The effect of two new immunosuppressive agents, FK506 and didemnin B, in murine pregnancy.

The purpose of this study was to investigate two promising immunosuppressive agents, didemnin B (DB) and FK506 (FK), during pregnancy to assess potential adverse maternal or fetal effects. Pregnant C3H mice were randomized into control and high- and low-dose treatment groups for each drug. Animals received daily injections from day 1 to day 16, and on day 17 of gestation the maternal condition, litter size, fetal resorption rates, and fetal/placental unit weights were determined. Immunoglobulin (IgG) levels were obtained for DB treatment groups. Delayed type hypersensitivity was assessed in virgin females. Both DB and FK had dose-dependent immunosuppressive activity in the DTH assay, and DB caused elevated IgG concentrations. High doses of DB caused diarrhea and maternal wasting with no fetal survival; with low-dose DB, maternal weight gain was depressed, but pregnancy outcome was not different from control animals. High-dose FK had no obvious detrimental effects on maternal health but caused resorption of all fetuses; administration of low-dose FK resulted in a higher number of resorptions, but fetuses that survived did not appear different from controls. We conclude that these immunosuppressive drugs can have adverse effects on pregnancy, but the maternal and fetal toxicity are dose-dependent.     

The effects of immunosuppressive agents on in vitro production of human immunoglobulins.

We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than two-fold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production.     

The effects of immunosuppressive agents on the function of human hepatocytes in vitro

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.     

Alterations in rat pulmonary macrophage function by the immunosuppressive agents cyclosporine, azathioprine, and prednisolone

Disturbances of the immune response of the lung induced by the action of immunosuppressive agents on the functional abilities of rat pulmonary alveolar macrophages (PAM) were analyzed following in vitro incubation or in vivo administration (for 30 days) of cyclosporinea, (CsA) azathioprine (Az) or prednisolone (Pr). Two major parameters were analyzed: oxygen consumption and superoxide release as indices of the overall state of oxygen metabolism of these cells reflecting the integrity of PAM oxidative mechanisms of microbicidal activity, and chemotaxis, an event clinically important for normal defense to infection. In vitro incubation with cyclosporine at concentrations as low as 10(-9) M caused a 52% inhibition of PAM superoxide release, but Az had no effect at concentrations up to 10(-6) M. Prednisolone caused a 38% inhibition of superoxide release; comparable levels of inhibition with Pr required concentrations at least 10-fold greater than with cyclosporine. Further experiments indicated that cyclosporine induced a 40% inhibition after contact with PAM for only 30 min. In vivo experiments indicated that cyclosporine (5 mg/kg), Az (20 mg/kg), or Pr (2 or 0.5 mg/kg) administered intraperitoneally had no effect on the number of PAM available for host defense, PAM oxygen consumption, or PAM superoxide release. However, PAM from cyclosporine-treated animals demonstrated complete inhibition of active migration or chemotaxis in modified Boyden chambers upon incubation with formylmethionyl-leucyl-phenylalanine (FMLP). The effect was apparently dampened by simultaneous administration of Pr with cyclosporine. These experiments suggest that with the exception of a marked effect on chemotaxis the in vivo effects of physiologic amounts of cyclosporine on PAM function are modest compared with the marked depression after in vitro addition.     

In vitro immunosuppressive effects of cytotoxic agents conjugated to antihuman lymphocyte globulin.

Antihuman lymphocyte globulin (ALG) was either coupled to the lymphocytoxic drug chlorambucil or covalently bound to the cytotoxic alkalating agent melphalan via a polyglutamic acid carrier. Both types of complexes strongly inhibited the proliferative response in human mixed lymphocyte cultures and the ability of mixed lymphocyte culture-activated T effector cells to lyse 51Cr-labelled lymphoblast target cells, and were more potent than ALG or drug alone. These experiments indicate that it is possible to bind cytotoxic agents to ALG without destroying either the properties of the drug or the ability of the antibody to bind to lymphoid cells.     

Quality of life after kidney transplantation. A prospective, randomized comparison of cyclosporine and conventional immunosuppressive therapy

The aim of this research is to compare the quality of life after kidney transplantation for patients treated with cyclosporine versus conventional immunosuppressive therapy. This evaluation assumes particular importance given the high cost of cyclosporine, the resistance of the government to cover these costs, and the absence in some series (including this one) of significant differences in patient and kidney survival. This study is based on a randomized, stratified, prospective trial and concentrates on nondiabetic patients from ages 19 to 56 at 1-year posttransplant. Patients on cyclosporine show significant advantages in physical, emotional, and social well-being. Differences for 5 out of 10 indicators of quality of life were significant at the P less than or equal to .05 level. Significant differences are found on health satisfaction, happiness measures, indices of overall life satisfaction and well-being, perceived adjustment of the family, and female (not male) vocational rehabilitation. The fewer number of episodes of rejection and infection are, in part, responsible for these advantages in quality of life for cyclosporine patients.     

