Kentaro Sugiyama

Immunosuppressive efficacy of mycophenolate mofetil when compared with azathioprine and mizoribine against peripheral lymphocytes from renal transplant recipients

Mycophenolate mofetil is currently used instead of azathioprine in clinical transplantation. However, comparative studies for the immunosuppressive potency of anti‐metabolites used for organ transplantation have not been well documented. We compared the pharmacological efficacy of mycophenolic acid (MPA), 6‐meraputopurine (6‐MP), and mizoribine (MZ) for inhibiting purine synthesis of peripheral blood mononuclear cells (PBMCs) in vitro by a mitogen assay procedure. PBMCs were obtained from 18 renal transplant recipients before operation and 18 healthy subjects. The inhibitory efficacy of 6‐MP against concanavalin A‐induced PBMC blastogenesis exhibited large variations between subjects in both recipients and healthy subjects. In contrast, the pharmacological efficacy of MPA on PBMC blastogenesis showed the smallest inter‐individual variation of all the purine synthesis inhibitors examined. Furthermore, the effects of MPA were almost similar in the recipients and healthy subjects. The pharmacological efficacy of MZ against PBMC blastogenesis was weaker than that of the other two agents and the inter‐individual variation of MZ IC50 against PBMCs of the patients was larger than that of MZ IC50 against PBMCs of healthy subjects. Reproducible immunosuppressive efficacy of MPA compared with other purinesynthesis inhibitors could be expected from the viewpoint of MPA pharmacodynamics against PBMCs in renal transplantation.

Correlation Between Pharmacological Efficacy of Cyclosporine A and Tacrolimus, Evaluated by Lymphocyte Immunosuppressant‐Sensitivity Test (LIST) with MTT Assay Procedure in Renal Transplant Recipients

Abstract The dose of calcineurin inhibitors in renal transplantation has been adjusted, based on the therapeutic drug monitoring data. However, the data do not always correlate with clinical drug efficacy. In vitro response of peripheral‐blood mononuclear cells to immunosuppressive drugs is reported to correlate with the recipient‐response to therapeutic efficacy of the drug. We report, here, usefulness of a lymphocyte immunosuppressant sensitivity test for the estimation of individual drug sensitivity in renal transplant recipients. The LIST we have developed includes MTT assay procedures without the use of radioisotope‐labeled compounds, which is convenient for general hospital use. Utilizing this procedure, we compared the pharmacological efficacy between cyclosporine A and tacrolimus in 41 renal transplant recipients.

Comparative study of the cellular pharmacodynamics of tacrolimus in renal transplant recipients treated with and without basiliximab.

Basiliximab is a recently developed immunosuppressive agent for the prevention of acute allograft rejection in renal transplant recipients. The combination use of basiliximab and a calcineurin inhibitor was suggested to be more effective in comparison to immunosuppressive therapy using calcineurin inhibitor without basiliximab. Cyclosporine has been generally administered with basiliximab for renal transplant recipients. However, in cases of tacrolimus-based immunosuppressive regimen, the clinical efficacy and safety of combined use of tacrolimus and basiliximab remains to be elucidated. This study evaluated the tacrolimus pharmacological efficacy using a lymphocyte immunosuppressant sensitivity test (LIST) with MTT assay procedures in 16 cases of renal transplant recipients treated by tacrolimus without basiliximab and in 13 cases treated by tacrolimus in combination with basiliximab. The rate of acute rejection episodes in the recipients treated with tacrolimus plus basiliximab was 1/13 (7.7%), whereas the rate in the recipients treated with tacrolimus without basiliximab was 6/16 (37.5%). The recipients were divided into two groups according to their peripheral blood mononuclear cell (PBMC) sensitivity to tacrolimus [i.e., including a tacrolimus high sensitivity group (IC50 <1.0 ng/ml) and a low sensitivity group (IC50 >1.0 ng/ml). In the recipients treated with tacrolimus without basiliximab, the rate of acute rejection episodes in the tacrolimus high sensitivity group was 1/10 (10.0%), which was significantly lower than the rate in the low sensitivity group of 5/6 (83.3%; p = 0.008). The incidence of cytomegalovirus infection was not significantly different between the tacrolimus high and the low sensitivity groups of the recipients treated with tacrolimus with and without basiliximab. Therefore, in the case of selected tacrolimus-based immunosuppressive therapy for renal transplant recipients, the tacrolimus pharmacological efficacy should be evaluated using LIST at a time just before the transplant procedure in order to accurately predict allograft rejection. The data also suggested that low tacrolimus sensitivity recipients should be treated with tacrolimus-based immunosuppressive therapy in combination with basiliximab.

