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BACKGROUND Methotrexate (MTX) is used as anchor drug for patients with early and established rheumatoid arthritis (RA). Vitamin K2 administration was also reported to be associated with decreased disease activity in RA. OBJECTIVES Immunosuppressive pharmacodynamics of vitamin K2 combined with MTX was investigated. METHODS Mitogen-activated peripheral blood mononuclear cells (PBMCs) were used to evaluate immunosuppressive pharmacodynamics of drugs in vitro. RESULTS Vitamin K2 alone dose-dependently suppressed T cell mitogen-activated proliferation of PBMCs of both healthy subjects and RA patients. 446.5 and 2232.5 ng/mL vitamin K2 significantly decreased the IC50 values of MTX on the proliferation of PBMCs of RA patients, with little influences on the pharmacodynamics of MTX in the healthy PBMCs. 4465 ng/mL vitamin K2 potentiated the pharmacodynamics of MTX in both RA patients and healthy PBMCs. The additional effects of vitamin K2 to potentiate the suppressive effects of MTX seemed not to be related to the regulation of CD4+ CD25+ T cells or CD4+ CD25+ Foxp3+ Treg cells. MTX alone at 100 ng/mL significantly decreased the percentage of CD4+ T cells in PBMCs of healthy subjects (p

OBJECTIVE To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. MATERIALS AND METHODS Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. RESULTS Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47 ± 4,910.02 ng/mL (mean ± SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p

Purpose We report the case of a male neonate with a respiratory disorder who developed adverse cardiorespiratory symptoms after the continuous infusion of midazolam. Methods To clarify the cause of cardiogenic shock, we performed whole exome sequencing and screened relative single-nucleotide variants of 2 cytochrome P450 (CYP) isoforms, CYP3A4 and CYP3A5, which play a dominant role in the metabolic elimination of midazolam. We measured endogenous cortisol 6β-hydroxylation clearance to phenotypically assess CYP3A activity. Findings The CYP3A activity level in the patient was significantly lower than the mean CYP3A activity level in healthy adults. Three intronic mutations in the CYP3A4 and CYP3A5 isoforms were detected in the patient. Implications Our findings suggest that the midazolam concentration in plasma was achieved at above the steady-state concentration during continuous infusion used to sedate neonates receiving mechanical ventilatory support. Evaluation of the drug-metabolizing ability based on CYP3A might be useful if adverse electrophysiologic variables or the induction of tachycardia occur because of delayed elimination.

Background Over 20 kinds of steroids, tacrolimus ointments, and cyclosporine capsules are usually recommended for the treatment of atopic dermatitis (AD), depending on the symptoms of patients. However, several side effects sometimes occur with the extensive use of these agents for the treatment of pediatric AD patients. The purpose of this study was to explore whether vitamin K2 could be a new immunosuppressive candidate for pediatric patients with AD. Methods The immunosuppressive efficacy of vitamin K2 was evaluated through a cell-culture procedure using mitogen-activated peripheral blood mononuclear cells (PBMCs) obtained from pediatric AD patients. Results The mean (SD) IC50 value of vitamin K2 for the proliferation of concanavalin A-activated PBMCs was 15.37 (30.05) μmol/L, while the value for tacrolimus was 0.10 (0.28) ng/mL (0.12 (0.35) nmol/L). There was a significant correlation between the IC50 values for vitamin K2 and those for tacrolimus (P = 0.0001, r = 0.8871). However, there was no significant correlation between the IC50 values of vitamin K2 and those of cyclosporine A or methylprednisolone. A significant correlation between the IC50 values of vitamin K2 or tacrolimus and blood eosinophil counts (P = 0.0099, r = 0.7086 and P = 0.0032, r = 0.7722, respectively) was observed. Conclusion Vitamin K2 -inhibited T-cell mitogen stimulated proliferation of PBMCs from pediatric AD patients in a dose-dependent manner. The PBMCs from pediatric AD patients were more sensitive to the immunosuppressive efficacy of vitamin K2 than the PBMCs from healthy subjects. The individual immunosuppressive pharmacological efficacy of vitamin K2 and of tacrolimus could be inferred from the blood eosinophil count of pediatric AD patients.

Recently, steroid reduction/withdrawal regimens have been attempted to minimize the side effects of steroids in renal transplantation. However, some recipients have experienced an increase/resumption of steroid administrations and acute graft rejection (AR). Therefore, we investigated the relationship between the individual lymphocyte sensitivity to steroids and the clinical outcome after steroid reduction/withdrawal. We cultured peripheral blood mononuclear cells (PBMCs) isolated from 24 recipients with concanavalin A (Con A) in the presence of methylprednisolone (MPSL) or cortisol (COR) for four days, and the 50% of PBMC proliferation (IC50) values and the PBMC sensitivity to steroids were calculated. Regarding the experience of steroid increase/resumption and incidence of AR within one year of steroid reduction/withdrawal, the IC50 values of these drugs before transplantation in the clinical event group were significantly higher than those in the event-free group. The cumulative incidence of steroid increase/resumption and AR in the PBMC high-sensitivity groups to these drugs before transplantation were significantly lower than those in the low-sensitivity groups. These observations suggested that an individual’s lymphocyte sensitivity to steroids could be a reliable biomarker to predict the clinical outcome after steroid reduction/withdrawal and to select the patients whose dose of steroids can be decreased and/or withdrawn after transplantation.

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10-100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10-100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

BACKGROUND Machine perfusion techniques offer a solution to the serious organ shortage. However, to assess the effects of machine perfusion, many detailed studies are required. In this study, an ex vivo reperfusion model using diluted autologous blood was confirmed to evaluate the utility of machine preservation for livers donated after cardiac death (DCD). In particular, beneficial effects of the oxygenated hypothermic machine perfusion (HMP) for DCD porcine livers are evaluated. MATERIAL AND METHODS Porcine livers were procured under warm ischemia time (WIT) of 60 min. The livers were preserved by hypothermic machine perfusion (HMP) or static cold storage (CS) for 4 h. After the preservation, the livers were perfused for 2 h using the ex vivo reperfusion model with diluted blood oxygenated by a membrane oxygenator at 35-38°C. RESULTS At 2 h of ex vivo reperfusion with 60 min of warm ischemic time (WIT), the portal vein pressure for CS was higher than HMP (18.8±15.9 vs. 7.5±3.9 [mmHg] in 60 min). Furthermore, LDH in CS was higher than HMP (528.5±149.8 vs. 194.1±32.2 [IU/L/100 g liver] in 60 min. P

Objectives The objective of this study was to investigate the influence of treatment with cholinesterase inhibitors (ChEIs) and calcineurin inhibitors (CNIs) on the occurrence of cramps in myasthenia gravis (MG) patients. Methods The frequency and duration of cramp and serum electrolytes were evaluated in 81 patients with MG. The patients were classified using Myasthenia Gravis Foundation of America postintervention status scores based on the treatment and the responsiveness to the treatment. Quantitative MG score, MG activities of daily living score, MG composite score, or MG quality of life 15 score was used to assess the health-related quality of life (QOL). Results Muscle cramps developed in 44 (54.3%) of 81 MG patients. The scores of MG activities of daily living, MG composite, or MG-QOL 15-item questionnaire in patients with cramp were significantly higher than those in patients without cramps (P = 0.002, P = 0.01, or P = 0.0022, respectively). The serum magnesium concentrations were lower in patients treated with CNI (n = 16) than in those not treated with CNI (n = 65) (P = 0.002). The probability of cramps was significantly higher in patients treated with ChEIs (≥180 mg/d) in addition to CNI than in patients who were treated with a low dose of ChEIs (≤60 mg/d) without concomitant CNI treatment (P = 0.017). Conclusions Our data suggested that treatment with a high dose of ChEI and CNI accelerated the probability of cramps and reduced the QOL in MG patients.

