Proteomics approach to identify the interacting partners of cellular prion protein and characterization of Rab7a interaction in neuronal cells.

The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).

Lipid-apheresis improves microcirculation of the upper limbs.

Lipid-apheresis (LA) is thought to improve microcirculation. However, limited data are available on the effects on peripheral microcirculation. We investigated upper limb microcirculation of 22 patients undergoing regular LA on a weekly basis before and after LA. Using standardized semiquantitative scales, we analyzed blood flow, vasomotor function, and erythrocyte aggregation by capillary microscopy. In addition, capillary blood flow in quiescence and under heat and cryo-stress was evaluated by photoplethysmographic and laser Doppler anemometry. Moreover, levels of vasoactive mediators adrenalin, noradrenalin, endothelin-1 (ET-1), atrial natriuretic peptide (ANP), asymmetrical dimethyl-arginine (ADMA), as well as total protein and fibrinogen were measured. We found a significant increase in blood flow, the number of perfused capillaries and an improvement of erythrocyte aggregation by capillary microscopy. Using laser Doppler anemometry, we were able to show that this increase was predominantly located in the superficial layer capillaries (Δ44.53 ± 135.81%, n.s.) and less so in deeper layer arterioles (Δ2.75 ± 24.84%, n.s.). Vascular response to heat and cryo stress was also improved after LA but failed to reach significance. LA significantly reduced levels of epinephrin (-33 ± 39.2%), ANP (-28.8 ± 20.2%), ADMA (-74.1 ± 23%), and fibrinogen (-45.4 ± 19.7%) when comparing before LA and after LA values. In summary, we found an improvement in the microcirculation of the upper limbs under LA, which may result from a decrease of vasoconstrictors, improvement of vasomotor function, and a decrease in blood viscosity or erythrocyte aggregation.

How delayed graft function impacts exposure to mycophenolic acid in patients after renal transplantation.

INTRODUCTION: Mycophenolic acid (MPA) plasma concentrations are highly variable on standard-dose mycophenolate mofetil therapy. At creatinine clearances below 25 mL/min, MPA clearance increases as a result of a higher nonprotein-bound fraction. Patients with delayed graft function (DGF) after renal transplantation are exposed to low total MPA concentrations, when risk of rejection is highest. This study investigated the influence of DGF on MPA exposure and on clinical outcome. METHODS: Adult renal transplantation patients treated with mycophenolate mofetil, corticosteroids, and either microemulsified cyclosporine (n = 459) or tacrolimus (n = 371) participated in a randomized controlled trial (the Fixed-Dose Concentration-Controlled [FDCC] Study). Abbreviated MPA areas under the curve (AUCs) were obtained on Day 3, Day 10, Week 4, and Month 3, to calculate MPA AUC0₋12. Free MPA AUC values were available for a subgroup of patients (n = 269). RESULTS: The overall incidence of DGF was 187 of 830 (23%) and did not differ between cyclosporine-treated (24%) and tacrolimus- (21%) treated patients. The incidence of biopsy-proven acute rejection at 12 months was significantly higher in patients with DGF (13.8% versus 21.4%). Patients with DGF had significantly lower dose-corrected MPA AUC on Day 3 and Day 10. Free MPA fraction and dose-corrected free MPA AUC were significantly higher in patients with DGF, from Day 3 until Month 3. The total number of patients with at least one opportunistic infection was significantly higher in patients with DGF (33.2%) compared with patients without DGF (25.8%) (P = 0.048). Patients with DGF developing opportunistic infections did not have higher total MPA AUC nor higher free MPA AUC compared with those without opportunistic infections. CONCLUSION: Patients with DGF have significantly lower dose-corrected MPA AUC in the first month after renal transplantation, presumably as a result of enhanced MPA clearance on account of the elevated MPA free fraction. Because patients with DGF have a higher rate of acute rejection and lower MPA exposure, higher dosing of mycophenolate mofetil in such patients may improve outcome. However, the already increased incidence of opportunistic infections in patients with DGF is a concern.

Amyloid-ß 1-42 levels are modified by apolipoprotein E ε4 in Creutzfeldt-Jakob disease in a similar manner as in Alzheimer's disease.

