33 In conclusion, the results of this analysis demonstrate the importance of IL28B genotype in HCV genotype 1 and 4 patients undergoing response-guided

therapy. In particular, our findings suggest that the combination of virologic response and IL28B genotype are both important considerations in optimizing treatment duration. In the future, the combination of virologic response and IL28B genotype may be helpful in identifying patients who may not benefit from the addition of a DAA, and alternatively, individuals who require prolonged DAA-based triple therapy. The authors thank Elisabeth Eder, Claudia Willheim, and Kerstin Zinober for technical help, and Drs. Bernhard Bauer, Fritz Renner, Martin Bischof, and Ludwig Kramer for referring their patients. Author contributions: Thomas-Matthias Scherzer: data collection and analysis, writing screening assay of the article. Harald Hofer, Petra Steindl-Munda, Andreas Maieron, Christian Datz. Hermann Laferl, Michael Strasser, and Rudolf Stauber: acquisition of data, critical revision of the article for important intellectual content. www.selleckchem.com/products/apo866-fk866.html Albert Friedrich Stättermayer and Emina Dulic-Lakovic: acquisition

of data. Peter Ferenci: study concept and design, principal investigator, analysis and interpretation of data, outlining and revising the article. Writing assistance: Support for third-party writing assistance for this article was provided by F. Hoffmann-La Roche Ltd. The study sponsor (F. Hoffmann-La Roche Ltd) was not involved in the analysis and interpretation of data. “
“Human 上海皓元 chronic cholestatic liver diseases are characterized by cholangiocyte proliferation, hepatocyte injury, and fibrosis. Yes-associated protein (YAP), the effector of the Hippo tumor-suppressor pathway, has been shown to play a critical role in promoting cholangiocyte and hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. Therefore, the aim of this study was to examine

whether YAP participates in the regenerative response after cholestatic injury. First, we examined human liver tissue from patients with chronic cholestasis. We found more-active nuclear YAP in the bile ductular reactions of primary sclerosing cholangitis and primary biliary cirrhosis patient liver samples. Next, we used the murine bile duct ligation (BDL) model to induce cholestatic liver injury. We found significant changes in YAP activity after BDL in wild-type mice. The function of YAP in the hepatic response after BDL was further evaluated with liver-specific Yap conditional deletion in mice. Ablating Yap in the mouse liver not only compromised bile duct proliferation, but also enhanced hepatocyte necrosis and suppressed hepatocyte proliferation after BDL. Furthermore, primary hepatocytes and cholangiocytes isolated from Yap-deficient livers showed reduced proliferation in response to epidermal growth factor in vitro.

2, 3 The etiology of non-B, non-C HCC has been poorly understood, although alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH), and hemochromatosis4, 5 are known as risk factors. In Japan, NAFLD has increased along with Westernization of lifestyle, and most NASH cases have developed due to such lifestyle-related diseases such as obesity, diabetes mellitus, and hyperlipidemia.6 Obesity and diabetes mellitus, as well as NAFLD, have also recently received increased attention as risk factors for HCC.1, 7-12 An increased risk of liver cancer with radiation dose among atomic bomb

survivors has been reported based on tumor registries, mortality studies, and pathology review,13-16 but hepatitis virus Selleckchem Metformin infection status was not taken into account. In three previous HBV studies at the Radiation Effects Research Foundation (RERF), the HBV surface antigen (HBsAg)-positive proportion increased with radiation dose.17-19 Previous research at RERF demonstrated no increase in the prevalence of anti-HCV antibody (anti-HCV Ab) with radiation dose,20 but reported

supermultiplicative effects between radiation exposure and chronic HCV infection in the etiology of HCC without cirrhosis.21 On the other hand, the cohort LY294002 study in workers at the Mayak nuclear facility demonstrated that the risk of liver cancer mortality was significantly associated with plutonium exposure,22 and that the incidence of HCC was marginally significantly associated with plutonium exposure.23 In the latest analysis, a significant plutonium dose-response relationship was observed for liver cancer mortality, with risk reasonably