Backgrounds Folic acid dose at ≦5 mg/week has been recommended for rheumatoid arthritis (RA) patients to decrease risk of methotrexate adverse effects. However, higher doses of folic acid is used in some cases. We examined the influence of high-dose folic acid on methotrexate efficacies and safety in Japanese RA patients. Methods 502 RA patients of four hospitals prescribed methotrexate and folic acid were included. These patients were divided into two subgroups according to the threshold of folic acid dose by 5 mg/week. Basic patient characteristics, methotrexate doses, and the efficacies or adverse effects of methotrexate were retrospectively compared between the two patient subgroups. Results The frequency of folic acid use at doses higher than 5 mg/week was significantly different between the four hospitals (P5 mg/week) dose of folic acid was significantly higher than that in the lower (≦5 mg/week) folic acid-treated subgroup (P=0.029). Folic acid dose between patients taking methotrexate less and more than 8 mg/week was not significantly different. Major conclusion Folic acid dose was dependent on the hospitals, while efficacies and hepatotoxicity of methotrexate was not basically different between patients taking less and more than 5 mg/week of folic acid.

Immunosuppressive therapy for prevention of acute rejection episode occasionally causes serious adverse effects, and thus it is important to develop new therapeutic approach for renal transplant recipients. This study evaluated the immunosuppressive pharmacodynamics of tetrandrine (TET) and/or methylprednisolone (MP) in hemodialysis patients in vitro by using the peripheral blood mononuclear cells (PBMCs) isolated from whole blood of hemodialysis patients. The median (range) of MP IC50 values against the proliferation of patients PBMCs was 7.04 (2.30~500.00) ng/mL. In contrast, the median (range) of MP IC50 values against the proliferation of healthy PBMCs was 4.44 (3.19~5.08) ng/mL. The median (range) of TET IC50 values against the proliferation of patients PBMCs was 1.61 (1.04~4.79) μM. Lower concentrations of TET (0.3-300 nM) were able to decrease the IC50 values of MP and thus potentiate the MP immunosuppressive effect on patient PBMCs. The median (range) of MP IC50 values in combination with 0.3, 3, 30, and 300 nM TET were 0.92 (0.49~8.39), 2.10 (0.45~20.00), 0.35 (0.092~1.05), and 0.14 (0.05~6.78) ng/mL, respectively. TET potentiates the MP immunosuppressive pharmacodynamics and thus, it was possible to use the combination of MP and TET to attenuate MP side effects. There were significant correlations between the IC50 values of TET and stimulation indices (P=0.04, r=0.58), the IC50 values of TET and the hemodialysis periods (P=0.04, r=0.57), or the IC50 values of MP combined with 0.3 nM TET and C-reactive protein concentrations (P=0.04, r=0.64), respectively. This article is protected by copyright. All rights reserved.

The ATP monitoring assay is a useful biomarker for risk monitoring to detect infection and rejection episodes in transplant recipients. Hemodialysis patients have a higher rate of infectious mortality. Infections in hemodialysis patients are mainly caused by venous catheters, uremia, malnutrition and inflammation. However, the risk of infection episodes has not been evaluated using a lymphocyte ATP monitoring assay in hemodialysis and chronic kidney disease (CKD) patients. We measured the ATP amounts in the peripheral CD4+ cells of CKD (N = 85) and dialysis patients (N = 17) using an "Immuknow" assay kit. These CKD patients were divided, according to kidney disease stage, into G3a, G3b, G4, and G5 groups. The ATP amounts in CD4+ cells of the dialysis patients and each of the CKD groups were compared with healthy subjects. In both the dialysis and CKD patients, the ATP amounts in CD4+ cells were lower than in healthy subjects. Furthermore, there were significant differences in the ATP amounts between healthy subjects and each of the CKD-G3a, CKD-G3b, and CKD-G4 groups (P

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α–induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

BACKGROUND Everolimus (EVL) plus tacrolimus (TAC) therapy is effective and safe in renal transplantation. However, the pharmacokinetic and pharmacodynamic information for EVL combined with TAC is limited. We investigated the pharmacodynamic drug-drug interaction between EVL and TAC at their therapeutic concentration range. MATERIAL AND METHODS Isolated peripheral blood mononuclear cells (PBMCs) from 22 healthy participants aged 22 to 24 years were cultured with concanavalin A (Con A) in the presence of EVL and/or TAC for 4 days, and the proliferation rate of the PBMCs was calculated. RESULTS TAC promoted the inhibitory efficacy of EVL against the mitogen-activated proliferation of PBMCs at the EVL therapeutic concentration range. When 0.175 ng/mL or more of TAC was combined with 30 ng/mL or more of EVL, the antagonistic effect of TAC on the inhibitory efficacy of EVL against the mitogen-activated proliferation of PBMCs was observed. Conversely, when 0.4 ng/mL TAC and 10 ng/mL or more of EVL were combined, the antagonistic effect of EVL on the inhibitory efficacy of TAC against the mitogen-activated proliferation of PBMCs was observed. CONCLUSIONS The pharmacodynamic synergistic efficacy of EVL and TAC in combination on mitogen-activated PBMCs was evident at the therapeutic concentration range, which is used in renal transplantation. However, these drugs antagonize each other to suppress the proliferation of activated PBMCs at concentrations higher than those clinically used.