The presence of apolipoprotein E (ApoE) ε4 allele is a risk factor for Alzheimer's disease (AD) and associated with a more pronounced reduction of amyloid-ß 1-42 (Aß1-42) in the cerebrospinal fluid (CSF). Because a decrease of Aß1-42 and increase of tau protein levels, both important biomarkers for AD, are also reported in Creutzfeldt-Jakob disease (CJD), we analyzed if a similar relationship can be observed in this rapid progressive dementia. Our study included 309 patients with sporadic CJD (147 neuropathologically confirmed and 162 probable cases). We analyzed the role of ApoE ε4 in sporadic CJD (sCJD), in particular the influence on the CSF-markers 14-3-3 protein, tau protein, neuron-specific enolase, S100 protein, Aß1-42, and Aß1-40. No differences in the ApoE ε4 allele frequency and ApoE genotype distribution between sCJD and published healthy controls were observed. The ApoE ε4 allele had no effect on disease duration or age at onset. We detected a dose-dependent ApoE ε4 effect on the decrease of Aß1-42 in sCJD. ApoE ε4 carriers with one ApoE ε4 allele showed significantly reduced Aß1-42 values (p < 0.0001) in comparison with non-carriers. ApoE ε4 allele is not a risk factor for sCJD but modifies the Aß1-42 levels in CSF in a similar manner as in AD. Based on our results in sCJD patients, we hypothesize that the ApoE ε4 effect on Aß1-42 values might not be disease-specific.

Mycophenolic acid displays IMPDH-dependent and IMPDH-independent effects on renal fibroblast proliferation and function.

The aim of this study was to elucidate the role of mycophenolic acid (MPA) in cellular pathways of renal fibrosis. Different assays were applied in a renal fibroblast model using COS-7 cells: assays for cell proliferation, scratch wound closure and collagen matrix contraction, gene quantification, and Western blotting. The results indicate that MPA treatment leads to inosine monophosphate dehydrogenase (IMPDH)-dependent inhibition of fibroblast proliferation and wound closure as well as an unexpected IMPDH-independent inhibition of collagen matrix contraction. Interestingly, the IMPDH-independent expression of CTGF after 6 hours incubation with MPA was significantly decreased; however, it became significantly increased and IMPDH-dependent after 24 hours of incubation and longer. Increased mRNA level of COL1A1, TGFbeta1, and TNFalpha was observed after MPA treatment. An unanticipated finding was the divergent and late MPA effect leading to a significant increase of TGFbeta1 and CTGF gene expression. The results suggest that long-term incubation with MPA alters signals located upstream of transforming growth factor-beta. Furthermore, the protein expression of the apoptotic marker ANXA5 was analyzed in the cell line to exclude apoptosis-related effects using 0.1 to 100 micromol/L MPA. Moreover, in COL4A3-deficient mice treated with different doses of mycophenolate mofetil, we found no significant differences in the gene expression of the same genes supporting the idea of a TGFbeta-independent pathway of tubulointerstitial fibrosis in this model for progressive renal disease. In conclusion, the current study indicates that MPA displays IMPDH-dependent and IMPDH-independent effects on renal fibroblast proliferation and function as well as complex signal transduction in COS-7-cells. Alternative inhibitory pathways may contribute to antifibrotic effect of MPA.

Low-density lipoprotein apheresis decreases ferritin, transferrin and vitamin B12, which may cause anemia in serially treated patients.

Clinical observations revealed an increased prevalence of iron deficiency anemia without chronic bleeding in patients treated with serial low-density lipoprotein (LDL) apheresis. Since several different proteins are adsorbed by LDL apheresis beside pro-atherogenic lipoproteins, we examined the modification of the full blood count, plasma iron, vitamin B12, folic acid, and hemolysis by LDL apheresis. Nineteen patients (55 (50-59) years, 4 female, 15 male) undergoing chronic LDL apheresis due to mixed dyslipidemia (N = 17), homozygous familiar hypercholesterolemia (N = 1) or isolated elevated lipoprotein(a) (N = 1) were included in this study. They were treated with direct adsorption of lipoproteins (DALI; N = 6), heparin-induced LDL-precipitation (HELP; N = 7) or double filtration plasmapheresis (DFPP; N = 6). The patients' full blood count, iron metabolism (plasma iron, ferritin, transferrin, transferrin saturation), vitamins involved in erythropoiesis (vitamin B12 and folic acid), and markers of hemolysis (haptoglobin and free hemoglobin) were analyzed directly before and after LDL apheresis. A single LDL apheresis session significantly decreased the levels (reduction in the median [25(th)-75(th) percentiles] of: ferritin 9.8 [1.3-18] %; P = 0.004), transferrin (12.1 [10.0-15.96] %; P = 0.0005), and vitamin B12 (17.8 [16.2-20.8] %; P = 0.0005). Thereby, transferrin and vitamin B12 were decreased in all (N = 19) and ferritin in 74% (N = 14) of the patients. Twelve out of 19 patients (63.2%) had mild anemia despite iron administration in 14 out of 19 patients (73.7%). LDL apheresis had no significant influence on full blood count, plasma iron, transferrin saturation, folic acid, or hemolysis. Similar changes were observed in all LDL apheresis methods used. LDL apheresis significantly decreases ferritin, transferrin, and vitamin B12, suggesting an influence of serial LDL apheresis on erythropoiesis.

