Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells

Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe’s equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity.     

Simultaneous LC-MS-MS determination of cyclosporine A, tacrolimus, and sirolimus in whole blood as well as mycophenolic acid in plasma using common pretreatment procedure

The purpose of the study was to develop rapid and simple procedure for simultaneous determination of cyclosporine A (CsA), tacrolimus (TCR), and sirolimus (SIR) in whole blood and mycophenolic acid (MPA) in plasma. Ascomycin (ASCO), cyclosporine D (CsD), and desmethoxysirolimus (DMSIR) were used as internal standards (IS) for TCR, CsA and MPA, and SIR, respectively. In the method development, six-level blood calibrators were used for CsA (range 47-1725 ng/ml), TCR (range 2.1-38.8 ng/ml), and SIR (range 2.4-39.6 ng/ml). Four-level calibrators were used for MPA (range 0.15-5.48 microg/ml). Four levels of quality control (QC) standards were used for blood samples, together with two levels of QC standards in plasma. All QC standards and calibrators were obtained from commercial sources. Sample preparation based on precipitation of 50 microl of sample in zinc sulfate-methanol-acetonitrile mixture containing IS, followed by centrifugation. HPLC was performed on ChromSpher pi column, 30 mm x 3 mm, in ballistic gradient of ammonium formate buffer-methanol at 0.8 ml flow rate. Following gradient elution profile was applied: 0-1.2 min at 30% methanol (divert valve to waste), 1.21-3.1 min 97% methanol (divert valve to detector), 3.11-3.7 min 30% methanol (divert valve to waste). ESI-MS-MS (MRM) was done on TSQ Quantum instrument with ESI source in positive ion mode. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes but MPA. For this compound sodium adduct was used. Following transitions were monitored: for CsA m/z 1220-1203; for CsD 1234-1217; for SIR 931.6-864.5 and 882.6; for DMSIR 902-834.5; for TCR 821.5-768.5 and 785.5; for ASCO 809.5-756; for MPA 343-211.6; for MPA-glucuronide 514-306 and 211.6. The limits of quantitation were: 1 ng/ml for TCR and SIR, 20 ng/ml for CsA, and 0.1 microg/ml for MPA. Post-column infusion experiments showed that no positive or negative peaks appeared after injection of matrix in the elution range of target compounds. General signal suppression caused by matrix ranged from 20-40%, and was caused mainly by zinc sulfate present in deproteinizing solution. Extracted samples were stable for 2 days at 4 degrees C and for at least 20 days at -20 degrees C. MPA was fully separated from its glucuronide, which was eluted at around 0.7-0.8 min and directed to the waste. Some mutual cross-contribution of CsD and CsA was observed (below 1%), other IS did not contribute to target compounds and vice versa. Observations of chromatograms from patients taken single therapy demonstrated that possible metabolites of CsA, TCR, or SIR did not interfere with target compounds or IS.     

自噬对高糖诱导的人冠状动脉内皮细胞凋亡的保护作用

目的:研究自噬对高糖诱导的人冠状动脉内皮细胞凋亡的影响。方法:将人冠状动脉内皮细胞,分别用常规培养基(正常对照组)、含30 mmol/L D-葡萄糖的高糖培养基(高糖组)、高糖培养基合并雷帕霉素(Rapamycin,RAPA;100 nmol/L)干预(RAPA组)和高糖培养基合并3-甲基腺嘌呤(3-Methyladenine,3-MA,5 mmol/L)干预(3-MA组)培养。利用CCK-8法检测细胞生长活力,使用流式细胞术检测细胞凋亡水平,western blot检测细胞自噬标记蛋白(Beclin1)的表达水平。结果:(1)高糖溶液刺激内皮细胞24 h后,细胞生长活力为正常组的55.0%(P0.01),自噬标记蛋白Beclin1的表达水平明显增加,凋亡水平为正常组的2.0倍;(2)与高糖组相比,RAPA组细胞生长活力明显增加,Beclin1的表达明显升高(P0.01),凋亡水平为高糖组的70.1%;(3)与高糖组相比,3-MA组细胞生长活力明显减少,Beclin1的表达明显降低(P0.01),凋亡水平为高糖组的1.42倍。结论:细胞自噬可能对高糖诱导的人冠状动脉内皮细胞具有凋亡保护作用。     