described by a linear function.24 However, liver cancer in those analyses included hepatoblastoma and intrahepatic cholangiocarcinoma as well as HCC. In addition, hepatitis virus infection status was not taken into account in a strict 上海皓元医药股份有限公司 and in-depth manner, although HCC accounted for most of the liver cancer. A lifespan study using B6C3F1 mice exposed to continuous low-dose-rate γ rays demonstrated that the incidence of HCC was significantly increased in male mice exposed to total doses equivalent to 8,000, 400, and 20 mGy and in females exposed to 8,000 mGy. However, the incidence of other liver tumors did not significantly increase except for that of hepatoblastoma in males exposed to 400 mGy.25 With the aim of determining whether radiation exposure is an independent risk factor for HCC, even after adjusting for hepatitis virus infection, alcohol consumption, body mass index (BMI), and smoking habit, we conducted a nested case-control study among atomic bomb survivors using stored sera. We also evaluated whether radiation, alcohol consumption, increase of BMI, and smoking habit contribute to increased risk for non-B, non-C HCC.

9 Interestingly, protumorigenic effects of MAT1A inhibition are reversed by blocking of DNA methyltransferases with 5–azacytidine, indicating that DNA methylation is a key mechanism of hepatocarcinogenesis induced by SAMe deficiency.10, 11 Overall, these observations suggest that MAT1A

and MAT2A are important epigenetic regulators whose expression is context–specific and is dependent on the stage of differentiation in the corresponding liver cells. Deregulation of MAT signaling is frequently observed during chronic liver disease progression and malignant transformation, but the mechanisms behind this tightly controlled regulation are largely unknown.5 Thus, a more detailed understanding of the MAT/SAMe metabolism and consecutive deregulation of DNA methylation BTK inhibitor cell line ultimately leading to carcinogenesis such find more as that provided by Yang and colleagues12 contributes significantly to our understanding of liver cancer and helps to identify new diagnostic, prognostic, and therapeutic targets. MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression as a part of the RNA interference machinery. miRNAs were first discovered in 1993 in Caenorhabditis

elegans. Since then, miRNA expression has been linked to virtually all known cellular processes, including proliferation, differentiation, and apoptosis.13 More recent studies have demonstrated MCE that miRNAs can act as disease modifiers and that aberrant regulation of several miRNAs contributes considerably to cancer initiation, propagation, and progression. Almost every type of human cancer has been associated with a specific pattern of deregulated miRNA activity, thereby promoting these molecules to attractive

targets for diagnostic and therapeutic interventions. miRNAs have also been associated with HCC development and progression by targeting a large number of critical oncogenic features (e.g., differentiation and metastasis) as well as key molecules involved in hepatocarcinogenesis.14 In liver cancer development, as well as that of other cancers, two functional subclasses of miRNAs have been discovered with either tumor–suppressive or oncogenic activity.15 With the advent of high–throughput technologies, current miRNA profiles are able to precisely dissect etiological subclasses and histological or clinical phenotypes in liver cancer.16 Additionally, a diagnostic and/or prognostic relevance could be attributed to several miRNAs. Although genomic analyses indicate that almost half of the known miRNAs are located on cancer–associated regions, the exact regulation of miRNAs during carcinogenesis still remains elusive.15 However, it seems abundantly clear that miRNAs not only contribute to epigenetic regulation during tumor development, but are also tightly regulated by epigenetic alterations such as DNA methylation.

9 Interestingly, protumorigenic effects of MAT1A inhibition are reversed by blocking of DNA methyltransferases with 5–azacytidine, indicating that DNA methylation is a key mechanism of hepatocarcinogenesis induced by SAMe deficiency.10, 11 Overall, these observations suggest that MAT1A

and MAT2A are important epigenetic regulators whose expression is context–specific and is dependent on the stage of differentiation in the corresponding liver cells. Deregulation of MAT signaling is frequently observed during chronic liver disease progression and malignant transformation, but the mechanisms behind this tightly controlled regulation are largely unknown.5 Thus, a more detailed understanding of the MAT/SAMe metabolism and consecutive deregulation of DNA methylation GDC-0068 concentration ultimately leading to carcinogenesis such Palbociclib mouse as that provided by Yang and colleagues12 contributes significantly to our understanding of liver cancer and helps to identify new diagnostic, prognostic, and therapeutic targets. MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression as a part of the RNA interference machinery. miRNAs were first discovered in 1993 in Caenorhabditis

