Sensitivity of baboon lymphocytes to cyclosporin A and FK 506: relative resistance of alloactivated cells to CyA

The interspecies differences in CyA pharmacokinetics necessitate the establishment of optimal immunosuppressive doses in the baboon, especially as its use as host for preclinical xenografts is anticipated. We assessed the immunosuppressive effects of CyA and FK 506 on lymphocytes from charma baboons, using human cells for comparison. At concentrations up to 100 mol/l, neither drug was toxic to lymphocytes. FK 506 inhibited baboon and human lymphocyte proliferation and IL-2 synthesis equally. In contrast, approximately four times higher doses of CyA were needed to inhibit baboon lymphocytes responding to alloantigens. This may explain the inadequate immunosuppression of baboon graft recipients treated with clinically acceptable doses of CyA. We propose that CyA whole blood target levels of ±1500 ng/ml should be used in this species and we provide evidence that chacma baboons are able to tolerate such doses without nephroxicity.     

FK506对许旺细胞体外增殖和分泌NGF的影响

目的 研究FK506促进许旺细胞体外增殖及对许旺细胞(SOs)分泌NGF的影响. 方法 将纯化的许旺细胞分6组:A组(空白对照组):含10%胎牛血清的DMEM/F12;B组:0.1 ng/mlFK506;C组:0.5 ng/ml FK506;D组:1.0 ng/ml FK506;E组:5.0 ng/mk FK506;F组:10 ng/ml FK506.将许旺细胞于倒置显微镜下观察并用S-100蛋白免疫组化染色鉴定;MTT法筛选FK506促SCs增殖的最佳作用浓度;流式细胞仪检测SCs周期;ELISA法检测培养72 h后SCs的NGF的分泌量. 结果 MTT法筛选:0.5 ng/ml FK506为促进SCs增殖的最佳作用浓度;当FK506浓度大于1.0 ng/ml时,SCs的生长活性逐渐下降并随着FK506浓度的逐渐增高,SCs的生长活性受抑制作用逐渐加强.流式细胞仪检测:含10%胎牛血清的DMEM/F12培养24 h、48 h、72 h,SCs S期百分比分别为:27.8%,39.3%和58.4%;0.5 ng/mlFK506培养24 h、48 h、72 h,SCs S期百分比分别为:54.2%、60.3%和94.6%.EUSA法检测FK506促SCs增殖后表达NGF的实验研究中发现:0.5 ng/ml FK506作用72 h后的SCs所分泌的NGF高达0.188 ng/ml. 结论 FK506应用于体外培养的SCs初期就能促进SCs增殖并使其保持良好的活性而高分泌NGF.     

Nephrotoxicity after orthotopic liver transplantation in cyclosporin A and FK 506-treated patients

Abstract Nephrotoxicity represents a serious side-effect of immunosuppression following orthotopic liver transplantation. In order to preserve the therapeutic potential of cyclosporin (CsA) and FK 506 in human liver transplantation and to differentiate the nephrotoxic action of either drug in a clinical setting, we evaluated the incidence of early and late nephrotoxicity in 121 patients, 60 randomly assigned to CsA- and 61 to FK 506-based immunosuppression. Early postoperative renal insufficiency (between PODO and 30; SCr 1.5-3 mg/dl) was observed to a similar extent in patients treated with CsA (36.7%) and FK 506 (42.6%). Early postoperative acute renal failure (ARF; SCr > 3 mg/dl) occurred in 18.3%, regardless of the immunosuppressive management. Approximately 50% of patients with ARF required hemodialysis (CsA: 11.7%; and FK 506: 8.3%). Mean onset of hemodialysis in CsA-treated patients was POD1 and in FK 506-treated patients, POD 6, which demonstrated a different time course of drug-pecific nephrotoxicity of CsA and FK 506 in early ARF. All patients with early postoperative ARF requiring hemodialysis survived more than 1 year (100% survival). New onset of late ARF (between POD 30 and 365), however, occurred in 6.5% under FK 506 and in 1.7% under CsA immunosuppression due to severe infections with the multiple organ failure syndrome. This observation was consistent with the assumption of overimmunosuppression rather than a primary nephrotoxic effect. Mortality of patients with late ARF requiring hemodialysis was 100%. Late renal insufficiency appeared in 23.3% of CsA- and in 29.4% of FK 506-treated patients, and represented a slowly progressing form of drug-pecific nephrotoxicity. These preliminary results demonstrated a similar outcome in terms of early and late nephrotoxicity, but longer follow-up will delineate its overall efficacy and toxicity in humans.     