Increased cyclosporine bioavailability induced by experimental nephrotic syndrome in rats

Components of whole blood and plasma are highly altered during the presentation of nephrotic syndrome. The present study was aimed to explore the influence of nephrotic syndrome on the pharmacokinetics of cyclosporine (CsA) (10 mg/kg) administered i.v. to control or puromycin-induced nephrotic rats (P-NS). We found an increase in CsA bioavailability in the nephrotic group compared with controls. The area under the curve of blood CsA versus time (AUCiv) increased from 27.7 +/- 5.3 to 60.6 +/- 13.8 mug.h.mL-1 in control and P-NS rats, respectively. The AUCiv augmentation was positively correlated with cholesterol levels. On the other hand, the total body clearance was significantly lower (0.38 +/- 0.06 vs. 0.17 +/- 0.03 L.(kg body mass)-1.h-1) and the volume of distribution at steady state (3.70 +/- 0.52 vs. 2.85 +/- 0.32 L/kg) was significantly smaller in nephrotic rats as compared with control. These pharmacokinetic changes lead to a longer terminal half-life of CsA in P-NS rats (11.8 +/- 1.6 vs. 6.9 +/- 0.91 h). We conclude that the physiopathologic changes induced by the nephrotic syndrome in P-NS animals result in a significant increase in CsA blood exposure by both the decrease in drug distribution and the reduction in elimination rate of CsA.     

In vitro prevention of cyclosporin-induced cell contraction by mycophenolic acid

Nephrotoxicity is a major side-effect of cyclosporin A (CsA), which induces a vasoconstrictive response in vascular smooth muscle and mesangial cells. Mycophenolic acid (MPA) is used in combination with low-dose CsA to reduce nephrotoxicity. We previously demonstrated that MPA affected mesangial cell contractile response to angiotensin II or KCl. Aims of the present study were to evaluate if MPA can prevent CsA-induced contraction of human mesangial and aortic smooth muscle cells (ASMC). Using a morphoquantitative approach, we evidenced that pretreatment with MPA (1 microM) prevented the reduction of cell area induced by CsA within 30 min in both cell types. We then compared the expression of three main cytoskeleton proteins: tubulin, alpha-smooth actin (SMA) and basic calponin, in ASMC and in mesangial cells treated with MPA and/or CsA. CsA alone did not significantly change the expression level of these proteins neither in mesangial cells nor in ASMC. MPA decreased the expression level of tubulin in both mesangial cells and ASMC. Surprisingly, MPA, which stimulated SMA and calponin expression in mesangial cells, exerted an inhibitory effect on both contractile protein expression in ASMC. In conclusion, our results evidenced opposite effects of MPA on calponin and SMA protein expression in ASMC and in mesangial cells, despite similar antiproliferative properties, suggesting that sarcomeric protein expression is controlled by different intracellular mechanisms in mesangial and smooth muscle cells. However, MPA interferes in both cell types with the constrictive properties CsA, which may partially explain the protective effects of MPA against CsA nephrotoxicity.     