elegans. Since then, miRNA expression has been linked to virtually all known cellular processes, including proliferation, differentiation, and apoptosis.13 More recent studies have demonstrated MCE that miRNAs can act as disease modifiers and that aberrant regulation of several miRNAs contributes considerably to cancer initiation, propagation, and progression. Almost every type of human cancer has been associated with a specific pattern of deregulated miRNA activity, thereby promoting these molecules to attractive

targets for diagnostic and therapeutic interventions. miRNAs have also been associated with HCC development and progression by targeting a large number of critical oncogenic features (e.g., differentiation and metastasis) as well as key molecules involved in hepatocarcinogenesis.14 In liver cancer development, as well as that of other cancers, two functional subclasses of miRNAs have been discovered with either tumor–suppressive or oncogenic activity.15 With the advent of high–throughput technologies, current miRNA profiles are able to precisely dissect etiological subclasses and histological or clinical phenotypes in liver cancer.16 Additionally, a diagnostic and/or prognostic relevance could be attributed to several miRNAs. Although genomic analyses indicate that almost half of the known miRNAs are located on cancer–associated regions, the exact regulation of miRNAs during carcinogenesis still remains elusive.15 However, it seems abundantly clear that miRNAs not only contribute to epigenetic regulation during tumor development, but are also tightly regulated by epigenetic alterations such as DNA methylation.

Trained clinical researchers and process improvement experts reviewed video recordings and conducted in-person observations to identify process milestones. These were Stem Cells inhibitor used to create maps of the process called Value Stream Maps which depict the time spent in direct patient care (Value Added Time (VAT)). We then constructed the metric Process Cycle Efficiency (PCE) by dividing Value Added Time by total preparation time for donor and recipient surgeries. RESULTS: Nine process milestones were identified: 1) Verification & placement of supplies/equipment 2) Nurse scrubs-in 3) Start counts 4) Resume supplies/equipment verification & placement 5) Arrival

of patient to OR 6) Start induction 7) Initiate vascular access 8) Start abdominal draping 9) Timeout to incision. Milestones 6 and 7 were considered VAT which ranged from 15–58 minutes for donors and 14–53 minutes for recipients. Total preparation time ranged from 70–1 86 minutes for donors and 58–167 minutes for recipients.

PCE which is a metric for the proportion of the Value Added Time ranged from 8–42% for donors and 22–42% for recipients. The time period from nurse scrubbing in to start of counts had the largest range of time for donors Estrogen antagonist (4–40min) and for recipients (9–50min). Delays were most frequently related to staff or equipment availability. The estimated cost associated with preoperative preparations is $8,500-$27,000 based on per-minute OR charges for the total time of preparation. CONCLUSIONS: These data show considerable variability in total preparation time and PCE, suggesting ample opportunities for process optimization and streamlining. Process optimization reduces variability and thereby reduces vulnerability to errors during preparation and can potentially improve the safety of subsequent intraoperative care and the overall cost of surgery. Disclosures: The following people have nothing medchemexpress to disclose: Donna Woods, Rebeca Khorzad, John R. Joseph, Elizabeth A. Pomfret, Mary Ann Simpson, Robert A. Fisher, Kathryn Waitzman, Amna Daud,

Daniela Ladner Purpose: Infcare Hepatitis is a real-time based computerized data system with clinical decision-support and quality assurance (QA) modules to assist clinicians in management of patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV). The main study objective was to assess the improvement of hepatocellular carcinoma (HCC) screening performance by annual ultrasonography (US) examinations in chronic HBV and HCV patients with cirrhosis followed in a Danish outpatient clinic after implementing the use of Infcare Hepatitis. Methods: Data was extracted from the Infcare Hepatitis database and all HBV and HCV patients registered with cirrhosis at both baseline (01 January 2011), prior to introduction of Infcare Hepatitis, and at two-years follow-up (31 December 2012) was included.