Inappropriately activated T cells mediate autoimmune diseases and T cell acute lymphoblastic leukemia (T-ALL). Glucocorticoid and chemotherapeutic agents have largely extended lives of these patients. However, serious side effects and drug resistance often limit the prognosis of considerable number of the patients. The efficient treatment of autoimmune diseases or T-ALL with drug resistance remains an important unmet demand clinically. Bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine have been applied for the treatment of certain types of autoimmune diseases and cancers, while studies on their action mechanisms and their further applications combined with glucocorticoids or chemotherapeutic agents remains to be expanded. This review introduced molecular mechanisms of tetrandrine and cepharanthine in T cells, including their therapeutic implications. Both tetrandrine and cepharnthine influence the growth of activated T cells via several kinds of signaling pathways, such as NF-κB, caspase cascades, cell cycle, MAPK, and PI3K/Akt/mTOR. According to recent preclinical and clinical studies, P-glycoprotein inhibitory effect of tetrandrine and cepharnthine could play a significant role on T cell-involved refractory diseases. Therefore, tetrandrine or cepharanthine combined with glucocorticoid or other anti-leukemia drugs would bring a new hope for patients with glucocorticoid-resistant autoimmune disease or refractory T-ALL accompanied with functional P-glycoprotein. In conclusion, bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine can regulate several signaling pathways in abnormally activated T cells with low toxicity. Bisbenzylisoquinoline alkaloids deserve to be paid more attention as a lead compound to develop new drugs for the treatment of T cell-involved diseases in the future.

Diabetes mellitus is one of the most common causes of chronic renal failure. Immunosuppressive efficacies of glucocorticoids, calcineurin inhibitors, and mycophenolic acid are possibly affected by insulin after renal transplantation in these patients.We investigated the effects of insulin on responses of mitogen-activated human peripheral blood mononuclear cells (PBMCs) to several immunosuppressive drugs.Antiproliferative efficacies of prednisolone, hydrocortisone, cyclosporine, tacrolimus, and mycophenolic acid against concanavalin A-stimulated PBMCs were evaluated in the presence of physiological (5 μunits/mL) and super physiological (50 μunits/mL) concentrations of insulin. Insulin-receptor expressions on PBMCs were evaluated by flow cytometry.Insulin itself had no effects on the mitogen-induced proliferation of PBMCs. The IC50 values of cyclosporine against the mitogen-activated PBMCs in the presence of 5 or 50 μunits/mL insulin were significantly higher than those of cyclosporine without insulin (p 0.05). The IC50 values of mycophenolic acid significantly increased by 50 μunits/mL insulin (p 0.01). Insulin receptors were detected on the mitogen-activated CD4(+)/CD14(+ )cells in PBMCs.These results indicate that insulin at even physiological concentration attenuates suppressive efficacies of several immunosuppressive drugs against mitogen-activated proliferation of human PBMCs, possibly via insulin receptors. Insulin used in dialysis patients accompanying diabetes mellitus is suggested to attenuate efficacies of immunosuppressive drugs after renal transplantation.

We estimated the pharmacological efficacy of vitamin K1 (VK1 ) and VK2 on the mitogen-activated peripheral blood mononuclear cells (PBMCs) of paediatric atopic dermatitis (AD) patients. VK2 suppressed the in vitro proliferation of T-cell mitogen-activated PBMCs of AD patients. In contrast, VK1 had little effect on the PBMC proliferation. The IL-2 production from the activated PBMCs of AD patients significantly increased (P

What is known and objective Renal transplant recipients receive immunosuppressive therapy to prevent acute rejection. We evaluated the immunopharmacological efficacy of vitamin K1 (VK1) and vitamin K2 (VK2) on T-cell mitogen-activated-peripheral lymphocytes of dialysis patients and healthy subjects. Methods The effects of VK1 and VK2 on the T-cell mitogen-stimulated proliferation of peripheral blood mononuclear cells (PBMCs) obtained from 12 healthy subjects and 12 dialysis patients were estimated. Seven cytokines produced from the activated PBMCs were measured by a BD Cytometric Beads Array kit. Regulatory T cells (Tregs) in PBMCs were analysed as CD4 + CD25 + FoxP3 + lymphocytes by flow cytometry. Results VK2 dose-dependently suppressed the concanavalin A-stimulated proliferation of PBMCs from healthy subjects and dialysis patients, whereas VK1 had no significant effect on the PBMC proliferation. VK1 and VK2 did not influence the production of most of the Th1/Th2/Th17 cytokines from the activated PBMCs of these subjects, although VK2 increased the IL-4 production from PBMCs of healthy subjects. The Treg percentages in the PBMCs of dialysis patients were markedly decreased compared to healthy PBMCs after the treatment with relatively low concentrations of VK2. What is new and conclusion The present data suggest that VK2 has immunosuppressive efficacy. VK2 may enhance the immunosuppressive efficacies of glucocorticoids while preventing osteoporosis caused by glucocorticoids.

Background Machine perfusion (MP) techniques are expected to prove useful for preserving the organ viability and recovering organ function for organ transplantation. Furthermore, an accurate assessment of organ viability using MP is important for expanding the donor criteria. In this study, an ex vivo reperfusion model (ERM) simulating transplantation using diluted autologous blood under normothermic conditions was evaluated for its utility of MP under subnormothermic conditions for livers donated after cardiac death (DCD). Methods The liver preservation methods for DCD porcine livers were evaluated using the ERM. This investigation was performed using a novel perfusion system developed by our research group. Porcine livers were procured with a warm ischemia time (WIT) of 60 minutes. The organs were then preserved using subnormothemic machine perfusion (SNMP) or static cold storage (CS) for 4 hours. We also compared these tissues with SNMP livers procured under a WIT of 0 minutes. After the preservation, the livers were reperfused for 2 hours using the ERM with diluted autologous blood oxygenated by a membrane oxygenator under NMP conditions. Reperfusion was evaluated based on perfusion flow dynamics and outflow of deviating enzymes. Results In the early stages of reperfusion, pressure in the blood vessels increased sharply in the CS group. Furthermore, the amount of aspartate aminotransferase accumulation was lower in the SNMP group than in the other groups. These results suggest ischemia-reperfusion injury is suppressed in SNMP conditions. Conclusion An ERM has use in evaluating the utility of MP for the DCD liver.

Glucocorticoids play significant roles in treatments of inflammatory and autoimmune diseases. Some patients show a poor or absent response even to high doses of glucocorticoids. The purpose of this study was to explore whether tetrandrine combined with glucocorticoids could be a new treatment strategy to resolve glucocorticoids resistance. Information on glucocorticoids sensitivity was usually obtained through mitogen-activated human peripheral blood mononuclear cells in cell culture procedures. Thus, human peripheral blood mononuclear cells was chosen as a model to study the immunosuppressive effect of methylprednisolone combined with tetrandrine, including the possible action mechanisms. Tetrandrine decreased the IC50 value of methylprednisolone significantly, but it showed little toxic effect on the concanavalin A-activated human peripheral blood mononuclear cells. Both tetrandrine and methylprednisolone inhibited the secretion of pro-inflammatory cytokines TNFα and IL-6 significantly and the combination showed stronger inhibitory ability. Tetrandrine and/or methylprednisolone did not increase the percentage of CD4+ CD25+ Foxp3+ regulatory T cells in CD4+ T cells. However tetrandrine with or without methylprednisolone significantly inhibited the function of drug efflux pump P-glycoprotein 170 of CD4+, CD8+ T cells and lymphocytes. Tetrandrine tended to suppress the phosphorylation of mitogen-activated protein kinase and this effect was potentiated by methylprednisolone. These tetrandrine effects were suggested to be beneficial for improving the immunosuppressive efficacy of glucocorticoids. Glucocorticoids combined with tetrandrine could be a new therapeutic approach to resolve glucocorticoids-resistance possibly via inhibiting the function of P-glycoprotein and blocking mitogen-activated protein kinase signaling pathway from but not affecting on CD4+ CD25+ Foxp3+ regulatory cells.