Retrospective analysis of long-term lipid apheresis at a single center.

We retrospectively analyzed 10 906 lipid apheresis sessions (heparin-induced lipoprotein precipitation, direct adsorption of lipoproteins, double filtration plasmapheresis, dextran sulfate adsorption, and immunoadsorption) in 38 patients who were consecutively treated in our department during the last 20 years. The incidences of major cardiovascular events (MACE) (death, cerebrovascular accident, myocardial infarction, limb amputation, and renal vascular involvement) were taken separately as primary end-points or as a combined end-point. The time-course of secondary end-points (coronary and extracranial status of arteries, left ventricular function, occlusive artery disease, and calculated glomerular filtration rate [cGFR]) were also evaluated, as well as the extent of the reduction in plasma lipids and lipoproteins and the incidence of therapy associated side-effects. MACE decreased from 7.02% events per patient per year at the start of lipid apheresis to 1.17% during lipid apheresis and the rate of myocardial revascularization decreased from 22.8% to 3.8% per patient per year. Classical (diabetes mellitus, arterial hypertension, and smoking history), as well as novel risk factors (cGFR < 60 mL/min, statin withdrawal, mixed hyperlipoproteinemia, and elevated lipoprotein (a)) were associated with an elevated risk for MACE. All applied methods had comparable effects. All lipid apheresis methods proved to be safe and suitable for long-term treatment. The present data demonstrate that treatment with lipid apheresis is very effective and leads to long-term reduction in cardiovascular mortality and morbidity.

Mycophenolic acid predose concentrations and renal function in a mouse model for progressive renal fibrosis.

Within the scope of this study, the potential antifibrotic effect of mycophenolate mofetil (MMF) on COL4A3-deficient mice as an animal model for progressive renal fibrosis was investigated regarding kidney function and survival. Thirty-five animals were randomly assigned to one of five groups and treated with doses of 0, 10, 50, 100, or 150 mg/kg MMF per day, respectively. When increasing somnolence was observed, indicating end-stage renal disease, the mice were euthanized and blood was obtained. Serum concentrations of creatinine, urea nitrogen, total protein, mycophenolic acid (MPA), and mycophenolic acid glucuronide (MPAG) were quantified. The kidney histology was examined using hematoxylin and eosin as well as trichrome staining. The mean overall survival was 65.9 (+/-6.1) days with no significant difference between the treatment groups (P > 0.05, Mantel-Cox test). Serum predose concentrations of MPA and MPAG showed considerable interindividual variability. There was no correlation between survival time and MPA or MPAG concentrations (P > 0.05, Spearman rank correlation). However, an apparent decrease in serum creatinine and urea nitrogen concentrations was observed at higher doses of MMF, eg, -54% for creatinine in the 150-mg/kg/day group compared with placebo. A highly significant reciprocal correlation between MPA concentrations and serum creatinine was demonstrated (P < 0.01, r = -0.655, Spearman rank correlation). In conclusion, MMF may be a candidate drug for preserving kidney function in progressive renal fibrosis.

The acyl glucuronide metabolite of mycophenolic acid induces tubulin polymerization in vitro.