6C-E). mRNA and protein levels PD-1/PD-L1 inhibitor of Fsp27 and Cideb were not affected by the knockdown of SREBP1c (Fig. 6D and Supporting Fig. 8C-E). Importantly, mRNA and protein levels of Cidea were significantly reduced in the SREBP1c-knockdown ob/ob hepatocytes treated with PAs (Fig. 6C-E). Consistently, the hepatic TAG level was reduced in SREBP1c knock-down hepatocytes treated with PAs (Fig. 6F). In contrast, the knockdown of SREBP1c did not affect Cidea mRNA and protein levels (Fig. 6C-E) and hepatic TAG accumulation in the presence of OAs (Fig. 6F). These data indicate that SREBP1c is an important mediator of saturated FA-induced Cidea expression

and hepatic lipid accumulation. During the course of our analysis, we noted that the increase in the Cidea protein levels was higher than the corresponding increase in its mRNA levels in the presence of both saturated and unsaturated FAs (Fig. 5D), which suggested that Cidea protein stability may be increased in the presence of FAs. To test this possibility, we first treated primary ob/ob hepatocytes with OAs or PAs and then incubated them with cycloheximide (CHX), which

inhibits protein synthesis. OA treatment significantly prolonged the half-life of Cidea, which was increased from 40 to 80 minutes (Fig. 7A,B). Consistent selleckchem with our previous study using adipocytes,33 the half-life of Fsp27 in ob/ob hepatocytes was also increased in the presence of OAs (Fig. 7A,B). Half-lives of Cidea and Fsp27 in ob/ob hepatocytes were also increased in the presence of PAs (Supporting Fig. 9A,B). In contrast, Cideb was a relatively stable protein; its stability was not affected by FA treatment (Fig. 7A). Because FAs are usually converted into TAGs and stored in LDs, we checked whether FA-induced Cidea stability medchemexpress in hepatocytes was dependent on lipid synthesis

by knocking down diacylglycerol O-acyltransferase (DGAT)1 and DGAT2, which are enzymes that catalyze the final step of TAG synthesis. Levels of DGAT1/2 in ob/ob hepatocytes were decreased by small interfering RNAs (siRNAs) specific for DGAT1/2 (Fig. 7C), and levels of Cidea and Fsp27 proteins and their half-life also decreased significantly (Fig. 7D-F and Supporting Fig. 9C). Similar results were observed in AML12 cells that overexpressed HA-Cidea (Supporting Fig. 9D-F). These data indicated that Cidea and Fsp27 were stabilized by treatment with FFAs and by lipid synthesis, which provided a positive feedback mechanism that promoted lipid storage and liver steatosis in hepatocytes. CIDE family proteins are important regulators of various aspects of lipid metabolism, including control of lipid storage and LD size in adipocytes (by Cidea and Fsp27)15, 19 and control of very-low-density lipoprotein (VLDL) lipidation in the liver (by Cideb).

Histopathologic analysis of mice to examine possible causes of death showed disease relapse with strong local tumor recurrence/intrahepatic metastases (Fig. 5C, left) accompanied by peritoneal carcinomatosis and small lung metastases (Fig. 5C, right lane). Since metastases could not be found in nonresected animals, the prolonged life span gained by resection obviously learn more allows for the outgrowth of circulating

micrometastases. As an important hallmark of R0-resection, the resection margin should be absolutely tumor-free. In orthogonally sectioned samples of tumors following resection, in all samples we achieved a minimum distance of at least 9 mm between tumor tissue and resection margin (Fig. 5D, left). Since satellites were detectable with a maximum visible distance of around 700 μm to the tumor margin, the performed resections were actually R0 (Fig. 5D, right). It is common sense in clinical oncology that satellite formation with vascular invasion during tumor progression can be regarded as a strong indication for local tumor recurrence after resection.[32, 33] Concepts for adjuvant therapies are recommended and deserve more detailed investigations to

overcome life-limiting disease relapse.[34] Therefore, we tested in our model the therapeutic efficacy of adjuvant www.selleckchem.com/products/atezolizumab.html gemcitabine treatment as used in recent clinical cholangiocarcinoma trials.[22] At first, gemcitabine treatment as a palliative approach in sham-operated mice showed no survival benefit compared to the sham-operated control group (Fig. 6), whereas tumor resection itself resulted in prolonged survival compared medchemexpress to both sham-operated groups. Compared to the resection

group, we observed a significant survival benefit by an adjuvant gemcitabine approach. Therefore, our data indicate a life-prolonging outcome if gemcitabine is used in the adjuvant setting after surgical R0-resection of ICC. Together, our results show that our established model reflects both genetic and clinical characteristics of human ICC and thus represents an excellent tool to investigate novel adjuvant ICC therapies in an animal model of highly predictive value. Intrahepatic cholangiocarcinoma is representative of tumor entities where resection of the primary tumor is the only option for cure, but frequent disease recurrence limits survival.[35] In general, clinical development of novel cancer therapies is expensive and time-consuming, since a large number of promising agents or strategies fail in late-stage clinical trials. Those disappointing results emphasize that preclinical animal models of reliable predictive value are urgently required, particularly for investigations of adjuvant systemic therapies. To meet these predictive requirements genetically engineered mouse models have been developed[36] and several new treatment strategies could be developed on the basis of these models.