Background Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT). Methods Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) − gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n = 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n = 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n = 6), grafts were preserved with 2 hours of SCS and rewarming up to 25°C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically. Results The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group. Conclusion RMP is expected to facilitate the recovery of the DCD liver grafts.

The lymphocyte immunosuppressant sensitivity test (LIST) with 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay procedure has been used to predict the pharmacological efficacy of immunosuppressive agents to prevent acute rejection episodes for renal transplant recipients. In this study, mycophenolic acid (MPA) pharmacological efficacies were evaluated by LIST at both prior to and just after renal transplantation. We compared the efficacies to the clinical outcome of these recipients. MPA's pharmacological efficacy was evaluated by LIST not only before the operation but also at 2, 4, and 6 weeks after transplantation in 16 renal transplant recipients. These recipients were divided into high- and low-sensitivity groups according to peripheral blood mononuclear cell (PBMC) sensitivity to MPA in vitro. The MPA sensitivities were compared to cytomegalovirus (CMV) infection and acute rejection episodes in these recipients under MPA immunosuppressive therapy. The rate of CMV infection episodes in the low-MPA pharmacological efficacy group categorized at 2 weeks after renal transplantation was 5/6 (83.3%), which was significantly higher than the rate of 1/10 (10.0%) ( p < 0.01) in the high-MPA sensitivity group. However, the MPA pharmacological efficacy evaluated both before and after transplantation had no relationship with the incidence of rejection episodes. These findings suggest that the MPA pharmacological efficacy evaluated by LIST at 2 weeks after operation is a useful biomarker for predicting the following occurrence of CMV infection episodes in renal transplant recipients.

Background: We have often observed that the blood trough concentration (Ct) of the once-daily, prolonged-release formulation of tacrolimus (Graceptor®; GRC) becomes unstable, and it is difficult to adjust it in the early period after transplantation. Consequently, we compared the relationships between pharmacokinetic parameters and influencing factors in a group of GRC-treated patients compared with those in a group of Prograf® (PRG)-treated patients. Methods: The study included 8 patients who were newly treated with GRC and 44 patients who were newly treated with PRG. We performed 24-hour therapeutic drug monitoring to compare the relationships between pharmacokinetic parameters, including the area under the curve (AUC), the ratio of the area under the trough level (AUTL)/AUC to indicate the relationships among AUCs, peak concentration (Cp), and Ct. The Cp/Ct values, and dose/body weight (UC/ [D/BW]) values reflected bioavailability and the influencing factors; number of days after transplantation and Dose/BW in the GRC-treated and PRG-treated patients. Results: The Cp/Ct values were higher and the AUTL/AUC and AUC corrected by dose/body weight (AUC/[D/BW]) values were lower with a low number of days after transplantation (particularly within 20 days) and a large dose than a high number of days after transplantation and a small dose in the GRC-treated patients. No such associations were observed in the PRG-treated patients. Conclusion: Care is required when using GRC as there is a tendency for Cp to increase owing to rapid absorption within 20 days after transplantation. Blood levels may be unstable and side effects may be more prevalent in some patients. Moreover, the utilization rate is low and the dose is high in such patients; therefore, unstable gastrointestinal function caused by a variety of factors may play a role in the early period after transplantation.

Background Graceptor is a new modified-release once-daily formulation of tacrolimus with an efficacy and safety profile similar to twice-daily tacrolimus (Prograf), as identified by clinical trials, offering a more convenient dosing regimen to improve adherence. The aim of this study was to analyze the safety of a 1:1 dose conversion from twice-daily Prograf to once-daily Graceptor in stable kidney transplant recipients. Methods We switched 33 Japanese patients who had undergone kidney transplantation ≥1 years before from twice-daily Prograf to once-daily Graceptor. The dose conversion ratio between Prograf and Graceptor was 1:1. We compared the following parameters: minimum tacrolimus concentration (C min ); concentration dose per weight (CDW); serum creatinine (sCr); blood urea nitrogen (BUN); total cholesterol (TC); high-density lipoprotein cholesterol (HDL-C); uric acid (UA); fasting blood sugar (FBS). Time points for measurements were 1 month before study start and 1 and 2 months afterward. Results The mean age of the subjects in this study was 46.5 ± 13.1 years. Mean C min decreased from 4.55 ± 1.79 to 3.20 ± 1.22 ng/dL. The mean CDW also decreased, from 99.8 ± 69.5 to 75.0 ± 55.1 mg/dL/kg over the 2 months. There were no significant changes in sCR, BUN, UA, and FBS. Mean TC increased from 187.5 ± 51.4 to 194.3 ± 43.4 mg/dL, and mean HDL-C changed from 53.7 ± 12.0 to 56.1 ± 11 mg/dL. There were no episodes of rejection or infection. Conclusions We conclude that switching from Prograf to Graceptor is safe and has the advantage of improving adherence. It could also have a beneficial effect in controlling glycemic levels and the adverse effects of tacrolimus. In many cases (25%–30%), the minimum concentration of tacrolimus decreased after changing tablets. With Graceptor, the ratio of area under trough level to area under the curve (AUC) is low compared with Prograf, resulting in low C min values of 1–2 ng/mL, and the AUC for Graceptor is very similar to that for Prograf.

The lymphocyte immunosuppressant sensitivity test (LIST) with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay procedure can predict the pharmacological efficacy of immunosuppressive agents. A previous study reported the pharmacological efficacy of tacrolimus evaluated by LIST just before renal transplantation significantly correlated with the incidence of acute rejection episodes. However, the pharmacological efficacy of tacrolimus has not been estimated after renal transplantation. Therefore, the present study evaluated the pharmacological efficacy of tacrolimus by LIST using the MTT assay procedure before and 1, 3, and 12 months after transplantation in 17 renal transplant recipients that received tacrolimus-based immunosuppressive therapy. The tacrolimus pharmacological efficacies before and after the procedure were also compared with incidence of acute rejection and cytomegalovirus (CMV) infection episodes. The individual values of tacrolimus 50% inhibition of lymphocyte proliferation (IC50) varied widely before transplantation, and the mean value of the IC50 was 126.4 ± 337.7 ng/ml. The patients were divided into two groups according to the tacrolimus IC50 values evaluated before transplantation. The rate of acute rejection episodes in the tacrolimus high-sensitivity group was significantly lower than that in the tacrolimus low-sensitivity group (p = 0.005). The tacrolimus IC50 deviation between patients expanded further at one and three months after surgery. However, the sensitivity deviation almost converged at 1 year after surgery. Moreover, the pharmacological efficacy of tacrolimus evaluated at 1, 3, and 12 months after transplantation did not significantly correlate with the incidence of acute rejection episodes. The pharmacological efficacies of tacrolimus evaluated at both before and after surgery were not significantly correlated with the episodes of CMV infection. These findings suggest that the pharmacological efficacy of tacrolimus evaluated with LIST before surgery is a useful biomarker for predicting the occurrence of acute allograft rejection in renal transplantation.

