Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.

BACKGROUND: Intraportal transplantation of pancreatic islets offers improved glycaemic control and insulin independence in type 1 diabetes mellitus, but intraportal thrombosis remains a possible complication. The thrombotic reaction may explain why graft loss occurs and islets from more than one donor are needed, since contact between human islets and ABO-compatible blood in vitro triggers a thrombotic reaction that damages the islets. We investigated the possible mechanism and treatment of such thrombotic reactions. METHODS: Coagulation activation and islet damage were monitored in four patients undergoing clinical islet transplantation according to a modified Edmonton protocol. Expression of tissue factor (TF) in the islet preparations was investigated by immunohistochemistry, immunoprecipitation, electron microscopy, and RT-PCR. To assess TF activity in purified islets, human islets were mixed with non-anticoagulated ABO-compatible blood in tubing loops coated with heparin. FINDINGS: Coagulation activation and subsequent release of insulin were found consistently after clinical islet transplantation, even in the absence of signs of intraportal thrombosis. The endocrine, but not the exocrine, cells of the pancreas were found to synthesise and secrete active TF. The clotting reaction triggered by pancreatic islets in vitro could be abrogated by blocking the active site of TF with specific antibodies or site-inactivated factor VIIa, a candidate drug for inhibition of TF activity in vivo. INTERPRETATION: Blockade of TF represents a new therapeutic approach that might increase the success of islet transplantation in patients with type 1 diabetes, in terms of both the risk of intraportal thrombosis and the need for islets from more than one donor.

Effect of fluvastatin on acute renal allograft rejection: a randomized multicenter trial.

BACKGROUND: Statin therapy has been reported to reduce the acute rejection rate following renal transplantation in a pilot study. The present study is the first randomized, double-blind and adequately powered study to examine the effect of statins on acute rejection of renal allografts. METHODS: A total of 364 patients were randomly assigned to receive either fluvastatin 40 mg or placebo in combination with conventional cyclosporine-based immunosuppressive therapy. The primary end point was treated first acute rejection. Secondary end points included biopsy-proven rejection, histological severity of rejection, occurrence of steroid-resistant rejection, and serum creatinine at three months following transplantation. RESULTS: Fluvastatin was well tolerated; no patients developed myositis or rhabdomyolysis. There was no difference in the acute rejection rate [86 (47.3%) fluvastatin vs. 87 (47.8%) placebo] and no significant difference in the severity of rejection, steroid resistant rejection or mean serum creatinine at three months (160 micromol/L vs. 160 micromol/L). Total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol and triglyceride levels increased following renal transplantation. With the exception of the increase in HDL-C, which was augmented, the increases in lipid parameters were significantly reduced by fluvastatin (total cholesterol +17.5% vs. 35.7%; LDL-C +6.3% vs. 46.7%; HDL-C +43.3% vs. 38.1%; triglyceride +52.2% vs 77.6%). CONCLUSIONS: Contrary to the reported effects of statins, fluvastatin had no effect on the incidence or severity of acute rejection following renal transplantation. There were no increases in adverse events. A significant and potentially beneficial alteration in the lipid profile was observed in the early post transplant period. We conclude that fluvastatin may be used safely to correct dyslipidemia in patients with end-stage renal failure through the peri-transplant period.

Laparoscopic or open surgery for living donor nephrectomy.

BACKGROUND: The anterior extraperitoneal approach for living donor nephrectomy has been used in more than 700 living cases in the unit and proved to be safe for the donor. In 1998, laparoscopic nephrectomy was introduced as an option when technically feasible. We found it essential to investigate the consequences of the new technique. SUBJECTS AND METHODS: One hundred living donor kidney transplantations were performed from 1998 to June 2000, 45 with laparoscopic, 55 with open nephrectomy. The donors took part in a structured interview 4 weeks after the donation and their responses were categorized in three classes. RESULTS: In each group, one recipient had delayed initial function. The serum creatinine levels after 3 and 7 days or the GFR values after 6 months did not differ. One graft has been lost following laparoscopic nephrectomy and four after open surgery. For the laparoscopy donors, the median number of post-operative days in hospital was 5.0 days (range 2-9), vs 6.0 (4-8) after open surgery (NS). The requirement of opoid analgesics post-operatively was 5.0 doses (1-22) vs 6.0 (1-38) (P=0.02); and after 4 weeks, 23 of 45 laparoscopic donors were free of pain vs eight of 55 open nephrectomy donors (P=0.0004). Approximately one-third of all donors felt some restriction of physical activity and the majority complained of impaired physical energy. There were no differences between the groups. The duration of sick-leave after laparoscopic surgery was median 6 (2-19) weeks vs 7 (1-16) (NS). CONCLUSIONS: Laparoscopic nephrectomy is safe. Less post-operative pain is a definite advantage for the donor.

Experience with laparoscopic donor nephrectomy at a European transplant centre.