In summary, our results using time-dependent covariate analysis establish for the first time the relationship between tumor progression and OS in HCC patients treated with sorafenib. In addition, we establish the correlation between progression pattern and PPS. Thus, these data need to be considered

in daily practice for informing patients about their life expectancy and also in research on trial design and analysis in HCC patients. We thank Mrs. Ingrid Rengel, Selleckchem MK-8669 Nuria Perez, and Jenny Brickman for contributions to this article. Additional Supporting Information may be found in the online version of this article. “
“We read with great interest Garg et al.’s article1 published in HEPATOLOGY. The authors conducted a randomized study to compare the efficacy of tenofovir disoproxil fumarate (TDF) therapy and placebo therapy in patients suffering from a severe spontaneous reactivation of chronic hepatitis B (CHB) presenting as acute-on-chronic liver failure. They reported a high overall mortality rate of 63.0% (17/27), with rates of 43% and 85% in the TDF and placebo arms, respectively. TDF significantly reduced this website the mortality rate and hepatitis B virus DNA levels and improved the

Child-Turcotte-Pugh and Model for End-Stage Liver Disease (MELD) scores in these patients at 3 months. It is noteworthy that some patients with cirrhosis were enrolled. First, we consider 3 months of observation to be too short for

determining the survival of these patients. We reexamined 96 patients with liver decompensation treated with lamivudine in our previous study in Taiwan.2 Eight patients (8.3%), two patients (2.1%), and three patients (3.1%) died within 1, 1 to 3, and 3 to 6 months of lamivudine treatment, respectively. In other words, 23.1% of the patients (3/13) who did not survive for 6 months died after 3 months of antiviral MCE treatment. Villeneuve et al.3 reported that 25% of their patients (1/4) without hepatocellular carcinoma died from hepatic failure within 3 to 6 months of the initiation of lamivudine treatment. Fontana et al.4 reported that patients with decompensated cirrhosis were still dying even after the first 6 months. Hence, the mortality rate is possibly underestimated in Garg et al.’s study.1 The mean baseline MELD scores (27 and 25 in the TDF and placebo arms, respectively) reflect the fact that the patients enrolled in their study had more severe liver disease. In HEPATOLOGY, Liaw et al.5 reported lower mortality rates for patients with CHB and decompensated liver disease who were treated with TDF (4.4% or 2/45) or entecavir (9.1% or 2/22) by 48 weeks.

ConA-induced hepatitis is dependent on NKT cell activity.21 Using an adoptive transfer approach, we found, as expected, that SCID mice that received liver NKT cells from PBS-treated mice exhibited typical liver necrosis (Fig. 6A) and elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 6B). In marked contrast, the histologic evidence of necrosis in the liver (Fig. 6A) and the ConA-induced elevation of the levels of serum ALT and AST

(Fig. 6B) were reduced significantly in the SCID mice that received liver NKT cells from IDEN-treated mice, indicating that IDENs have a direct effect on induction of anergy in liver NKT cells. APCs play an essential role in Pexidartinib price liver NKT cell activation by presenting lipid-related antigen on an APC CD1d molecule–dependent and independent manner.22 First, we tested whether DCs take up IDENs. Both CD11c+ DCs and F4/80+ macrophages from the livers of naïve mice took up IDENs rapidly (as

early as 2 hours) and continued to take up IDENs over 24 hours (Fig. 7A, Supporting Fig. 7). We then tested whether IDEN treatment has an effect on NKT cell activation of mice. FACS analysis of liver leukocytes suggested GSK1120212 order that the expression of MHCII and CD86 by the DCs (Fig. 7B) was reduced in mice that had been administered IDENs. On coculture of DCs purified from the livers of mice that had been administered IDENs or vehicle with carboxyfluorescein succinimidyl ester–labeled liver NKT cells isolated from mice that had been administered IDENs or vehicle in the presence of α-GalCer, DCs from the livers of mice that had been administered IDENs exhibited a reduced ability to stimulate the proliferation of NKT cells, regardless of the source of the NKT cells (Fig. 7C). RT-PCR analysis further indicated that

