Proteomic Profiling of Exosomal Proteins for Blood-based Biomarkers in Parkinson's Disease.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder and is characterized by loss of dopaminergic neurons. Biomarkers for tracking disease progression are useful indicators of the pathological conditions or the effects of therapeutic interventions on disease progression, but there are currently no known biomarkers in the blood that correlate with the progression of PD. Several studies have suggested that exosomes reflect intracellular changes that occur in response to pathological conditions and are an effective source of biomarkers for disease progression. To identify candidate biomarkers of disease progression in PD, we isolated exosomes from plasma of PD patients at Hoehn and Yahr (HY) stages II and III and performed protein profiling of the exosomes using two-dimensional differential gel electrophoresis (2D-DIGE). The expression levels of three proteins (clusterin, complement C1r subcomponent, and apolipoprotein A1) in PD patients at HY stages II and III were significantly decreased compared to healthy subjects (p < 0.05). Apolipoprotein A1 in PD patients at HY stage III was significantly decreased compared to HY stage II and correlated with progression of PD (r < -0.77, p < 0.01). Fibrinogen gamma chain in plasma was also decreased in PD patients at HY stages II and III compared to healthy subjects. Therefore, these three exosomal proteins (clusterin, complement C1r subcomponent, and apolipoprotein A1) and fibrinogen gamma chain in plasma may be biomarker candidates for the diagnosis of PD. In particular, the expression levels of apolipoprotein A1 in exosomes may be useful for tracking the progression of PD.

A selective peroxisome proliferator-activated receptor δ agonist PYPEP suppresses atherosclerosis in association with improvement of the serum lipoprotein profiles in human apolipoprotein B100 and cholesteryl ester transfer protein double transgenic mice.

OBJECTIVE: Although peroxisome proliferator-activated receptor (PPAR) δ agonists have been shown to improve the serum lipoprotein profiles in humans, the impact of the changes in these lipoprotein profiles on atherosclerosis remains to be elucidated. The aim of this study was to investigate the relationship between the selective PPARδ agonist-induced alterations of serum lipoprotein profiles and the development of atherosclerosis in human apolipoprotein B100 and cholesterol ester transfer protein double transgenic (hApoB100/hCETP-dTg) mice with human-like hypercholesterolemic dyslipidemia. METHODS: hApoB100/hCETP-dTg mice fed an atherogenic diet received a novel PPARδ agonist (PYPEP) or vehicle for 18 weeks, followed by evaluation of atherosclerosis. Serum samples were collected during the treatment period at least at 3-week intervals to determine the lipoprotein levels and the levels of an inflammatory marker, macrophage chemotactic protein-1 (MCP-1), and to analyze the lipoprotein profile by fast protein liquid chromatography. The cholesterol efflux capacity of high-density lipoprotein (HDL) was examined using [(3)H]-cholesterol labeled macrophages. RESULTS: Compared with vehicle treatment, PYPEP treatment caused increases in the serum levels of HDL cholesterol and apolipoprotein A-I (ApoA-I), as well as reductions in the serum non-HDL cholesterol and MCP-1 levels. The HDL fraction from the PYPEP-treated group maintained its cholesterol efflux capacity and showed an increased population of smaller HDL particles. PYPEP substantially suppressed atherosclerotic lesion progression, and the lesion areas had significant correlations with non-HDL cholesterol, HDL cholesterol, ApoA-I and MCP-1 by Pearson's correlation analysis. A multiple regression analysis revealed that non-HDL cholesterol and ApoA-I were significantly associated with the atherosclerotic lesion area. CONCLUSION: A novel PPARδ agonist, PYPEP, suppressed atherosclerotic lesion progression by improving the serum lipoprotein profiles, including increased levels of ApoA-I and functional HDL particles, as well as a reduced non-HDL cholesterol level, in hApoB100/hCETP-dTg mice with human-like hypercholesterolemic dyslipidemia.

Carbamazepine differentially affects the pharmacokinetics of fexofenadine enantiomers.

AIM: This aim of this study was to characterize the impact of the P-glycoprotein (P-gp) inducer, carbamazepine, on fexofenadine enantiomer pharmacokinetics. METHODS: Twelve healthy volunteers initially received a 60mg dose of fexofenadine alone. Subsequently, a 100mg dose of carbamazepine was administered three times daily (300mg day(-1) ), and on day 7, fexofenadine was co-administered. RESULTS: Carbamazepine significantly decreased the area under the plasma concentration-time curve and the amount excreted into the urine of (S)- and (R)-fexofenadine. The P-gp inducer showed a greater effect on the pharmacokinetic parameters of (S)-fexofenadine. CONCLUSION: This study indicates that carbamazepine may alter the pharmacokinetics of fexofenadine enantiomers.