Effects of arsenic disulfide on proliferation, cytokine production, and frequencies of CD4(+), CD8(+), and regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

Influence of arsenic disulfide (As2S2) on human immune cells has little been investigated. Effects of As2S2 on proliferation, cytokine production, and frequencies of CD4(+) T, CD8(+) T and CD4(+)CD25(+)Foxp3(+) regulatory T cells in mitogen-activated human peripheral blood mononuclear cells were examined. Anti-proliferative effects of As2S2 on peripheral blood mononuclear cells activated by T-cell mitogen were assessed by a colorimetric assay. Cytokine concentrations in the culture medium were measured with beads-array procedures followed by flow cytometry. CD4(+) T cells, CD8(+) T cells and CD4(+)CD25(+)Foxp3(+) regulatory T cells were stained with fluorescence-labeled specific antibodies followed by flow cytometry analysis. As2S2 at 1-10μM significantly suppressed mitogen-activated proliferation of peripheral blood mononuclear cells (p<0.05). As2S2 at 10μM inhibited production of IL-6, -10, -17A, tumor necrosis factor-α, and interferon-γ from the activated peripheral blood mononuclear cells, though the effects were not statistically significant. As2S2 at 10μM significantly suppressed the frequencies of CD4(+) T and CD8(+) T cells (p<0.05), whereas significantly enhanced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (p<0.05). The data suggest that As2S2 attenuates T cell-mediated immunity by not only suppressing the proliferation of T cells and cytokine release but also increasing the frequency of regulatory T cells. T cell-mediated autoimmunity contributes to bone marrow failure in myelodysplastic syndrome (MDS), and thus the above As2S2 effects are beneficial for the treatment of MDS patients.

Influence of anticancer agents on cell survival, proliferation, and CD4+CD25+Foxp3+ regulatory T cell-frequency in human peripheral-blood mononuclear cells activated by T cell-mitogen

Many anticancer agents currently used are considered to be cytotoxic not only to cancer cells but also to functional immune cells. To learn more about the immunosuppressive adverse influence of chemotherapeutic drugs in cancer chemotherapy, we examined the effects of arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate on the survival, proliferation, cytokine production, and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Arsenic trioxide, dacarbazine, and 5-fluorouracil increased trypan-blue stained (dead) cell rates and suppressed the mitogen-activated proliferation of PBMCs significantly at 1-100μM (p<0.05). Methotrexate also significantly increased the percentages of dead cells and suppressed the mitogen-activated PBMC-proliferation at concentrations of more than 0.05μM (p<0.01). Arsenic trioxide significantly inhibited the production of interferon γ, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 5μM (p<0.05). In contrast, the anticancer agents significantly increased Treg cell-frequency in the activated PBMCs at concentrations of more than 0.1μM for methotrexate, 5μM for arsenic trioxide and 5-fluorouracil, and 50μM for dacarbazine, respectively (p<0.05). These agents did not significantly influence the production of transforming growth factor (TGF) β from the activated PBMCs at a concentration range of 0.05-50μM. Our data suggest that the anticancer agents: arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate attenuate T cell mediated immunity by not only inhibiting the proliferative response of T cells but by also increasing the frequency of Treg cells, which may result in the suppression of the effector T cell function.

Comparison of suppressive potency between prednisolone and prednisolone sodium succinate against mitogen-induced blastogenesis of human peripheral blood mononuclear cells in-vitro

Clinically, both prednisolone and prednisolone sodium succinate are widely used as immunosuppressive agents for the treatment of various allergic disorders. However, whether prednisolone sodium succinate itself has immunosuppressive or anti‐inflammatory effects is unclear, and prednisolone sodium succinate may exhibit its efficacy only after hydrolytic conversion to prednisolone in‐vivo. If this is the case, the impairment of prednisolone sodium succinate conversion to prednisolone in some clinical conditions may attenuate the efficacy of prednisolone sodium succinate. We therefore compared the pharmacological efficacy of prednisolone with that of prednisolone sodium succinate in‐vitro using human peripheral blood mononuclear cells (PBMCs). PBMCs were obtained from 5 healthy subjects and 1 patient with pneumonia. The cells were incubated in the presence of concanavalin A and the cell growth was estimated by 3‐(4,5‐dimethyl thiazo‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Both prednisolone and prednisolone sodium succinate dose‐dependently suppressed PBMC blastogenesis. Mean (s.d.) prednisolone and prednisolone sodium succinate IC50 (concentration of drug that gave 50% inhibition of cell growth) values were 580.0 (1037.9) and 3237.1 (4627.3) nm, respectively. The ratio of prednisolone IC50/prednisolone sodium succinate IC50 ranged from 0.005 to 0.230. Thus, prednisolone sodium succinate potency was markedly lower than that of prednisolone. After incubation of PBMCs with 100 μm prednisolone sodium succinate, 22.7–42.9 μm prednisolone was liberated into the culture medium, as determined by HPLC. The ratio of prednisolone liberation from prednisolone sodium succinate was not affected by the presence of fetal bovine serum or PBMC, or both, in the culture medium. These results suggested that the PBMC‐suppressive effects of prednisolone sodium succinate might be due, at least partially, to prednisolone liberated from prednisolone sodium succinate into the culture medium. Prednisolone sodium succinate can be converted to prednisolone in the absence of serum or PBMCs, but the ratio of this conversion was very slow (t£frac12; > 4 days). Therefore, impairment of the enzymatic conversion of prednisolone sodium succinate to prednisolone in some pathological conditions such as liver diseases may result in attenuation of the clinical efficacy of prednisolone sodium succinate.