MPA increases idarubicin-induced apoptosis in chronic lymphatic leukaemia cells via caspase-3

The caspase family of protease is speculated to have a crucial role in apoptosis. The effect of treatment with Idarubicin (IDA) and Medroxyprogesterone acetate (MPA), used alone or in combination, on the activation of Caspase-3 in canine Chronic Lymphatic Leukaemia (CLL) cells was investigated, in order to clarify the mechanism of chemo- and hormone-therapy mediated apoptosis. Caspase activity was determined by a quantitative fluorimetric assay. Apoptosis was monitored by propidium iodide (PI) and nucleosomes assay. Treatment of CLL cells for 24 h with MPA 5 microM did not significantly activate caspase-3 but its activity was increased almost 5-fold more with IDA 1 microM (P < 0.05) than control. Treatment of CLL cells with IDA 1 microM in equimolecular association with MPA was able to increase the activation of caspase-3 induced by IDA of the 61.2% (P < 0.05) in comparison with IDA alone. The activation of caspase-3 was confirmed evaluating apoptosis by PI and nucleosomes assay. Furthermore, both caspase-3 activation and apoptosis triggered by IDA alone or in combination with MPA were significantly inhibited by specific caspase-3 inhibitor AC-DEVD-CMK. These findings provide an explanation for IDA and MPA induced-apoptosis mechanism.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Blood flow conditions in the proximal pulmonary arteries and vena cavae: healthy children during upright cycling exercise

Diagnostic testing in patients with congenital heart disease is usually performed supine and at rest, conditions not representative of their typical hemodynamics. Upright exercise measurements of blood flow may prove valuable in the assessment of these patients, but data in normal subjects are first required. With the use of a 0.5-T open magnet, a magnetic resonance-compatible exercise cycle, and cine phase-contrast techniques, time-dependent blood flow velocities were measured in the right (RPA), left (LPA), and main (MPA) pulmonary arteries and superior (SVC) and inferior (IVC) vena cavae of 10 healthy 10- to 14-yr-old subjects. Measurements were made at seated rest and during upright cycling exercise (150% resting heart rate). Mean blood flow (l/min) and reverse flow index were computed from the velocity data. With exercise, RPA and LPA mean flow increased 2.0 +/- 0.5 to 3.7 +/- 0.7 (P < 0.05) and 1.6 +/- 0.4 to 2.9 +/- 0.8 (P < 0.05), respectively. Pulmonary reverse flow index (rest vs. exercise) decreased with exercise as follows: MPA: 0.014 +/- 0.012 vs. 0.006 +/- 0.006 [P = not significant (NS)], RPA: 0.005 +/- 0.004 vs. 0.000 +/- 0.000 (P < 0.05), and LPA: 0.041 +/- 0.019 vs. 0.014 +/- 0.016 (P < 0.05). SVC and IVC flow increased from 1.5 +/- 0.2 to 1.9 +/- 0.6 (P = NS) and 1.6 +/- 0.4 to 4.9 +/- 1.3 (P < 0.05), respectively. A 56/44% RPA/LPA flow distribution at both rest and during exercise suggests blood flow distribution is dominated by distal pulmonary resistance. Reverse flow in the MPA appears to originate solely from the LPA while the RPA is in relative isolation. During seated rest, the SVC-to-IVC venous return ratio is 50/50%. With light/moderate cycling exercise, IVC flow increases by threefold, whereas SVC remains essentially constant.     

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.     

Cyclosporine A dosing-time-dependent effects on plasma creatinine and body weight in male Wistar rats treated for 3 weeks.