A sensitive column-switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies.

A simple and sensitive column-switching HPLC-UV method was developed for the simultaneous determination of aripiprazole, a novel atypical antipsychotic drug, and its active metabolite, dehydroaripiprazole in human plasma. Aripiprazole, its active metabolite and 7-[5-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]pentyloxy]-3,4-dihydro-2(1H)-quinolinone (OPC-14558) as an internal standard were extracted from 1 mL of plasma using a mixture of chloroform/n-heptane (3:7, v/v), and the extract was injected into a column I (TSK BSA-ODS/S precolumn, 5 µm) for cleanup and column II (C(18) STR ODS-II analytical column, 5 µm) for separation. Peaks were detected with an UV detector set at a wavelength of 254 nm, and the total time for chromatographic separation was ∼20 min. Mean absolute recoveries were 74.0 and 74.7% for aripiprazole and dehydroaripiprazole, respectively. Intra- and inter-day CVs were less than 7.5 and 7.1% for aripiprazole concentrations ranging from 2 to 600 ng/mL, and 9.2 and 4.5% for dehydroaripiprazole concentrations ranging from 2 to 160 ng/mL. The validated concentration ranges for this method were 1-500 ng/mL and the limits of detection were 0.5 ng/mL for both aripiprazole and dehydroaripiprazole. This method was applied to pharmacokinetic study in human volunteers and patients taking aripiprazole.

Selective CB1 cannabinoid receptor antagonist, SR141716A, attenuates liver injury induced by Concanavalin A.

AIM: The aim of this study was to investigate the hepatoprotective activity of a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, in a Concanavalin A (Con A)-induced mouse liver injury model and to determine whether SR141716A has an effect on the production of inflammatory cytokines and chemokines induced by Con A. RESULTS: Injection of Con A (20 mg/kg) to mice developed hepatitis determined by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation and necrosis in the liver. Pretreatment with SR141716A (30 mg/kg) significantly reduced plasma AST and ALT level, protected against necrosis in the liver, and significantly reduced plasma cytokine and chemokine levels, including TNFalpha, IFN-gamma, CXCL9, MIP1-alpha, and IL-10 and no change decreased in IL-4. CONCLUSIONS: The selective CB1 antagonist, SR141716A, exerts a hepatoprotective effect on Con A-induced liver injury in mice by attenuating the increase in cytokine and chemokine levels and inhibiting hepatocyte injury. These findings raise the possibility of using CB1 antagonists as anti-inflammatory drugs for treating hepatitis as well as other inflammatory diseases.

Synthesis and structure-activity relationships of oxamyl dipeptide caspase inhibitors developed for the treatment of liver disease.

The P4 region of a series of oxamyl dipeptide caspase inhibitors was optimized by the combination of anti-apoptotic activity in the Jurkat/Fas (JFas) cellular assay and membrane permeability in the PAMPA assay. Two highly potent anti-apoptotic agents with moderate membrane permeability, 29 and 36, showed strong in vivo efficacy in a murine model of alpha-Fas-induced liver injury.

[Application of mesenchymal stem cells to liver regenerative medicine].

Stem cell-based therapy has received attention as a possible alternative to organ transplantation, owing to the ability of stem cells to repopulate and differentiate at the engrafted site. We transplanted bone marrow-derived mesenchymal stem cells (BMSCs) into liver-injured rats to test the therapeutic effect. Rat bone marrow cells were cultured in the presence of hepatocyte growth factor (HGF). RT-PCR and immunocytochemical analysis indicated that the BMSCs expressed the albumin mRNA and the production of protein after cultivation with HGF for 2 weeks. The BMSCs appeared to differentiate into hepatocyte-like cells in response to the culture with HGF. After labeling with a fluorescent marker, the BMSCs were transplanted into CCl(4)-injured rats by injection through the caudal vein. The liver was excised and blood samples were collected 4 weeks later. Engraftment of the transplanted BMSCs was seen with significant fluorescence in the injured liver. Transplantation of the BMSCs into liver-injured rats restored their serum albumin level and suppressed transaminase activity and liver fibrosis. Therefore, BMSCs were shown to have a therapeutic effect on liver injury. Recently, we have been trying to use mesenchymal stem cells isolated from dental papilla of discarded human wisdom teeth. Autologous transplantation of mesenchymal stem cells from bone marrow and dental papilla could be ethically and functionally promising for stem cell-based therapy.

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Orally-administered caspase inhibitor PF-03491390 is retained in the liver for prolonged periods with low systemic exposure, exerting a hepatoprotective effect against alpha-fas-induced liver injury in a mouse model.