Renal grafts from live donors represent an important source for transplantation of end stage renal failure patients. Postoperative short- and long-term comfort is essential. Laparoscopic nephrectomy was performed in 22 cases. The left kidney was preferred for optimal length of the vessels. One procedure was converted to open surgery because of venous bleeding. Warm ischemia time varied between 4 and 7.5 min. Urine production started peroperatively in all cases, and the renal function was excellent. Shoulder pain 1-3 days postoperatively was observed in seven patients; the rest were comfortable on peroral non-opioid analgesia. The patients were discharged at postoperative days 3-9, and returned to work 2-4 weeks later as compared to 4-8 weeks after open nephrectomy at our centre. Laparoscopic donor nephrectomy in the hands of experienced laparoscopic and transplant surgeons is a safe operation with less discomfort to the living kidney donor.

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.

Single dose anti-CD4 monoclonal antibody for induction of tolerance to cardiac allograft in high- and low-responder rat strain combinations.

Repeated administration of monoclonal antibodies (mAb) directed against the CD4 lymphocyte receptor may induce specific, long-lasting unresponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression. We assessed the effect of a single dose of murine anti-rat depleting anti-CD4 mAb (OX-38) on allograft survival in high- and low-responder rat strain combinations. Isogenic strains of DA (RT1av1), PVG (RT1c), AUG (RT1c), and WF (RT1u) rats were used. Recipients in antibody treated groups were given one dose of 5 mg/kg OX-38 mAb on the day of transplant, a dose which was shown to effectively deplete (or block) circulating CD4+ T cells. Other groups were treated for 10 days with cyclosporin A (CsA) and/or Linomide, a novel immunomodulator, which is the first compound able to fully eliminate the effect of CsA in the rat cardiac allograft model. The DA strain was identified as a low-responder to the allogeneic haplotype RT1c (PVG or AUG), but not to RT1u (WF), and developed true tolerance following RT1c grafting and OX-38 or low-dose CsA (5 mg/kg) induction, as verified by the response to retransplantation of a graft from the same donor strain or a third-party challenge. PVG recipients of DA grafts were characterized by high response and only modest (OX-38; median 9.5 days) or moderate (CsA; 23.5 days) prolongation of graft survival. Contrasting graft survival results were obtained in the low-responder combination, either very early rejection (at 10 days) or permanent graft survival (> 100 days). Linomide challenge affected CsA treatment in the high-responder combination but not tolerance induction in the low-responder combination, or the effect of OX-38. It was concluded that in rat heart transplantation a single-dose anti-CD4 mAb therapy may induce permanent donor-specific unresponsiveness in a low-responder strain combination, and that anti-CD4 mAb seems to be unique among immunosuppressive agents while being resistent to challenge by Linomide.

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.

Activation of alloreactive natural killer cells is resistant to cyclosporine.

BACKGROUND: We have previously shown that cytotoxic T lymphocytes (CTL) with alloreactivity were induced when Wistar Furth (WF; RT1u) rats were immunized with allogeneic Brown Norway (BN; RT1n) cells. In contrast, when BN rats were immunized with WF cells, the allospecific response was confined to alloreactive natural killer (NK) cells, and no CTL activity was observed. In this study, the effect of cyclosporine (CsA) on the activation of alloreactive NK cells in vivo was analyzed. METHODS: Distinct peritoneal effector cells from rats immunized with allogenic cells with or without concomitant CsA and/or interleukin (IL) 2 treatment were tested for specific cytolytic activity. Furthermore, the presumptive role of NK cells in rejection immunity was addressed in a cardiac graft model. RESULTS: The results showed that doses of CsA that completely inhibited the activation of alloreactive CTL, only marginally affected the activation of alloreactive NK cells. We also showed that CsA treatment failed to prolong graft survival in BN recipients of WF hearts. Treatment of BN rats with CsA/IL-2 during immunization with allogeneic WF cells resulted in concomitant induction of alloreactive NK cells and alloreactive CTL. CONCLUSIONS: We have demonstrated that CsA failed to suppress the activation of alloreactive NK cells. Consequently, the cardiac graft survival in the donor-recipient combination known to activate alloreactive NK cells was not significantly prolonged by CsA treatment, emphasizing the involvement of NK cells as effectors in organ rejection. Furthermore, the parallel emergence of alloreactive NK cells and CTL only in the presence of CsA/IL-2 indicated that CsA interfered with alloreactive NK cell-associated suppression of CTL activated by allogeneic tissue.

Allogeneic heart transplantation activates alloreactive NK cells.

