients significantly. Some grafts showed neovascularization in the subendocardial region adjacent to organized intraventricular clots, suggesting the release of angiogenic factors from such clots; such growth factors may contribute to the atherosclerotic vessel wall reaction in this model. We conclude that CsA promotes the development of graft atherosclerosis in heterotopically transplanted syngeneic cardiac grafts in the rat. We furthermore found that cilazapril has a beneficial effect on the degree of atherosclerosis in CsA-treated recipients.

Induction of transforming growth factor-beta during cardiac allograft rejection.

The polypeptides of the transforming growth factor-beta (TGF-beta) family are potent endogenous immuno-regulators. Using a rat cardiac allograft transplant model, we investigated the expression of the precursor forms of TGF-beta 1, TGF-beta 2, and TGF-beta 3 and the latent TGF-beta binding protein (LTBP) by immunohistochemistry. The activity of TGF-beta in the extracts from transplanted as well as normal hearts was measured using a bioassay, and Northern blot analysis was performed on RNA extracts. The transplanted hearts were analyzed both during acute rejection up to 6 days and during chronic rejection up to 6 mo after transplantation and compared with normal controls. The animals of the chronic rejection group received cyclosporin A for immunosuppression. The TGF-beta bioactivity dramatically increased in the transplanted allografts during the chronic rejection process compared to the normal hearts, and so did the immunostaining as well as the mRNA levels for TGF-beta 1 and, to a lesser extent, the immunostaining for TGF-beta 2. TGF-beta 3 expression remained unchanged and was only found in the myocardium in trace amounts. During the acute rejection process up to 6 days after transplantation, TGF-beta immunoreactivity increased only slightly, whereas the TGF-beta mRNA was severalfold increased. Control animals treated with cyclosporin A showed a similar pattern at day 6 with regard to TGF-beta expression. LTBP was induced simultaneously with TGF-beta 1 and occurred within interstitial spaces of the myocardium. The TGF-beta was produced by macrophage-like infiltrating lymphocytes. In conclusion, highly elevated levels of TGF-beta and LTBP were found during chronic rejection of cardiac allografts in rats. The induction of TGF-beta may counteract the rejection process and could be useful for new therapeutic approaches in the prevention of allograft rejection.