Cyclosporine A (CsA) nephrotoxicity was assessed in 120 male Wistar rats (350 +/- 50 g) entrained to a 12-h cycle (light-dark 12:12); plasma creatinine level and body weight were examined in controls and in rats that had been treated daily with oral CsA or vehicle alone (olive oil-ethanol 90:10) for 21 days; daily dosing (40 mg/kg) was at one of six equally spaced given times during the 24-h cycle. The variations observed in both indexes were shown to be circadian dosing stage dependent. Nephrotoxicity was present as early as the third day of treatment with CsA; plasma creatinine level was enhanced by about 50% in rats dosed around the time of the change from darkness to light: at 22 HALO, 146.7 +/- 4.5 mumol/L, against 92.0 +/- 2.8 mumol/L for controls (p less than 0.05); and at 2 HALO, 148.3 +/- 10.0 mumol/L, against 95.0 +/- 4.3 mumol/L for controls (p less than 0.05). Thereafter, a remission episode was observed between days D5-D9. The more drastic effects were seen on days D16 and D21, in animals dosed in the beginning of the dark span (14 HALO): 185 +/- 10 mumol/L for CsA and 98.0 +/- 5.3 mumol/L for controls (p less than 0.01) and, to a lesser extent, in rats treated at the early resting phase (2 HALO): 152.4 +/- 31 mumol/L for CsA and 95.0 +/- 4 mumol/L for controls (p less than 0.05). The normal increase in body weight during the 21-day period (about 14 +/- 8% in controls) was impeded in CsA-administered rats, especially those dosed at the beginning of the activity span (14 HALO) that even suffered weight reduction. Differences in percentages of survivors were noticed, depending on dosing stage. About 40% of the animals in every time CsA-treatment group died, except for those dosed at the end of the resting period (10 HALO), when all animals died. In surviving rats, the cessation of CsA dosing resulted in a reversible effect on the study variables.     

Nephrotoxicity of cyclosporin A in hereditary hypertriglyceridemic rats

It has been suggested that cyclosporin A (CsA) nephrotoxicity can be reduced by the concomitant administration of omega-3 fatty acids or vitamin E. The present study was designed to establish whether the effect of the above substances can also be demonstrated in rats with hereditary hypertriglyceridemia (HTG) whose sensitivity to the nephrotoxic effect is greater than in control AVN rats. CsA administration at a dose of 10 mg/kg/day to HTG rats resulted in a significant rise (p<0.001) in serum levels of creatinine (from 66.0+/-7.6 to 108.4+/-11.6 micromol/l) and urea (from 8.3+/-0.7 to 22.3+/-18 mmol/l) which was not found in AVN rats. The baseline values of systolic blood pressure (SBP) were significantly higher in HTG rats. However, in both strains CsA administration was associated with a similar SBP increase which was not prevented by omega-3 fatty acids (EPAX) or vitamin E administration. Concomitant administration of CsA with EPAX at a dose of 600 mg/kg b.w./day in HTG rats prevented the rise in the serum levels of creatinine (65.4+/-14.7 micromol/l) and reduced the increase in the serum urea levels (11.9+/-7.6 mmol/l). Concomitant administration of CsA and vitamin E (at a dose of 25 mg/kg/day) also reduced the increase (p<0.05) in the serum levels of creatinine (70.7+/-14.3 micromol/l) and urea (9.8+/-3.4 mmol/l) compared to the effects elicited by the administration of CsA alone (p<0.05). Administration of CsA alone or in combination with EPAX or vitamin E did not have a marked effect on diuresis, proteinuria, urinary osmolality, urinary excretion of urea, creatinine and potassium. Under all experimental conditions, the rate of urinary excretion of sodium in HTG rats was significantly lower (p<0.01) than in AVN rats. The results obtained support the assumption that omega-3 fatty acids and vitamin E at the doses used reduce CsA nephrotoxicity in rats with hereditary hypertriglyceridemia whose sensitivity to the nephrotoxic effect of CsA is significantly higher than in AVN rats.     

The Emit® 2000 Cyclosporine Specific Assay, Extended Range: development of an application protocol for the V-Twin® analyzer

We evaluated a new protocol for measurement of cyclosporine A (CsA) 2 H after dose (C2) on the V-Twin? analyzer. Imprecision, recovery, and linearity were determined using CsA-spiked blood pools. Accuracy was evaluated using specimens from renal, cardiac, and liver transplant patients, and results were compared with those from liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the Abbott TDx?/TDxFLx? assay. Cross-reactivity and interferences were assessed in the presence of 800 ng/mL CsA. Imprecision coefficients of variation were 3.3%-4.8% (within run) and 5.9%-8.7% (total). Recovery was within 10% of the expected values. Linearity was 350-2,000 ng/mL. Calibration was stable for ≥ 2 weeks. Method comparison showed regression statistics: V-Twin? = 1.01 × LC tandem MS + 36.1, r = 0.971; V-Twin? = 1.13 × Abbott - 92.4, r = 0.969. Metabolite cross-reactivity and interference (endogenous substances and drugs) were within ±10%. The C2 protocol on the V-Twin? analyzer provides acceptable assay performance and accurate determination of whole blood CsA drawn at 2 H after dose.     