Potentiation by nitric oxide of cyclosporin A and FK506-induced apoptosis in renal proximal tubule cells

Proximal tubular epithelial cells (PTEC) exhibit a high sensitivity to undergo apoptosis in response to proinflammatory stimuli and immunosuppressors and participate in the onset of several renal diseases. This study examined the expression of inducible nitric oxide (NO) synthase after challenge of PTEC with bacterial cell wall molecules and inflammatory cytokines and analyzed the pathways that lead to apoptosis in these cells by measuring changes in the mitochondrial transmembrane potential and caspase activation. The data show that the apoptotic effects of proinflammatory stimuli mainly were due to the expression of inducible NO synthase. Cyclosporin A and FK506 inhibited partially NO synthesis. However, both NO and immunosuppressors induced apoptosis, probably through a common mechanism that involved the irreversible opening of the mitochondrial permeability transition pore. Activation of caspases 3 and 7 was observed in cells treated with high doses of NO and with moderate concentrations of immunosuppressors. The conclusion is that the cooperation between NO and immunosuppressors that induce apoptosis in PTEC might contribute to the renal toxicity observed in the course of immunosuppressive therapy.     

Human pancreatic carcinoma cells are sensitive to photodynamic therapy in vitro and in vivo.

BACKGROUND: The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model. METHODS: Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis. RESULTS: PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session. CONCLUSION: PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.     

In-vitro effects of cyclosporin A, FK506, 6-mercaptopurine, and prednisolone on lymphokine-activated killer cells

In transplant recipients, immunosuppressive regimens are deleteriouson natural killer (NK) and lymphokine-activated killer (LAK)cells, which beyond their well-known antitumoral activity displaymany important biological functions. In order to find whichregimens could preserve NK and LAK functions we tested the influenceof CsA, FK506, 6-mercaptopurine and prednisolone on HLA-unrestrictedcytotoxicity during an in-vitro IL-2 activation. For each drug we obtained periphal blood samples from 11 healthyvolunteers. Non-adherent PBMC were incubated 2 days with eitherCsA, FK506, 6-mercaptopurine or prednisolone, whose concentrationsranged from 0 to 10 µg/ml, in order to screen infratherapeutic,therapeutic, and supratherapeutic doses. Thereafter, 100 IUof IL-2 were added for a further 3-day culture. Before and afterthe culture, we analysed (1) the cell subsets by direct immunofluorescence staining with anti-CD3/CD16/CD56 antibodies, (2) the LAKactivity with the lysis of Daudi cells, (3) the cell proliferationwith a 24-h incorporation of thymidine. Cyclosporin and FK506 did not impair the LAK cytotoxicity northe number of LAK cells, whereas both prednisolone and 6-mercaptopurinedecreased the LAK cytotoxicity, the number of CD3^– CD16⁺CD56⁺ cells, and the thymidine uptake. As a whole, the LAK cytotoxicitywas correlated with the number of CD3^– CD16⁺ CD56⁺ cellsbut not with the number of CD3^– CD16^– CD56^–cells, and it also increased with the incorporation of thymidine.This latter was correlated with the number of CD3^– CDl6⁺CD56⁺ cells, but not with the number of CD3⁺ CD16^– CD56^–cells. We conclude that in vitro the LAK activity is impaired by prednisoloneand 6-mercaptopurine, but not by CsA or FK506, and that thedeficiency of the LAK cytotoxicity seems to be related to thedecreased number of IL-2-activated NK cells.     

HMGB1 release in co-cultures of porcine endothelial and human T cells

High mobility group box-1 (HMGB1) protein, primarily from the nucleus, is released into the extracellular milieu either passively by necrotic or damaged cells, or actively by secretion from monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory stimulator by promoting cytokine (for example, tumor necrosis factor-alpha) production, and also has pro-coagulant activity. The signaling pathway initiated by receptor for advanced glycation end-product (RAGE), which is the HMGB1 receptor, also induces complement activation. Recent studies have implicated HMGB1 in acute cardiac allograft rejection, and have identified infiltrating T cells and other damaged cells as its main sources. HMGB1 blockade using the anti-HMGB1 antibody HMGB1 box-A (amino-terminal region) and soluble RAGE rescues mice from acute rejection. We therefore studied the release of HMGB1 in co-cultures of porcine aortic endothelial cells (PAEC) and human leukocytes. Human T cells, but not B cells, monocytes or neutrophils, stimulated significant HMGB1 release in culture with PAEC; this activity required cell-cell contact and was dose-dependent, as determined by Western blotting. The released HMGB1 originated from both cell types, as immunofluorescent microscopy showed that it was present in the cytosol of PAEC in contact with T cells, and had disappeared from the T-cell nuclei. These results demonstrate that direct interactions between PAEC and T cells might be a key factor in triggering HMGB1 release, which suggests that HMGB1 is associated with graft rejection in the early phase.     