A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents

Difficulty in preventing rejection of fetal pig islet-like cell clusters (ICCs) transplanted into pigs using traditional forms of immunotherapy has been reported. An in vitro study of the efficacy of seven different immunosuppressive agents to inhibit proliferation of pig peripheral blood mononuclear cells (PBMC) was performed, and a comparison was made between the human and pig to determine if the efficacy of these agents differed between species. The efficacy of cyclosporine (CsA), azathioprine (Aza), methylprednisolone (MP), FK506, rapamycin (RAP), mycophenolate mofetil (MMF) and deoxymethylspergualin (MeDSG) to inhibit pig and human PBMC proliferation in mitogenic experiments using phytohaemagglutinin (PHA) as a stimulus was performed. Further, allogeneic pig mixed lymphocyte reactions (MLR) were used to determine the activity of these agents in a model more comparable to the allograft rejection process. It was found that pig PBMC stimulated with PHA or in a MLR were inhibited by the agents tested, with the exception of MeDSG that was ineffective in mitogenic experiments. The inhibitory effects of these agents differed between PHA and MLR, the respective (50% inhibitory concentration) IC50 values for pig PBMC being 1.7 and 0.08 microg/ml for CsA, 1.4 and 4.4 microg/ml for Aza, 0.11 and 0.002 microg/ml for MP, 3.0 and 2.8 ng/ml for FK506, 2.1 and 0.3 ng/ml for RAP and 10.8 and 454 ng/ml for MME Pig PBMC were less sensitive than human PBMC to the antiproliferative effects of CsA, Aza, FK506, RAP and MMF, but not MP on PHA stimulation, the ratio of the pig to human IC50 values being 19, 11, 13, 2.3, 1.4, and 0.4, respectively. These data suggest that the doses of most immunosuppressive agents administered to prevent rejection in pigs need to be higher than those used to achieve therapeutic benefit in humans.     

Evidence that the immunosuppressive effects of FK506 and cyclosporine are identical.

The fungal metabolite FK506 was discovered because it shared an important property, the ability to inhibit production of IL-2, with another well-known immunosuppressive fungal metabolite, CsA. FK506 has, since its isolation, also been shown to share other immunosuppressive effects with CsA. This study was performed to further investigate the in vitro immunological properties of FK506, in comparison with and in combination with CsA, to evaluate the plausibility that their mechanisms of action were identical or similar, in spite of their different molecular structures. The ability to inhibit several responses of human peripheral blood lymphocytes to mitogenic and alloantigenic stimulation was explored. Synergistic effects of the two drugs were extensively studied, since this would provide additional information regarding their mechanisms of action. Also, similar immunosuppressive properties would enable the use of the two drugs in combination. We found that FK506 and CsA had very similar mechanisms of action and additive effects were recorded.     

Isolation and in vitro characterization of a novel immunosuppressive cyclosporine metabolite

A novel metabolite (M-E) was identified by high-performance liquid chromatography in the serum of cyclosporine-treated renal transplant recipients during a second wave of immunosuppressive activity after disappearance of the initial wave due to the direct effect of CsA. M-E was identified in human serum and porcine bile both by HPLC and by a preparative thin-layer chromatography (TLC). It demonstrated homogeneity with characteristic retention times on C8 and C18 column HPLC systems using a variety of elution systems, and distinctive TLC mobility (Rf 0.35). Metabolite E (M-E) was documented to be a CsA metabolite by radioactive tracer studies, by crossreactivity with a polyclonal sheep antibody in radioimmunoassay, and by the presence of a characteristic mass spectrum. Further, in vitro immunosuppressive assays documented effects of M-E similar to those of CsA. The relative activity of M-E versus CsA was quantitated by potency ratios: for inhibition of normal human mixed lymphocyte culture reactions, the ratio was 0.79 +/- 0.23. Interindividual differences were observed in patient susceptibility to MLR inhibition not only by CsA, as previously reported by others, but also by M-E. There was a lesser effect of M-E compared with CsA in inhibiting proliferation of, and IL-2 generation by, C3H murine splenocytes stimulated with concanavalin A: the potency ratios for both systems were about 0.5, possibly reflecting an interspecies variability in generation of or susceptibility to M-E. These studies suggest that heretofore unidentified metabolites--including, but not limited to, M-E--may play an important role in the immunosuppressive effect of CsA in man.