A study about drug combination therapy of Schisandra sphenanthera extract and Rapamycin in healthy subjects

To assess the effect of the drug combination of Schisandra sphenanthera extract (SchE) and Rapamycin (RAPA), 18 healthy subjects were given oral treatments of RAPA alone and with SchE. Pharmacokinetic investigations and indexes of hepatic and renal functions, as well as other indices of oral RAPA administration (2 mg), were performed both before and after the SchE treatment period. Whole-blood RAPA concentrations were determined by enzyme-linked immunosorbent assay. The research found that the mean whole-blood RAPA AUC(0-∞), C(max), and t(max) increased almost 2-, 2.1-, and 1.3-fold, respectively, and CL/F (-38.0%) decreased almost 1.6-fold in these subjects when RAPA was administered with SchE compared with oral RAPA administered alone. The results of this study proved that SchE can increase the oral bioavailability of RAPA and will add important information to the interaction area between drugs and herbal products.     

FK506, transforming growth factor-beta1 and mesangial matrix synthesis: parallels and differences compared with cyclosporine A

Cyclosporine A (CsA)-induced glomerulosclerosis is a well-described side effect of CsA treatment. Current evidence indicates that FK506 causes similar morphologic changes. Recently, we demonstrated that CsA up-regulates the expression of transforming growth factor-beta1 (TGF-beta1), its receptors type I (TbetaR-I) and type II (TbetaR-II), as well as related matrix protein synthesis in mesangial cells (MCs). Here, we assessed the effect of FK506 on the expression of TGF-beta1, TbetaR-I, TbetaR-II, fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) in MCs. Resting MCs were incubated with/without FK506. Time- and concentration-dependent expression was measured at the mRNA and protein level. Compared to untreated controls, FK506 stimulated TGF-beta1 mRNA (maximum at 8 h, 100 ng/mL: 2.13+/-0.15-fold, P<0.005) and protein expression (maximum at 96 h, 100 ng/mL: 1.96+/-0.29-fold, P<0.005). In contrast, TbetaR-I and TbetaR-II protein expression remained unchanged. Concerning matrix protein synthesis, FK506 slightly increased FN production (96 h, 100 ng/mL: 1.38+/-0.28-fold, P<0.05), but not PAI-1 production. These results indicate that, comparable to CsA, FK506 induced glomerulosclerosis is also due to a direct effect on mesangial matrix production, which is at least in part mediated via up-regulation of TGF-beta1 expression. The fact that, unlike CsA, FK506 does not increase the expression of TbetaR-I, TbetaR-II, and PAI-1, deserves further investigation.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment.     

Human apolipoprotein A-I kinetics within triglyceride-rich lipoproteins and high density lipoproteins.