Effects of vitamin K3 and K5 on proliferation, cytokine production, and regulatory T cell-frequency in human peripheral-blood mononuclear cells.

AIMS The effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. MAIN METHODS Anti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. KEY FINDINGS VK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10-100μM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10-100μM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10μM (p<0.001). SIGNIFICANCE Our data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells.

Correlation between the pharmacological efficacy of cyclosporine and tacrolimus as evaluated by the lymphocyte immunosuppressant sensitivity test (LIST) and the MTT assay procedure in patients before and after renal transplantation.

OBJECTIVES Cyclosporine and tacrolimus are calcineurin inhibitors that are used to prevent acute rejection in renal transplant recipients. The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of these immunosuppressive agents for renal transplant recipients. There is a correlation between cyclosporine and tacrolimus pharmacological efficacy as evaluated by LIST by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay procedure prior to renal transplantation. However, the LIST can also evaluate patients before and after the transplantation. MATERIALS AND METHODS The present study examined the relationship between cyclosporine and tacrolimus pharmacological efficacy by LIST using the MTT assay in 16 renal transplant recipients at 1, 3 and 12 months after transplantation, as well as before the operation. RESULTS The relationship of cyclosporine and tacrolimus pharmacological efficacy gave a significant Kendall and Spearmans coefficient correlation in these transplant recipients by the LIST using the MTT assay procedure immediately prior to renal transplantation (rk = 0.711, rs = 0.877, p < 0.01). Furthermore, correlations between the cyclosporine and tacrolimus IC50 values were also observed with a significant Kendall and Spearmans coefficient correlation at 1 and 12 months after transplantation (rk1month = 0.65, rs1month = 0.829, p < 0.01, and k12month = 0.433, rs12month = 0.603, p < 0.01, respectively). However, no statistically significant relationship was observed between the pharmacological efficacies of the calcineurin inhibitors at 3 months after transplantation (rk3month = 0.117, rs3month = 0.1, p > 0.05). CONCLUSIONS Both cyclosporine and tacrolimus exhibit pharmacological efficacy by the inhibition of calcineurin. However, the correlation between cyclosporine and tacrolimus pharmacological efficacies may be altered, due to immunosuppressive therapy or clinical events at 3 months after renal transplantation.

Cyclosporine pharmacological efficacy estimated by lymphocyte immunosuppressant sensitivity test before and after renal transplantation

Objective:  Lymphocyte immunosuppressant sensitivity test (LIST) is useful for predicting the pharmacological efficacy of immunosuppressive agents. In this study, the pharmacological efficacy of cyclosporine was estimated by LIST before and after renal transplantation.

Comparison of suppressive potency between azathioprine and 6-mercaptopurine against mitogen-induced blastogenesis of human peripheral blood mononuclear cells in-vitro.

Azathioprine (AZ) is a prodrug of 6‐mercaptopurine (6‐MP), but little is known about the relative suppressive efficacy of these agents against blastogenesis of human peripheral blood mononuclear cells (PBMCs) in‐vitro. We compared the pharmacological efficacy of AZ and 6‐MP against T cell mitogen‐induced blastogenesis of PBMCs in‐vitro. PBMCs were obtained from seven healthy subjects. The potency of AZ and 6‐MP to suppress PBMC‐blastogenesis in‐vitro was compared using 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) assay procedures. Production of 6‐MP from AZ in PBMC culture was examined by high‐performance liquid chromatography. Both AZ and 6‐MP dose‐dependently suppressed PBMC blastogenesis. Mean ± s.d. IC50 (concentration of drug that gave 50% inhibition) values for AZ and 6‐MP were 230.4 ± 231.3 and 149.5 ± 124.9 nM, respectively. Thus, the potencies of AZ and 6‐MP to suppress PBMC blastogenesis were not significantly different. A significant correlation was observed between the IC50 values of AZ and 6‐MP (P < 0.01, n = 6). After incubation of PBMCs with 100 μM AZ for 4 days, 35.3–92.5 μM 6‐MP was liberated into the culture medium. The ratio of 6‐MP liberation from AZ was influenced by the presence of PBMCs, but not by the medium or fetal bovine serum. The results suggest that the suppressive potency of the prodrug AZ and its converted form 6‐MP against blastogenesis of human PBMCs in‐vitro is similar, although PBMCs appeared to convert AZ to 6‐MP. AZ is suggested to be effective after conversion to 6‐MP to express immunosuppressive efficacy in‐vitro.