OBJECTIVES: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) forms covalent protein adducts and possesses antiproliferative properties independent of IMPDH inhibition. The underlying mechanism is unknown. Disorganized tubulin polymerization prevents cell cycle progression. We investigated whether AcMPAG interacts with tubulin polymerization. DESIGN AND METHODS: AcMPAG (1.0-100 microM) was incubated with bovine tubulin in the presence of GTP. Polymerization was followed at 340 nm. The time until onset and the extent of polymerization were determined. MPA (100 microM), phenolic glucuronide MPAG (100 microM), and paclitaxel (10 microM) served as controls. RESULTS: MPAG was without effect. The AcMPAG effect on tubulin polymerization was dose dependent and significantly stronger (about 2.5-fold) than that of MPA (n=4; p<0.05), but weaker than paclitaxel. CONCLUSIONS: MPA and AcMPAG can induce tubulin polymerization in the presence of GTP with AcMPAG being significantly stronger. This property of AcMPAG may contribute to its IMPDH independent antiproliferative effect.

Prenatal programming of metabolic syndrome in the common marmoset is associated with increased expression of 11beta-hydroxysteroid dehydrogenase type 1.

OBJECTIVE: Recent studies in humans and animal models of obesity have shown increased adipose tissue activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which amplifies local tissue glucocorticoid concentrations. The reasons for this 11beta-HSD1 dysregulation are unknown. Here, we tested whether 11beta-HSD1 expression, like the metabolic syndrome, is "programmed" by prenatal environmental events in a nonhuman primate model, the common marmoset monkey. RESEARCH DESIGN AND METHODS: We used a "fetal programming" paradigm where brief antenatal exposure to glucocorticoids leads to the metabolic syndrome in the offspring. Pregnant marmosets were given the synthetic glucocorticoid dexamethasone orally for 1 week in either early or late gestation, or they were given vehicle. Tissue 11beta-HSD1 and glucocorticoid receptor mRNA expression were examined in the offspring at 4 and 24 months of age. RESULTS: Prenatal dexamethasone administration, selectively during late gestation, resulted in early and persistent elevations in 11beta-HSD1 mRNA expression and activity in the liver, pancreas, and subcutaneous-but not visceral-fat. The increase in 11beta-HSD1 occurred before animals developed obesity or overt features of the metabolic syndrome. In contrast to rodents, in utero dexamethasone exposure did not alter glucocorticoid receptor expression in metabolic tissues in marmosets. CONCLUSIONS: These data suggest that long-term upregulation of 11beta-HSD1 in metabolically active tissues may follow prenatal "stress" hormone exposure and indicates a novel mechanism for fetal origins of adult obesity and the metabolic syndrome.

Pharmacokinetics of mycophenolic acid and its glucuronide metabolites in stable adult liver transplant recipients with renal dysfunction on a low-dose calcineurin inhibitor regimen and mycophenolate mofetil.

Low-dose calcineurin inhibitors (CNIs) in combination with a fixed dose (2 g/d) of mycophenolate mofetil (MMF) are a strategy to minimize exposure to cyclosporine (CSA) or tacrolimus (TAC) and thus reduce CNI-related side effects. This study compared the pharmacokinetics (PK) of mycophenolic acid (MPA) and its glucuronide metabolites in stable adult liver transplant recipients with moderately impaired renal function converted from a standard to a low-dose CNI regimen in combination with a fixed dose of MMF. Full 12-hour PK profiles of MPA, free MPA, the aryl glucuronide (MPAG), and the acyl glucuronide (AcMPAG) were obtained from 30 stable liver transplant patients on low-dose CNI (CSA, n = 12; TAC, n = 18) therapy at least 3 months after initiation of low-dose therapy. Predose CSA and TAC concentrations (quantified by liquid chromatography-tandem mass spectrometry) ranged from 17 to 35 and 1.1 to 3.7 microg/L, respectively. The PK variables for MPA, MPAG, AcMPAG, and free MPA displayed wide interindividual variability. Of note was the observation that there were no significant differences in the exposure to MPA, MPAG, and free MPA between the CSA and TAC groups. MPA area under the concentration-time curves (AUCs) ranged from 31.8 to 102.1 (median: 52.9) mg.h(-1).L(-1) in the CSA group and from 22.9 to 144.8 (median: 55.9) mg.h(-1).L(-1) in the TAC group. The AcMPAG AUC on patients under low-dose CSA therapy was higher than that observed under patients on low-dose TAC therapy, although this did not quite reach statistical significance (P = 0.057). Patients receiving CSA had a significantly higher AcMPAG Cmax but not AcMPAG AUC, suggesting that only peak CSA concentrations on a low-dose CSA regimen are sufficient to impair the biliary excretion of AcMPAG. In summary, the influence of CSA on the exposure to MPA was attenuated in stable adult liver transplant recipients on a low-dose CNI therapy in combination with a fixed dose of MMF as compared with patients on a standard CNI therapy. Dose adjustment according to drug concentration measurements is recommended to optimize dosing of MMF and to maintain adequate immunosuppression in patients converted to low-dose CNI therapy.

Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European consensus conference.

In 2007, a consortium of European experts on tacrolimus (TAC) met to discuss the most recent advances in the drug/dose optimization of TAC taking into account specific clinical situations and the analytical methods currently available and drew some recommendations and guidelines to help clinicians with the practical use of the drug. Pharmacokinetic, pharmacodynamic, and more recently pharmacogenetic approaches aid physicians to individualize long-term therapies as TAC demonstrates a high degree of both between- and within-individual variability, which may result in an increased risk of therapeutic failure if all patients are administered a uniform dose. TAC has undoubtedly benefited from therapeutic drug monitoring, but interpretation of the blood concentration is confounded by the relative differences between the assays. Single time points, limited sampling strategies, and area under concentration-time curve have all been considered to determine the most appropriate sampling procedure that correlates with efficacy. Therapeutic trough TAC concentration ranges have changed since the initial introduction of the drug, while still maintaining adequate immunosuppression and avoiding drug-related adverse effects. Pharmacodynamic markers have also been considered advantageous to the clinician, which may better reflect efficacy and safety, taking into account the between-individual variability rather than whole blood concentrations. The choice of method, differences between methods, and potential pitfalls of the method should all be considered when determining TAC concentrations. The recommendations of this consensus meeting regarding the analytical methods include the following: encourage the development and promote the use of analytical methods displaying a lower limit of quantification (1 ng/mL), perform careful validation when implementing a new analytical assay, participate in external proficiency testing programs, promote the use of certified material as calibrators in high-performance liquid chromatography with mass spectrometric detection methods, and take account of the assay and intermethod bias when comparing clinical trial outcomes. It is also important to consider that TAC concentrations may also be influenced by other factors such as specific pharmacokinetic characteristics associated with the population, drug interactions, pharmacogenetics, adverse events that may alter TAC concentrations, and any change in the oral formulation that may result in pharmacokinetic changes. This meeting emphasized the importance of obtaining multicenter prospective trials to assess the efficacy of alternative strategies to TAC trough concentrations whether it is other single time points or area under the concentration-time curve Bayesian estimation using limited sampling strategies and to select, standardize, and validate routine biomarkers of TAC pharmacodynamics.

Increased frequency of positive family history of dementia in sporadic CJD.

OBJECTIVE: To analyze whether a positive family history of dementia (PFHD) is more common in sporadic CJD (sCJD) than in healthy/population controls and to study associated risk factors. PATIENTS/METHODS: Six hundred and eighty-five sCJD patients and 659 sex-/age-matched controls were included. A PFHD in parents/grandparents/siblings was evaluated. The PRNP M129V polymorphism and ApoE genotype in sCJD with/without PFHD were determined by PCR. RESULTS: PFHD was found in 12.1% of sCJD patients and 5.6% of controls (p<0.001). No significant difference in M129V polymorphism was found between sCJD with and without PFHD. Thirty-six percent of sCJD patients with PFHD, 26% without PFHD and 19% of healthy controls had at least one ApoE4 allele. Compared to controls, ApoE4 allele frequency (p=0.005) and proportion of ApoE4 allele carriers (p=0.019) were significantly higher in sCJD with PFHD. INTERPRETATION: A higher frequency of the ApoE4 allele in sCJD with a PFHD could be indicative of an additional risk factor in CJD.

Regulation of IL2 and NUCB1 in mononuclear cells treated with acyl glucuronide of mycophenolic acid reveals effects independent of inosine monophosphate dehydrogenase inhibition.