Myasthenia gravis (MG) is an autoimmune disorder generally mediated by antibodies against the acetylcholine receptors of the skeletal muscles. Depending on the disease burden, MG patients may experience chronic dysregulation of both the hormonal stress axis and the immune system, consequently, aggravating the disease itself but also leading to secondary psychopathological abnormalities. A long-term clinical course requires long-term glucocorticoid (GC) therapy, which may change the psychological state by affecting the pituitary-adrenocortical system in MG patients. In this study, we investigated the function of the pituitary-adrenocortical system in MG patients who were treated with prednisolone (PSL) and evaluated their quality of life by using the Medical Outcomes Study 36-item Short-Form Health Survey and the 28-item general health questionnaire (GHQ-28). ACTH and cortisol levels in the plasma of patients who were treated with PSL (PSL[+] group, n = 18) were lower than those in the plasma of patients who were treated without PSL (PSL[-] group, n = 29; P < 0.05 and P < 0.01, respectively). In the PSL(+) group, we confirmed that cortisol levels negatively correlated with daily PSL dosages (P < 0.05). The anxiety and depression scores from the GHQ-28 in the PSL(+) group were lower than those in the PSL(-) group (P < 0.05, respectively). There was no significant correlation between cortisol levels and corticotropin levels in plasma of the PSL(-) group. However, we confirmed that corticotropin levels positively correlated with cortisol levels in plasma (P < 0.01) and negatively correlated with anxiety/insomnia scores from the GHQ-28 (P < 0.05) in the PSL(+) group. In conclusion, low-dose GC treatment complemented the pituitary-adrenocortical system and improved the psychological state in MG patients.

Bacterial colonization on skin or tonsil may influence the clinical response of patients with psoriasis to immunosuppressive drugs. However, few studies have investigated the effects of bacterial superantigens on therapy in these patients. Recently, combination therapy with topical glucocorticoids (GC) and vitamin D3 (VD3) appears to be more effective than GC or VD3 monotherapy for psoriasis. We evaluated the suppressive effects of betamethasone butyrate propionate (BBP), three VD3 derivatives (calcipotriol, maxacalcitol and tacalcitol), cyclosporin and BBP plus VD3, on concanavalin A (ConA)- or streptococcal pyrogenic enterotoxin A (SPEA)-stimulated proliferation of peripheral blood mononuclear cells (PBMC) obtained from 35 psoriasis patients. Drug concentrations effecting 50% inhibition concentration of ConA- or SPEA-stimulated PBMC proliferation were estimated. Cytokine levels of tumor necrosis factor-α, γ-interferon, interleukin-1b, -2, -4, -5, -6, -8 -10 and -12p70 in PBMC culture supernatants were measured with bead-array procedures. Suppression of PBMC proliferation by BBP was significantly lower when PBMC were stimulated by SPEA than when stimulated by ConA. In contrast, the suppressive effects of calcipotriol and tacalcitol increased significantly when PBMC were stimulated by SPEA than when stimulated by ConA. The suppressive effect of BBP on SPEA-stimulated PBMC proliferation was improved significantly by adding 1-1000 ng/mL calcipotriol, compared with BBP alone. Cytokine levels in PBMC culture supernatants were not significantly different between ConA- and SPEA-stimulated PBMC. Calcipotriol and BBP in combination markedly suppressed SPEA-stimulated PBMC proliferation. SPEA produced by colonization of hemolytic streptococci may reduce the efficacy of BBP but not VD3 derivatives in the treatment of psoriasis.

The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of immunosuppressive agents. We herein estimated the mycophenolic acid efficacy using LIST before and 1, 3, and 12 months after the operation in 15 renal transplant recipients. The pharmacological efficacy of mycophenolic acid as assessed before transplantation showed small individual variations, whereas the variability greatly increased at 1 and 3 months after transplantation. Furthermore, the individual IC50 variation among these subjects at 1-year after operation was closely similar to that observed before surgery. No significant implications of these individual differences in the mycophenolic acid sensitivity were noted in regard to the clinical courses of these recipients.

Successful immunosuppressive therapy is critical for liver transplantation; however, a considerable number of patients experience fatal rejection or alternatively exhibit serious infection resulting from excessive immunosuppression. The in vitro tacrolimus response of peripheral blood mononuclear cells (PBMCs) before transplantation was compared to the clinical outcome up to 4 weeks after operation in 28 living-donor liver transplant recipients treated with tacrolimus. The tacrolimus IC50 values against concanavalin A-induced PBMC blastogenesis in vitro were calculated. These recipients were classified into two groups with the mean tacrolimus IC50 (0.18 ng/ml) as the cutoff point, after which the clinical outcome between the patient groups was compared. The allograft rejection incidence in the low-sensitivity group (IC50 < 0.18 ng/ml; n = 16) was 6/12 (50.0%), which was significantly higher than the incidence of 2/16 (12.5%) in the high-sensitivity group (IC50 > 0.18 ng/ml; n = 12) ( p = 0.0297). In contrast, the infection incidence in the high-sensitivity group was 6/16 (37.5%), which was significantly higher than that of the low-sensitivity group (1/12; 8.3%) ( p = 0.0401). These data suggest that patients exhibiting a low PBMC sensitivity to tacrolimus have a risk of rejection, whereas highly sensitive patients have a risk of infection in living-donor liver transplantations under tacrolimus therapy.

Leptin is a small peptide hormone which centrally regulates weight. Leptin receptor (OB-R) is expressed in hematopoietic cells, the central nervous, and immune systems. OB-R bears a homology similar to members of the class Iota cytokine family, and therefore, leptin appears to modulate immune responses. The aim of this study was to examine the effect of plasma leptin, soluble OB-R (sOB-R), and the free leptin index (FLI), the ratio between leptin and sOB-R levels, on the sensitivities of peripheral blood mononuclear cells (PBMCs) to prednisolone and cyclosporine in 16 healthy subjects and seven nephrotic syndrome (NS) patients. The NS patients had significantly higher serum sOB-R and lower FLI, compared with the healthy subjects (respectively, P=0.0026, P=0.0383). Whereas, the NS patients had significantly lower PBMC sensitivity to prednisolone (P=0.0049). PBMCs sensitivity to cyclosporine was not significantly different between the healthy subjects and the NS patients. In addition, when the data from all subjects were analyzed, there was a significantly positive correlation between plasma sOB-R concentrations and the IC50 values of prednisolone (P=0.0478). In contrast, plasma leptin concentrations and FLIs did not correlate significantly with the prednisolone and cyclosporine IC50 values. From these observations it can be suggested that plasma leptin has little effect on PBMC sensitivity to immunosuppressive drugs in NS patients. Molecular background(s) for the influence of sOB-R on the PBMC sensitivity to glucocorticoids remain(s) to be elucidated.