The ability of natural killer (NK) cells to recognize and reject transplants has so far been shown in hematopoietic grafts only. This study was designed to ascertain whether NK cells may also be involved in the rejection of transplanted organs. In most rat strain combinations, immunization with allogeneic cells induces a T cell response with cytotoxic T lymphocyte (CTL) activation. We have previously found one exception to this. In contrast to Wistar Furth rats (WF, RT1u), which manifest allospecific CTL activation in response to immunization with Brown Norway (BN, RT1n) cells, BN rats immunized with repeated intraperitoneal (i.p.) injections of allogeneic WF spleen cells manifest activation of alloreactive NK effector cells. The alloreactive NK cells were of the TCR-, CD3-, CD8+, and NKR-P1 intermediate phenotype and killed target cells with alloselectivity. In this study we used a heart transplantation model to study the rejection response of BN rats receiving WF grafts. NK cell infiltration was greater in WF hearts transplanted to BN recipients than in BN hearts transplanted to WF recipients. Furthermore, the extent of T cell infiltration was less in BN recipients. In WF rats transplanted with allogeneic BN hearts, CTL were activated in response to i.p. challenge with allogeneic BN cells, whereas BN rats transplanted with allogeneic WF hearts and i.p. challenged with allogeneic WF cells, manifested activation of alloreactive NK cells but no measurable activation of classic CTL. The alloreactive NK cells killed their allogeneic targets with specificity and with potency comparable to that of CTL. Furthermore, WF grafts were rejected in BN recipients as efficiently as were BN grafts in WF recipients. These results not only show cardiac allografts to be able to activate alloreactive NK cells, but also suggest that NK cells may be involved in the rejection of solid organ transplants and function as classic CTL in certain donor-recipient combinations.

Bioavailability of cyclosporine in rats after intragastric administration: a comparative study of the L2-phase and two other lipid-based vehicles.

The purpose of this study was to evaluate formulations based on surface active dietary lipids only as oral vehicles for cyclosporine. The absolute bioavailability of cyclosporine from two new lipid vehicles was determined in rats after intragastric administration and compared to that of Sandimmun oral solution, which contains non-ionic surface active substances in addition to dietary lipids. In the new vehicles, cyclosporine was dissolved in two different mixtures of glycerides from long-chained fatty acids. One mixture forms an L2-phase, an oil with very low interfacial tension towards water, and was administered both as the oily L2-phase and as a predispersed emulsion formulation. The other mixture forms a liquid crystalline phase and was administered only as an aqueous dispersion. The mean bioavailability of cyclosporine from Sandimmun was 8% while it was 34% from the L2-phase, 38% from the predispersed L2-phase and 27% from the dispersed liquid crystalline phase. The coefficients of variation in area under the blood concentration curve after administration of the two formulations based on the L2-phase were quite low (31% for the L2-phase and 24% for the predispersed L2-phase) and comparable to that after intravenous administration (24%), while the dispersed liquid crystalline phase gave a higher variability (91%), comparable to that of Sandimmun oral solution (101%). The low variabilities found with the two L2-phase vehicles suggest that this formulation is "self-emulsifying' in the gastrointestinal tract. Since the L2-phase is based on dietary lipids only, it is expected to be well tolerated and could prove to be a good vehicle for long-term clinical use of oral cyclosporine.

Moderate additive immunosuppressive effect of thalidomide combined with cyclosporin A in rat cardiac transplantation.

Thalidomide is an immunomodulating agent shown to prolong graft survival in experimental skin, renal, cardiac and bone marrow transplantation. The main purpose of the present study was to investigate the possible additive effect of combining thalidomide with cyclosporin A (CyA). Members of our group have previously created a basis for such studies by demonstrating the ability of Linomide to abolish the effect of CyA. The additional effect of combined treatment with a second drug is thereby more readily evaluated, compared with using subtherapeutic dose levels to induce early rejection. Cardiac grafting was performed in three rat strain combinations (BN to WF, DA to Lew, and BN to Lew). Rats were given no treatment, or thalidomide, CyA and/or Linomide in single, double or triple drug therapy. Except for a consistent beneficial effect of CyA as single drug treatment, graft survival varied depending on the rat strain combination used. In the DA to Lew combination, the expected effects of Linomide were seen, and thalidomide was shown to prolong graft survival significantly (P = 0.004) when added to CyA and Linomide. However, there was no effect of thalidomide when given alone. In WF recipients of BN hearts, thalidomide tended to prolong graft survival (P = 0.07), and surprisingly Linomide manifested a marked immunosuppressive effect (P = 0.0002) and did not counteract the effect of CyA. When transplanting BN grafts to Lew recipients, Linomide reduced significantly but did not abolish completely the effect of CyA. Neither Linomide nor thalidomide had any beneficial impact on graft survival on their own. To sum up, thalidomide was shown to have a minimal or moderate immunosuppressive effect additive to that of CyA. The effects of the two immunomodulating drugs, thalidomide and Linomide, varied depending on the rat strain combination used, and were similar with respect to prolongation of graft survival when used as single drug treatment in BN to WF grafting, a fact which may indicate them to have a similar mechanism of action, both having been shown to exert similar effects on levels of tumour necrosis factor alpha.

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.

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)