The aim of the present study was to investigate whether the acyl glucuronide of mycophenolic acid (AcMPAG) directly affects gene expression independent of guanosine (G) depletion. Human native mononuclear cells from healthy volunteers were studied. A concentration of 100 micromol/L (50 mg/L) AcMPAG, which provided effective inhibition of cell proliferation according to dose-response curves, was selected for gene expression analysis on microarray, verified by quantitative real-time polymerase chain reaction on the LightCycler. Differentially regulated genes on the microarray were 114 inosine monophosphate dehydrogenase-independent genes involved in cell proliferation, signal transduction, chemokine stimulation, endocytosis, vesicle transport, cell adhesion, and cytoskeleton. For verification, 16 genes, which were directly or indirectly related to cell proliferation, were selected for quantitative real-time polymerase chain reaction. SCNM1, ANP32E, CXCL13, CALM1, DKFZp451J0118, TPM3, CDC42, YWHAE, CXCL3, RDX, NDUFA3, and SOD1 showed no significant difference between the studied groups (P > 0.05). CCL1 gene expression was significantly regulated (P < 0.05) only in the mononuclear cell group treated with AcMPAG, whereas YWHAZ gene expression was significantly regulated only in the group treated with AcMPAG in presence of G and 8-aminoguanosine. The difference of interleukin 2 (IL2) and nucleobindin 1 (NUCB1) expression was significant between control and AcMPAG (P < 0.05) however not significant between AcMPAG in presence and absence of G and 8-aminoguanosine (P > 0.05). The expression of interleukin 2 and nucleobindin 1 revealed an effect of AcMPAG on gene expression independent of inosine monophosphate dehydrogenase inhibition.

Long-term pharmacokinetics of mycophenolic acid in pediatric renal transplant recipients over 3 years posttransplant.

Data on exposure to mycophenolic acid (MPA), the active moiety of mycophenolate mofetil (MMF), in pediatric renal transplant recipients beyond the first year posttransplant are scarce. The authors therefore analyzed the long-term pharmacokinetics of MPA in 25 pediatric patients treated with 600 mg MMF/m body surface area twice a day in conjunction with cyclosporine A and prednisone. Plasma samples for 12-hour pharmacokinetic profiles were collected on day 7, and after 3, 9, 24, and 36 months posttransplant. Both the actual and the dose-normalized MPA-area under the concentration-time curve (AUC0-12) increased approximately 2-fold between day 7 and month 9 but stabilized thereafter. Both the actual and the dose-normalized MPA-AUC0-12 at months 24 and 36 were comparable to that at month 9. Presuming a therapeutic window of 30-60 mg h/L, 15 (60%) of 25 patients at day 7 had an MPA-AUC0-12 <30 mg h/L, indicating potential underexposure, whereas the proportion of patients with an MPA-AUC0-12 <30 mg h/L between months 3 and 36 was low (5%-17%). These data suggest that the recommended MMF dose of 600 mg/m body surface area twice a day in conjunction with cyclosporine A leads to MPA underexposure early posttransplant in a significant subset of patients, indicating a need for a higher initial MMF dose. Dose-normalized MPA exposure increases in the first 9 months posttransplant, consistent with a reduced MPA metabolism and increased enterohepatic recycling of MPA.

Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer.

OBJECTIVES: Unequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer. DESIGN AND METHODS: Paired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis. RESULTS: Densitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (>3-fold, P<0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (>3-fold, P<0.05) compared to normal tissue. CONCLUSIONS: CLIC1 and TPD52 were significantly (P<0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers.

AcylMPAG plasma concentrations and mycophenolic acid-related side effects in patients undergoing renal transplantation are not related to the UGT2B7-840G>A gene polymorphism.