Many epidemiological studies have shown that coffee consumption reduces the risk of type 2 diabetes mellitus (T2D), although the reasons as to why remain unclear. In this study we investigated the effect of caffeine on pancreatic beta-cell damage in rats using the diabetogenic agent, streptozotocin (STZ). Wistar rats were given intraperitoneal injections of saline or caffeine (10, 50 or 100 mgkg−1). After 15 min, the rats were injected with a citrate buffer or 65 mgkg−1 STZ. Three days after injection, an oral glucose tolerance test (OGTT) was performed on the rats. Furthermore, three days after the OGTT, the pancreas was isolated and homogenized, followed by determination of insulin content. STZ treatment significantly increased the plasma glucose level compared with the control at all times during the OGTT, which was significantly diminished by caffeine pretreatment at all doses. STZ treatment significantly decreased the plasma insulin level, however, which was not recovered by caffeine pretreatment. Pancreatic insulin content was significantly reduced by STZ treatment compared with the control, which was significantly recovered by caffeine pretreatment at a dose of 100 mgkg−1 (P < 0.01). We showed that caffeine protects pancreatic beta-cells against STZ toxicity. Further investigation will be required to understand the protective effect of caffeine against beta-cell destruction in T2D.

The clinical efficacy of calcineurin inhibitors administered to renal transplant patients is considered to be a strong function of the area under the concentration time curve (AUC). Interestingly, monitoring timings of blood concentrations for two similar calcineurin inhibitors, cyclosporine (CYA; Neoral®) and tacrolimus (TAC; Prograf®) are different. Namely, CYA blood concentration is usually monitored at 2 h after administration (C2) substituted for peak concentration (Cp) and TAC at trough concentration (Ct). In the literature, data describing such characteristics of CYA and TAC have been presented in the past. However, each of these patient groups had different backgrounds. We have attempted to examine the behavior of blood concentration curves simultaneously for both CYA and TAC by establishing controlled groups of renal transplant patients with similar clinical backgrounds. Furthermore, we have analyzed the correlation with Cp and Ct versus AUC implementing area under the trough level (AUTL), or area above the trough level (AATL) as new pharmacokinetic parameters, such that C2 for CYA and Ct for TAC have been verified using controlled clinical data. We have also found distinct differences in the pharmacokinetics between CYA and TAC with the relationships between AUC, Cp, and Ct.

Over-expression of P-glycoprotein (P-gp) in lymphocytes is implicated in the failure of immunosuppressant therapy. We investigated P-gp function in peripheral-blood CD3+, CD4+, and CD8+ cells of 14 healthy subjects and 12 patients with systemic lupus erythematosus (SLE). P-gp function was estimated by the transporter activity of the cells based on the efflux of Rhodamine-123 (Rh123) from the cells in the presence or absence of a P-gp inhibitor, cyclosporine A. P-gp function in the CD8+ cells of the healthy subjects was significantly higher than that of the SLE patients (p=0.0318), whereas the function in CD3+ cells and CD4+ cells were not significantly different between the healthy subjects and the SLE patients. The patients were divided into two subgroups according to their clinical response to glucocorticoid (GC) therapy, i.e., a high-response group (HR) (n=6) and a low-response group (LR) (n=6). In contrast, P-gp function in CD4+ cells of the LR group was significantly higher than that of the HR group (p=0.0432). Further, no significant differences in the P-gp function in CD3+ and CD8+ cells were observed between the two groups. The data showed a relationship between clinical sensitivity to GC therapy and P-gp function of CD4+ cells in SLE patients. Thus, the estimation of P-gp function in peripheral-blood CD4(+) cells might be useful for the estimation of clinical response to GC therapy.

Leukemia is a type of cancer of the body’s blood-forming tissues, including the bone marrow and the lymphatic system. Treatments for leukemia are complex, depending upon the type of leukemia and other factors. Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) and accounts for approximately 10-15% of all cases of AML in adults. Arsenic and its compounds are widely distribut‐ ed in the environment and have been used medicinally for over 2,000 years. In fact, investigators from China and the USA have demonstrated that treatment with ATO (As2O3, AsIII) results in complete remission in 90% of relapsed APL patients since mid-1990s. Moreover, As2S2 or As4S4, also known as realgar, has been gaining increasing attention and is traditionally used to treat certain types of hematological disorders including chronic myeloid leukemia (CML), AML, myelodysplastic syndrome (MDS) and MDS/AML in China. In this chapter, we first highlight the pharmacokinetics of ATO and realgar in leukemia patients and/or healthy volunteer. We will further summarize the detailed mechanisms underlying the cytocidal effects of these arsenic compounds. We also provide detailed insight into potential future clinical applica‐ tion of those promising candidates endowed with potent antitumor activities in view of combination with arsenic compounds.

The success of chemotherapy in cancer treatment is frequently limited by intrinsic or acquired multidrug resistance due to increased expression of a plasma membrane P-glycoprotein . P-glycoprotein is an ATP-dependent transporter that effluxes many lipophilic anticancer agents out of cells, thereby reducing intracellular drug concentration and results in cancer cell survival. It has been reported that certain kinds of natural compounds and their derivatives inhibit P-glycoprotein function, and overcome the multidrug resistance phenotype.

Successful immunosuppressive therapy is critical for liver transplantation. However, a considerable number of patients show clinical resistance to the therapy and experience rejection episodes, or alternatively exhibits serious adverse effects of drugs. We examined the in vitro response of peripheral blood mononuclear cells (PBMCs) to immunosuppressive drugs in cirrhosis patients awaiting liver transplantation. We evaluated the suppressive efficacy of prednisolone, methylprednisolone, cyclosporine, and tacrolimus on the in vitro blastogenesis of PBMCs obtained from 22 cirrhosis patients and 31 healthy subjects. In vitro drug concentrations giving 50% inhibition of PBMC blastogenesis (IC50s) were calculated. Two out of these 22 patients received liver transplantation from living donors, and their clinical courses were surveyed until 5 weeks after operation. The median IC50 values for prednisolone, cyclosporine, and tacrolimus against blastogenesis of PBMCs from cirrhosis patients were significantly lower than those of PBMCs from healthy subjects (p < 0.01). However, large individual differences were observed in the IC50 values of the immunosuppressive drugs examined, especially in the cirrhosis patients. One recipient exhibiting high PBMC sensitivity to tacrolimus (IC50 = 0.001 ng/ml) showed good clinical course without rejection until 5 weeks after liver transplantation. The other recipient exhibiting relatively low PBMC sensitivity to taclolimus (IC50 = 0.30) showed allograft rejection at 1 week after operation. We concluded from these observations that PBMCs of cirrhosis patients are vulnerable to the immunosuppressive effects of prednisolone and calcineurin inhibitors. However, large individual variations in the IC50 values suggest that patients exhibiting relatively lower sensitivity to these drugs may have risks of rejection, whereas highly sensitive patients are possibly able to reduce the dose of immunosuppressive drugs to avoid serious drug-adverse effects, after liver transplantation.