the expression of IL-12 and tumor necrosis factor α, which are critical for activation of DCs and DC-mediated activation of NKT cells, was significantly lower in DCs sorted from the livers of α-GalCer–injected mice that had been administered IDENs (Fig. 7D). Additionally, the levels of the immunosuppressive cytokine IL-10 were higher. DCs also can activate NKT cells in a CD1d-independent manner through Toll-like receptor (TLR)-induced release of soluble mediators, including IL-12 and type I IFNs.23,24 We found that IDENs suppressed 上海皓元医药股份有限公司 the expression of IL-12 and IFN-β in TLR-stimulated DCs (Fig. 7E) and that IFN-γ release was reduced greatly when TLR ligand–treated DCs were cocultured with liver NKT cells in the presence of IDENs (Fig. 7F). Thus, IDENs can also induce NKT cell anergy through modification of the ability of DCs to stimulate NKT cell anergy in the context of both glycolipid presentation and TLR-mediated pathways. To further determine whether IDEN-associated PGE2 plays a role in the inhibition of production of IL-12, the effects of IDENs isolated from indomethacin-treated mice on the production of TLR-stimulated DCs was evaluated. ELISA results (Fig.

5, 0.75, 1, 1.5, 2, 2.5, 3,

4, 6, 8, 12, 16, 24, 48, 72, and 96 hours postdose). For telaprevir concentration analysis, blood samples were drawn on day 1 and day 8, period 2 (sampling timepoints: predose, 0.5, 1, 2, 2.5, 3, 4, 6, and 8 hours post-morning dose). The effect of telaprevir on tacrolimus PK was studied at steady-state telaprevir. During period 1, volunteers were admitted to the CRU on day −1 and discharged on day 3. On day 1, a single 2-mg oral dose of tacrolimus (4 capsules Prograf, 0.5 mg) was administered 2.5 hours after the start of the standard, medium-fat breakfast. There was a minimum washout of 14 days between day 1, period 1 and day 1, period 2. During period 2, volunteers were admitted

to the CRU on day 7 and SP600125 concentration discharged on day 11. From days 1 to 13 of period 2, telaprevir 750 mg q8h was administered 0.5 hours after the start of a meal or snack. On day 8, a single 0.5-mg oral dose of tacrolimus (1 capsule Prograf, Selleck Seliciclib 0.5 mg) was administered 2.5 hours after the start of a standard, medium-fat breakfast (i.e., 2 hours post-telaprevir dose). Volunteers returned for a follow-up visit on day 23 (±3 days). Approximately 4 mL of blood was drawn by way of direct venipuncture or indwelling catheter at each timepoint and processed for analyzing whole blood tacrolimus concentrations and plasma telaprevir concentrations. When tacrolimus was administered alone, blood samples were collected for tacrolimus analysis on day 1, period 1 (sampling timepoints: predose, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 上海皓元医药股份有限公司 24, 48, 72, 96, and

120 hours postdose). When tacrolimus was coadministered with telaprevir, blood samples were collected for tacrolimus analysis on day 8, period 2 (sampling timepoints: predose, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 24, 48, 72, 96, 120, and 144 hours postdose). Similarly, for telaprevir concentration analysis, blood samples were drawn on day 8, period 2 (sampling timepoints: predose, 0.5, 1, 2, 2.5, 3, 4, 6, and 8 hours post-morning dose). Whole blood concentrations of both cyclosporine and tacrolimus and plasma telaprevir concentrations were analyzed using validated assay methods. Briefly, cyclosporine, telaprevir, and their internal standards were extracted from samples using liquid/liquid extraction. Tacrolimus and its internal standard were extracted from samples using protein precipitation followed by solid-phase extraction. After evaporation under nitrogen, the residue of each analyte was reconstituted and analyzed using liquid chromatography followed by tandem mass spectrometry with selected ion monitoring in the positive ion mode. Calibration curves for each analyte was generated using weighted (1/x2) linear least-squares regression. The lower limit of quantitation for the cyclosporine assay in whole blood was 0.