Stable isotope methodology was used to determine the kinetic behavior of apolipoprotein (apo) A-I within the triglyceride-rich lipoprotein (TRL) fraction and to compare TRL apoA-I kinetics with that of apoA-I in high density lipoprotein (HDL) and TRL apoB-48. Eight subjects (5 males and 3 females) over the age of 40 were placed on a baseline average American diet and after 6 weeks received a primed-constant infusion of [5,5,5-(2)H(3)]-l-leucine for 15 h while consuming small hourly meals of identical composition. HDL and TRL apoA-I and TRL apoB-48 tracer/tracee enrichment curves were obtained by gas chromatography;-mass spectrometry. Data were fitted to a compartmental model to determine the fractional secretion rates of apoA-I and apoB-48 within each lipoprotein fraction. Mean plasma apoA-I levels in TRL and HDL fractions were 0. 204 +/- 0.057 and 134 +/- 15 mg/dl, respectively. The mean fractional catabolic rate (FCR) of TRL apoA-I was 0.250 +/- 0.069 and HDL apoA-I was 0.239 +/- 0.054 pools/day, with mean estimated residence times (RT) of 4.27 and 4.37 days, respectively. The mean TRL apoB-48 FCR was 5.2 +/- 2.0 pools/day and the estimated mean RT was 5.1 +/- 1.8 h. Our results indicate that apoA-I is catabolized at a slower rate than apoB-48 within TRL, and that apoA-I within TRL and HDL fractions are catabolized at similar rates.     

Enhanced sensitivity of human hepatoma cells to 5-fluorouracil by small interfering RNA targeting Bcl-2

This study was designed to reveal whether the apoptosis induced in human hepatocellular carcinoma (HCC) cell lines by 5-fluorouracil (5-FU) could be enhanced by transfecting Bcl-2 small interfering RNA (siRNA). Bcl-2 siRNA and control siRNA were transfected into cells following treatment with or without 5-FU. Suppression of Bcl-2 expression was confirmed by Western blotting; cell viability was evaluated by MTS assay, and the occurrence of apoptosis in cells was evaluated by apoptosis assay. Expression of Bcl-2 protein after transfection of 20 nM Bcl-2 siRNA was significantly lower than that of control. Incubation of all cell lines with Bcl-2 siRNA reduced cell viability 96 h after 5-FU treatment compared with all other controls: Huh-7 (P < 0.01), Huh-7 with hepatitis C replicon (P < 0.01), HepG2 (P < 0.01), HLE (P < 0.05). Moreover, the proportion of apoptosis in control siRNA, Bcl-2 siRNA, control siRNA prior to 5-FU treatment, and Bcl-2 siRNA prior to 5-FU treatment groups were (4.6 +/- 2.3)%, (7.5 +/- 0.5)%, (6.0 +/- 2.1)%, and (19.5 +/- 0.86)%, respectively. The Bcl-2 siRNA prior to 5-FU treatment group showed the strongest effect of inducing apoptosis. In conclusion, the combination Bcl-2 siRNA and 5-FU might represent a new therapeutic option for HCC.     

Medroxyprogesterone acetate prevents the cardioprotective and anti-inflammatory effects of 17beta-estradiol in an in vivo model of myocardial ischemia and reperfusion

Previous studies demonstrated the protective effects of estrogen administration in models of cardiovascular disease. However, there is a discrepancy between these data and those from the recent clinical trials with hormone replacement therapy in menopausal women. One possible explanation for the divergent results is the addition of progestin to the hormone regimen in the Women's Health Initiative and the Heart and Estrogen/Progestin Replacement Study trials. The aim of the present study was to examine the effects of a combination of 17beta-estradiol (E(2), 20 microg) and medroxyprogesterone acetate (MPA, 80 microg) on infarct size in New Zealand White rabbits. Infarct size as a percentage of the area at risk was significantly reduced by administration of E(2) 30 min before induction of myocardial ischemia compared with vehicle (19.5 +/- 3.1 vs. 55.7 +/- 2.6%, P < 0.001). However, E(2) + MPA failed to elicit a reduction in infarct size (52.5 +/- 4.6%), and MPA had no effect (50.8 +/- 2.6%). E(2) also reduced serum levels of cardiac troponin I, immune complex deposition in myocardial tissue, activation of the complement system, and lipid peroxidation. All these effects were reversed by MPA. The results suggest that MPA antagonizes the infarct-sparing effects of E(2), possibly through modulation of the immune response after ischemia and reperfusion.     

Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge

During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% +/- 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% +/- 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% +/- 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 +/- 9) and DHT- (36 +/- 15) but not vehicle- (4 +/- 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.