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.

Successful immunosuppressive therapy is critical for the treatment of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and nephrosis. However, a considerable number of patients have shown clinical resistance to therapy. Bacterial infection might influence the clinical response of patients to immunosuppressive drugs, but few studies have been carried out to investigate the effect of bacterial superantigens on the efficacy of the drugs in these patients. We evaluated the suppressive efficacy of prednisolone, methylprednisolone, cyclosporine, and tacrolimus on the blastogenesis of PBMCs obtained from 12 ANCA-associated vasculitis patients (ANCA patients), eight patients with nephrotic syndrome, and eight healthy subjects. PBMC-stimulation index was calculated from the formula: [3H]thymidine incorporated in the presence of stimulant (dpm)/[3H]thymidine incorporated in the absence of stimulant (dpm). In vitro drug concentrations giving 50% inhibition (IC50s) of PBMC blastogenesis stimulated with concanavalin A (con A) or toxic shock syndrome toxin 1 (TSST-1) derived from Staphylococcus aureus (S. aureus) were calculated. The IC50 values for the four drugs evaluated in TSST-1-stimulated PBMCs were significantly higher than those evaluated in con A-stimulated PBMCs in both ANCA patients and nephrosis patients (p

Background:Staphylococcus aureus-producing superantigens (SAgs), such as staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin-1 (TSST-1), are frequently observed in atopic dermatitis (AD). However, little has been done to establish the association of immune responses to SAgs and the therapeutic response to immunosuppressive drugs in AD. Therefore, we investigated the prevalence and role of SAgs in the pathophysiology and immunosuppressive drug sensitivity in AD patients. Methods: We classified 29 patients into two groups on the basis of their clinical AD scores: a low-score group (n = 14) corresponding to mild to moderate patients and a high-score group (n = 15) corresponding to severe patients. We estimated the plasma anti-SEB or TSST-1 IgE of these patients and healthy subjects by ELISA. We also estimated individual drug sensitivity by determining drug concentrations that would give 50% inhibition (IC50) of peripheral-blood mononuclear cell (PBMC) proliferation in vitro. Results: The levels of plasma anti-SEB or TSST-1 IgE in the severe patients were significantly higher than those in the mild to moderate patients (p < 0.05 and p < 0.01, respectively). When stimulated with concanavalin A in vitro, PBMCs in the severe patients exhibited low sensitivity to the suppressive efficacy of tacrolimus (FK506) as compared to the mild to moderate patients (p < 0.01). Furthermore, there was a significant correlation between the IC50s of FK506 and plasma anti-TSST-1 IgE levels (p < 0.01). Conclusions: We showed that PBMCs in severe AD patients exhibited lower sensitivity to FK506, and had higher plasma levels of anti-TSST-1 IgE as compared to the mild AD patients. SAgs appear to be one of the causes of decreased PBMC sensitivity to FK506, and therefore an alternative treatment would be useful based on the individual drug sensitivity data and anti-TSST-1 IgE levels.

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.

Overexpression of multidrug resistance (MDR) protein, P-glycoprotein (P-gp), on lymphocytes has been suggested to be implicated in the failure of glucocorticoid (GC) therapy in patients with ulcerative colitis (UC). However, whether the overexpression of P-gp in a class of patients with inflammatory bowel disease (IBD) is intrinsic or related to the administration of GC is unknown. Relative amounts of MDR1 mRNA expressed in peripheral blood mononuclear cells (PBMCs) were measured using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique in 25 UC patients having no history of GC administration, 25 UC patients having experienced GC therapy, 19 patients with Crohn's disease (CD) with no history of GC therapy, and 27 healthy subjects. Relative amounts of MDR1 mRNA expressed in PBMCs were compared among the groups. The relationship between the amounts of MDR1 mRNA expressed, as well as the total dose of GC administered or the period of GC therapy in UC patients, was examined. The relative amounts of MDR1 mRNA expressed in PBMCs were not significantly different between the healthy subjects and CD patients or UC patients having no history of GC therapy. However, the mean MDR1 mRNA amount in PBMCs of UC patients having experienced GC therapy was significantly greater than that in PBMCs of UC patients with no history of GC administration (p = 0.0375). The amounts of MDR1 mRNA in PBMCs of UC patients having experienced GC therapy significantly correlated with the total dose of GCs administered (p = 0.0175). Overexpression of MDR1 mRNA in PBMCs of IBD patients is not intrinsic. However, high-dose administration of GCs for the treatment of UC may result in an increased expression of MDR1 mRNA, which may impair successful GC therapy in these patients.

We investigated peripheral-blood mononuclear cell (PBMC) response to immunosuppressive drugs and its influence on glucocorticoid therapy in ulcerative colitis (UC). IC50s of immunosuppressive drugs on in vitro blastogenesis of PBMCs stimulated with concanavalin A were estimated in 76 UC and 146 healthy subjects. Individual differences in IC50s for prednisolone, methylprednisolone, cyclosporine, and tacrolimus on blastogenesis of PBMCs from UC patients were spread from 11.0 to 1000, 0.6 to 1000, 0.01 to 1000, and 0.001 to 4.6 ng/ml, respectively. Normal upper thresholds for IC50s of these drugs were estimated from the mean + 2 S.D. of the IC50s of healthy PBMCs, and the patients exhibiting IC50s over these levels were arbitrarily considered as resistant. The incidences of resistance to glucocorticoids and cyclosporine in UC were significantly higher than those in healthy subjects (p < 0.0005). In 14 UC patients, there was a significant correlation between amounts of prednisolone (p < 0.05) or period of prednisolone administration (p < 0.05) for UC treatment and prednisolone IC50. The results showed that large individual deviations in PBMC response to the drugs were observed in UC, and UC patients exhibiting low PBMC sensitivity to prednisolone required a high prednisolone amount as well as long period of prednisolone administration for treatment. Thus, the drug sensitivity tests could be informative to single out refractory patients to the immunosuppressive therapy.