In a mouse model of alpha-Fas-induced acute liver injury, the orally-administered caspase inhibitor PF-03491390 (formerly named IDN-6556) was retained in the liver for prolonged periods with a low systemic exposure. Reductions in the elevated plasma levels of alanine aminotransferase (ALT) revealed that the retention of PF-03491390 in the liver exerted a hepatoprotective effect, even when pre-administered to mice 4 h before alpha-Fas insult. Prolonged retention of PF-03491390 in the liver after oral administration has the benefit of low systemic exposure, making this a beneficial agent for the treatment of liver diseases.

Protection against CCl4-induced injury in liver by adenovirally introduced thioredoxin gene.

Antioxidation therapy is a promising strategy for treating or preventing oxidative stress-related liver diseases. The human thioredoxin (TRX) gene was inserted into an adenovirus vector (Adv-TRX), which was administered to mice. The mice were treated with 1 ml/kg CCl4 48 h after the infection. Blood samples were taken and the liver was excised 24 h after the CCl4 treatment. Serum ammonia, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined, and liver sections were stained with hematoxylin and eosin. RT-PCR analysis showed that the introduced TRX gene was expressed only in the liver. Adv-TRX decreased the serum ammonia, AST, and ALT levels. Hematoxylin-eosin staining indicated that the CCl4-induced injury was significantly prevented by the Adv-TRX infection. The gene delivery of TRX, which plays a central role in intracellular redox control, was shown to be effective in protecting the liver against oxidative stress-induced injury.

Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats.

BACKGROUND/AIMS: The autologous transplantation of bone marrow cells is a promising treatment for liver disease. Pluripotent bone marrow stem cells can differentiate into hepatocytes, but few reports address the therapeutic effect of transplanting these stem cells into damaged liver in vivo. Here, we transplanted bone marrow-derived mesenchymal cells (BMMCs) to test their effect in liver-injured rats. METHODS: Rat bone marrow cells were cultivated for 2 weeks in the presence or absence of hepatocyte growth factor (HGF), labeled with a fluorescent marker, and transplanted by injection into CCl(4)-injured rats. Blood samples collected 4 weeks later were analyzed for albumin production and transaminase levels. The amount of fibrosis was determined by histology. RESULTS: RT-PCR analysis detected alpha-fetoprotein and albumin mRNAs in BMMCs cultured with HGF for 2 weeks. Albumin protein was also produced in the BMMC cultures by a subpopulation of cells. Transplantation of the BMMCs into liver-injured rats restored their serum albumin level and significantly suppressed transaminase activity and liver fibrosis. These effects were not seen when the BMMCs were cultured without HGF. CONCLUSIONS: The transplantation of BMMCs cultured with HGF effectively treats liver injury in rats. This is a promising technique for autologous transplantation in humans with liver injury.

Stimulation of hepatocyte survival and suppression of CCl4-induced liver injury by the adenovirally introduced C/EBPbeta gene.

Gene therapy has attracted attention as a potentially effective alternative to liver transplantation for the treatment of hepatic failure. We chose the C/EBPbeta gene, which plays vital roles in liver regeneration, as a candidate for gene therapy, and examined its effect on hepatocyte survival and the suppression of liver inflammation. C/EBPbeta gene overexpression significantly maintained hepatocyte viability during 12 days of the culture. Urea synthesis ability, which is a liver-specific function, in Adv-C/EBPbeta-infected hepatocytes was stably maintained during the culture, but the activity per cell was significantly lower than that in non-infected cells. On the contrary, DNA synthesis activity in Adv-C/EBPbeta-infected hepatocytes was significantly higher than that in non-infected cells. COX-2 was induced in Adv-C/EBPbeta-infected hepatocytes, and the addition of NS398, a specific inhibitor of COX-2, suppressed the viability-maintenance effect. COX-2 was thus shown to be involved in the survival effect of C/EBPbeta gene. The introduction of the C/EBPbeta gene into liver-damaged mice significantly suppressed the serum AST and ALT activities. These results indicate that C/EBPbeta appears to be a survival factor under stressful conditions, and the introduction of the gene has therapeutic function against liver injury.

Maintenance of hepatocyte functions by coculture with bone marrow stromal cells.

Rat hepatocytes and bone marrow stromal cells (BMSCs) were cocultured with the aim of maintaining differentiated hepatocyte functions. After BMSCs were expanded to a confluent monolayer, freshly isolated hepatocytes were cultured with them, separated by a semipermeable membrane. The BMSCs significantly increased the urea synthesis and albumin secretion activities of the hepatocytes. Conditioned medium prepared from a BMSC monoculture had the same effect. Further study showed that interleukin-6 was involved in the maintenance of urea synthesis and another factor in the maintenance of albumin secretion.