Patient and graft survival in the European Multicentre Liver Study - FK 506 vs cyclosporin A

Abstract A prospective randomised study was conducted to evaluate the efficacy and safety of FK 506 administered with corticosteroids compared with a cyclosporin A-based immunosuppressive regimen in patients undergoing primary liver transplantation. 545 patients were recruited in eight European centres, of whom 267 were randomised to FK 506 therapy and 273 to cyclosporin A-based therapy. The estimated Kaplan-Meier patient and graft survival figures of 82.9% and 77.5% respectively in the FK 506 group were higher than the comparable figures in the cyclosporin A group (77.5% and 72.6%, respectively). These differences did not reach statistical significance. Retransplantation rates, time to first rejection episode and number of rejection episodes were all lower ( P < 0.001) in the FK 506 group. The infection rates were comparable between the two groups. During the study, the dose of FK 506 was reduced; this did not compromise efficacy and reduced the associated toxicity. FK 506 provides effective immunosuppression in patients undergoing primary liver transplantation and is associated with a lower incidence of rejection.     

Immunosuppression with FK506 in rat arterial allografts: fate of allogeneic endothelial cells

PURPOSE: This study clarified the efficacy of low-dose FK506 and the possibility of discontinuing the use of FK506. METHODS: Fresh carotid arteries were allografted from ACI rats to Lewis rats. FK506 (0.2 mg/kg/day) was given intramuscularly from day 3 after transplantation until the rats were killed (group III), or it was given for 4 weeks and then discontinued (group IV). Isogeneic (group I) and allogeneic (group II) models served as untreated control groups. Grafts were harvested on days 0, 1, 3, 7, 14, 21, 28, 70, and 105 after transplantation. Histological evaluation and measurement of the endothelial cell (EC)-covered area were performed by means of scanning electron microscopy. RESULTS: In group I, ECs were denuded immediately after transplantation and subsequently regenerated within 2 weeks. In group II, after denudation and regeneration of ECs, massive leukocyte adhesion and subsequent destruction of regenerated ECs, followed by intimal hyperplasia, were observed. In group III, FK506 suppressed rejection almost completely, without intimal hyperplasia. In group IV, severe rejection and denudation of regenerated intima appeared 2 weeks after the use of FK506 ended. CONCLUSION: The denudation and regeneration of ECs may play an important role in the process of rejection and graft performance. FK506 proved to be successful in rat arterial allografting, and ECs of donor origin could survive on the allograft as long as FK506 was effective; however, cessation of the use of FK506 resulted in severe destruction of intima. To prevent allograft failure, long-term administration of an immunosuppressant is essential.     

Direct activation of human responder T cells by porcine stimulator cells leads to T cell proliferation and cytotoxic T cell development

Abstract: Functional activities of highly purified T human responder lymphocytes reactive against porcine stimulator cells were studied to evaluate whether porcine stimulator cells can directly activate a xenospecific cellular immune response. Mixed lymphocyte culture (MLC) tests revealed that CD4+ human responder cells proliferate when stimulated in vitro with porcine cells, a reaction similar to that of alloresponses regarding magnitude and tempo. A direct pathway of activation was verified by the requirement for adherent porcine stimulator cells and the partial blocking by monoclonal antibodies against porcine major histocompatibility complex (MHC) class II antigens. An analysis of proliferative CD4+, CD3+, CD16-, and 56- human T cell clones revealed that some clones were seemingly recognizing allele-specific determinants, whereas others could be restimulated by a wide range of porcine stimulator cells. Cytotoxic T cells (CTLs) were generated following direct recognition of pig cell ligands by human T cells in the absence of autologous antigen-presenting cells (APCs). Although the identification of target antigen(s) on the pig cell recognized by the CTL warrants some discussion, the pattern of killing exhibited by the CTLs indicates the recognition of porcine polymorphic determinant(s). The implications of these findings for cellular reactivity against porcine transplants are discussed.     

FK506促进乳鼠雪旺细胞增殖的实验研究

目的观察FK506对体外培养的乳鼠雪旺细胞增殖的影响。方法将纯化的雪旺细胞分两组,一组设为对照,另一种加含终浓度为0.5ng/mlFK506的DMEM/F12培养液培养,用MTT法检测不同时间点的OD值并绘制生长曲线,另在培养第48、72小时后用3H-胸腺嘧啶核甙测定法检测其DPM值。结果经含0.5ng/mlFK506培养液培养的雪旺细胞,其对数生长期提前出现,并且在峰值上高于对照组;接种后48h和72h,3H-胸腺嘧啶核甙检测DPM值实验组也高于对照组,差异有统计学意义(P<0.05)。结论FK506有促进雪旺细胞分裂增殖的作用,可能是其促进周围神经再生的重要机制之一。     