Mycophenolic acid (MPA) is metabolized primarily by glucuronidation to form the biologically inactive 7-O-glucuronide conjugate (MPAG), which is the major urinary excretion product. MPA is also converted to acyl-glucuronide metabolite (AcylMPAG), which has been suggested to be involved in the generation of MPA-related adverse events such as diarrhea or leucopenia. This conversion of MPA to AcylMPAG is catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). We studied the impact of the -840G>A polymorphisms in the UGT2B7 gene on the pharmacokinetics of AcylMPAG. We also investigated whether the plasma concentrations of AcylMPAG are correlated with MPA-related toxicity to further evaluate its potential clinical significance. In a randomized, controlled trial, comparing fixed-dose mycophenolate mofetil (MMF) with concentration-controlled MMF therapy, patients undergoing renal transplantation were treated with a calcineurin inhibitor, MMF, and corticosteroids. Informed consent was obtained from 332 patients for genotyping. In all patients, blood samples were drawn (three samples within the first 2 hours after administration) on Day 3, Day 10, Week 4, and Months 3, 6, and 12 to measure MPA and AcylMPAG plasma concentrations. The pharmacokinetics of AcylMPAG were correlated with the -840G>A single nucleotide polymorphism (SNP) in the UGT2B7 gene. Heterozygosity for the -840G>A SNP in the UGT2B7 gene was found in 145 patients (145 of 332 [44%]) and 93 (93 of 332 [28%]) patients were homozygous for the -840G>A allele. No difference was found in the dose-normalized AcylMPAG trough (C0) levels and dose-normalized AcylMPAG areas under the concentration-time curve (AUCs) at each visit between carriers and noncarriers of the -840G>A SNP. Also, metabolic ratios, expressed as AcylMPAG/MPA and AcylMPAG/MPAG, were not related to UGT2B7 genotype. The dose-normalized AcylMPAG-C0 and AcylMPAG AUC were higher in the cyclosporine-treated group compared with the tacrolimus-treated patients at each visit. There was no difference in AcylMPAG concentrations (trough or AUC) or AcylMPAG/MPAG ratio between patients with compared with patients without diarrhea. None of the -840G>A UGT2B7 SNPs was disproportionately present among the patients with diarrhea. There was a higher incidence of diarrhea in tacrolimus-treated patients [26 of 163 (16.0%)] compared with cyclosporine-treated individuals [five of 51 (9.8%)], although AcylMPAG concentrations were lower in tacrolimus-treated patients. In this study, we have found no influence of the -840G>A UGT2B7 SNP on AcylMPAG exposure in patients undergoing renal transplantation. There also was no association between this variant genotype and the incidence of diarrhea or leucopenia, two adverse events for which a role for AcylMPAG has been suggested.

Physiological role of the cellular prion protein (PrPc): protein profiling study in two cell culture systems.

The physiological role of the cellular prion protein (PrP (c)) is still not fully understood. Current evidence strongly suggests that PrP (c) overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrP (c) in PrP (c)-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/or the level of PrP (c) expression seem to be critical for the fluctuation between PrP (c)'s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrP (c) itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrP (c) into PrP (c)-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrP (c)-deficient cells) endogenous PrP (c). Protein profiling following transient PrP (c) overexpression in HEK 293 cells revealed a major PrP (c) involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrP (c) into mouse neuronal PrP (c)-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrP (c) overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrP (c) expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrP (c) in cell physiology.

Effect of the antioxidant idebenone on adverse events under mycophenolate mofetil therapy in a rat model.

BACKGROUND: Diarrhea and anemia are side effects of mycophenolic acid (MPA), but underlying mechanisms are not fully understood. Gene expression of major-alpha-hemoglobin and catalase was suppressed in livers of mycophenolate mofetil (MMF)-treated rats, suggesting MPA attenuates cellular defense against reactive oxygen species (ROS). We investigated whether the antioxidant idebenone might alleviate MPA-related side effects. METHODS: Rats were treated as follows: group 1: controls; group 2: idebenone; group 3: MMF; and group 4: MMF/idebenone. Blood was collected weekly to determine cell counts, hemoglobin, MPA, plasma albumin, total protein, creatinine, and urea concentrations. On day 28 RNA was extracted from liver, kidneys, and bone marrow (BM). Colon and jejunum were examined histologically. RESULTS: High-dose MMF-treated rats developed diarrhea, dehydration, and weight loss. After a week, a significant decrease (P=0.001) in erythrocyte count and hemoglobin concentration was observed that was not influenced by idebenone. Degenerative changes in the jejunum were slightly attenuated by idebenone. Idebenone did not influence MPA-induced suppression of catalase. A significant suppression of major-alpha-hemoglobin and the erythropoietin (EPO)-receptor in BM of MMF-treated groups and almost complete absence of hemopoietic progenitor cells were observed. EPO-mRNA was markedly upregulated in the MMF-group and even more in the MMF/idebenone-group. CONCLUSION: Idebenone showed minimal benefit on MMF-related diarrhea and anemia. BM of MMF-treated rats revealed erythroid aplasia as a possible reason for anemia. Marked upregulation of EPO-mRNA presumably reflects a compensatory mechanism. Because ROS have the potential to suppress EPO expression, it can be hypothesized that enhanced EPO-mRNA expression in MMF/idebenone-treated rats is caused by antagonism of ROS.