Introduction: Aromatase inhibitor use for postmenopausal hormone-sensitive breast cancer patients often results in drug-induced osteoarthropathy, while its accurate mechanism has not been clarified. We investigated the implication of female hormones and several cytokines in osteoarthropathy complicated with aromatase inhibitor treatment, and the efficacy of vitamin E on the severity of osteoarthropathy, in breast cancer patients. Methods: Sixty two breast cancer patients treated with aromatase inhibitor for average of 1.77 years were included. These patients were orally administered vitamin E (150mg/day) for 29.8 days to alleviate aromatase inhibitor-related osteoarthropathy. Severity of osteoarthropathy was scored, and the patients were grouped based on the severity or vitamin E efficacy. Serum estradiol, progesterone, vitamin E, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), IL-2, IL-4, IL-6, IL-10, and IL-17A concentrations were measured by ELISA or beads array procedures followed by flow cytometry. Results: There was no significant difference in serum concentrations of the biomarkers between the severe and the mild osteoarthropathy groups before vitamin E administration. The osteoarthropathy scores significantly decreased after vitamin E administration (p=0.0243), while serum-estradiol concentrations did not change. The serum-estradiol concentrations before vitamin E administration in the group sensitive to the vitamin E efficacy were significantly lower, as compared with those in the insensitive group (p=0.0005). The rate of the highly sensitive patients to the vitamin E efficacy in those exhibiting low serum-estradiol concentrations was significantly higher than that in the high serum-estradiol group (p=0.0004). In the sensitive group, serum-estradiol concentrations after taking vitamin E were significantly higher than those before taking vitamin E (p=0.0124). Conclusions: Vitamin E administration seemed to be a potential way for relieving osteoarthropathy complicated with aromatase inhibitor use. Using serum-estradiol concentration, it would be possible to select out the breast cancer patients who will respond well to the vitamin E therapy for osteoarthropathy complicated with aromatase inhibitor.

A reliable biomarker to differentiate high-risk recipients who will experience a decrease in allograft function after glucocorticoid withdrawal has not been established in renal transplantation. We examined the clinical significance of peripheral blood lymphocyte sensitivity to glucocorticoids in vitro for the differentiation of the high-risk patients after glucocorticoid reduction/withdrawal in renal transplant recipients.The study included 44 renal transplant recipients with stable allograft function. Peripheral lymphocyte responses to suppressive effects of cortisol, methylprednisolone, cyclosporine, and tacrolimus in mitogen assay procedures in vitro were examined. Clinical outcome after glucocorticoid reduction/withdrawal was retrospectively compared between recipients with lymphocytes normally sensitive to the drugs and those with hyposensitivity. The receiver-operating characteristic (ROC) curve analysis was undertaken for setting the cutoff IC50 values of the drugs against the T cell mitogen-induced lymphocyte proliferation to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal.The median (range) IC50 value for cortisol in the recipients who showed decreased renal function due to glucocorticoid withdrawal was 10,000 (570.9-72,279.3) ng/mL (n = 9), which was significantly higher than the value of 351.6 (2.0-10,000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 35) (P0.001). Similarly, the median (range) IC50 value for methylprednisolone in the recipients who showed decreased renal function after glucocorticoid withdrawal was 69.1 (21.5-1442.7) ng/mL (n = 9), which was significantly higher than the value of 13.8 (0.7-1000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 30) (P0.003). In contrast, there was no significant difference in the median IC50 values of cyclosporine and tacrolimus between the 2 recipient subgroups. The ROC curve analyses for the IC50 values of the immunosuppressive drugs estimated the cutoff value of cortisol and methylprednisolone to be 3580.0 and 21.5 ng/mL, respectively. The ROC AUCs for cortisol and methylprednisolone were 0.83 and 0.84, respectively. According to the cutoff IC50 value, the incidence of decreased allograft function in the low cortisol sensitivity (IC503580.0 ng/mL) subgroup was 7 of 13 patients, which was significantly higher than that of the higher sensitivity subgroup of 2 of 31 (P = 0.0012). A similar case was observed using the cutoff IC50 value of methylprednisolone (P = 0.0012), whereas recipient grouping according to the cutoff IC50 values of cyclosporine and tacrolimus failed to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal.Glucocorticoid pharmacodynamics in lymphocytes of individual patient origin is a reliable biomarker for differentiation of renal transplant recipients who will experience a safe reduction/withdrawal of glucocorticoid.

Cellular pharmacodynamics of cyclosporine (INN, cyclosporin) is considered to be closely implicated in clinical efficacy of the drug in kidney transplantation and other immunologic disorders. We applied this strategy to patients with minimal change nephrotic syndrome to predict individual clinical efficacy of cyclosporine.Drug sensitivity tests were carried out with peripheral blood mononuclear cells from 31 patients with minimal change nephrotic syndrome. The 50% lymphocyte-mitosis inhibition of cyclosporine on in vitro blastogenesis of peripheral blood mononuclear cells stimulated with concanavalin A were estimated, and interpatient variations of 50% lymphocyte-mitosis inhibition were evaluated. The relationship between cyclosporine-50% lymphocyte-mitosis inhibition and clinical outcomes indicated a decrease of urinary protein and the period required for complete remission under cyclosporine therapy was examined in 14 patients. We also evaluated the correlation between cyclosporine-50% lymphocyte-mitosis inhibition and interleukin-2 production and percentages of interleukin 2 receptor-positive peripheral blood mononuclear cells in vitro.Cyclosporine 50% lymphocyte-mitosis inhibition on peripheral blood mononuclear cell blastogenesis deviated largely between patients from 0.2 to 86.0 ng/mL. We found a statistically significant negative correlation between cyclosporine-50% lymphocyte-mitosis inhibition in vitro and decreasing rates of urinary protein at 1 week after onset of cyclosporine administration (r = -0.655, P.02). When we arbitrarily divide the 14 patients who received cyclosporine therapy according to their median 50% lymphocyte-mitosis inhibition of cyclosporine into two groups, that is, a high-sensitivity group (50% lymphocyte-mitosis inhibition18.1 ng/mL, n = 6) and a low-sensitivity group (50% lymphocytemitosis inhibition18.1 ng/mL, n = 8), the period required for complete remission was significantly shorter in the high-sensitivity group (P.03). The 50% lymphocyte-mitosis inhibition of cyclosporine on interleukin-2 production in culture medium was correlated with 50% lymphocyte-mitosis inhibition of the drug on peripheral blood mononuclear cell blastogenesis (r = 0.806, P.02). Decreasing rates of interleukin-2R-positive cells by cyclosporine treatment in vitro were negatively correlated with peripheral blood mononuclear cells blastogenesis in the presence of the drug (r = -0.694, P.02).Peripheral blood mononuclear cell response to cyclosporine in vitro is closely related to clinical efficacy of the drug in minimal change nephrotic syndrome. Peripheral blood mononuclear cell resistance to cyclosporine was correlated with ability of the cells to express interleukin 2 and interleukin 2R.