Pronounced renal vasoconstriction and systemic hypertension in renal transplant patients treated with cyclosporin A versus FK 506

This prospective study investigated hypertension and renal vasoconstriction developing during the 1st year after renal transplantation in patients randomly allocated to treatment with FK 506 (n = 28) or CyA (n = 13). Starting doses were 0.2–0.3 mg/kg per day for FK 506 and 5–8 mg/kg per day for CyA; doses were subsequently adjusted to trough levels (5–15 ng/ml for FK 506 and 100–150 ng/ml for CyA). We compared 24-h ambulatory blood pressure measurement, antihypertensive treatment, serum creatinine, and resistance index (RI), measured by Doppler ultrasound at the level of the interlobar artery. Until month 2 of treatment, FK 506-treated patients had a significantly lower RI (8 %) and better renal graft function, as evidenced by significantly lower serum creatinine values. Some 13 % of FK 506-treated patients, compared to 70 % of CyA-treated patients (P < 0.01), needed additional antihypertensive drugs after transplantation to keep blood pressure stable. FK 506 treatment, at the above-mentioned dosages, was associated with a significantly higher number of infections (urinary tract infection, pyelonephritis, and pneumonia). We conclude that CyA produces greater renal vasoconstriction and systemic hypertension than FK 506, as reflected in higher renal interlobar artery RI values and a greater need for antihypertensive treatment. After 2 months of treatment and a reduction in CyA trough levels, the renal effects (i. e., lower RI and lower creatinine values), but not the systemic hypertensive effects, disappear. Received: 25 March 1997 Received after revision: 25 September 1997 Accepted: 8 October 1997     

Effect of FK506 treatment on allocytolytic T lymphocyte induction in vivo: differential effects of FK506 on L3T4+ and Ly2+ T cells.

Although FK506 has been widely investigated as a potent suppressor of organ allograft rejection in animals, little is known about the effect of FK506 on T cell responses to allografts in vivo. In the present study, we have studied the effect of FK506 on the induction of allocytolytic T lymphocyte using mice primed with alloantigens and treated with FK506. FK506 suppressed the CTL induction of spleen cells and peritoneal exudate cells (PEC) in a dose-dependent manner. Time-course kinetic studies indicated that the CTL activity was markedly dependent on the time of administration of FK506 to the mice. Lymphocytes from these FK506-treated animals were found to be reactivated upon exposure to the same alloantigens in a secondary mixed lymphocyte culture (MLC). Furthermore, FK506 was shown to have a differential effect on the activation of helper (L3T4+) and cytotoxic (Ly2+) T cell subpopulations. L3T4+ T cells from the mice primed with alloantigens and treated with FK506 had normal helper activity in the generation of CTL in MLC, whereas Ly2+ T cells from these mice were profoundly suppressed CTL activity upon reexposure to the same alloantigens in a secondary MLC. Exogenous IL-2 or L3T4+ T cells could overcome the immunosuppressive effect of FK506 on the CTL induction of Ly2+ T cells in a secondary MLC. Finally, we have demonstrated that this FK506 effect appeared to be antigen nonspecific since Ly2+ T cells from alloprimed FK506-treated mice failed to induce CTL against the third-party alloantigens as well as the same alloantigens in a secondary MLC.     

The in vitro activity and specificity of human endothelial cell-specific promoters in porcine cells

Abstract: The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is generally agreed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications. Luciferase assays were used to evaluate the activity and specificity of human endothelial-cell specific promoters in porcine aortic-, microvascular- and nonendothelial cells. The promoters for human Flk-1 (fetal liver kinase-1), Flt-1 (fms-like tyrosine kinase), ICAM-2 (intercellular adhesion molecule-2), thrombomodulin and vWf (von Willebrand factor) supported similar levels of luciferase expression in human and porcine aortic endothelial cells, with the Flk-1 promoter being the strongest followed by the thrombomodulin promoter. Relative to the activity of the CMV promoter, the human endothelial cell-specific promoters all showed less activity in porcine kidney microvascular endothelial cells than in liver or brain microvascular endothelial cells. The thrombomodulin and Flk-1 promoters exhibited similar activity in liver and kidney microvascular endothelial cells, whereas the Flk-1 promoter was stronger in aortic and brain microvascular endothelial cells. Human endothelial cell-specific promoters also showed some degree of specificity in pig, because they supported less luciferase activity in porcine nonendothelial cell lines. Based on the in vitro data and previously published in vivo data, the human Flk-1 and thrombomodulin promoters are good candidate promoters for strong endothelial cell-specific gene expression in transgenic pigs.