Magnitude of Urine Albumin and KIM-1 Changes Can be Used to Differentiate Glomerular Injury From Tubular Injury in Rats.

Emerging urinary kidney safety biomarkers have been evaluated in recent years and have been shown to be superior to the serum parameters blood urea nitrogen (BUN) and creatinine (sCr) for monitoring kidney injury in the proximal tubule. However, their potential application in differentiating the location of the initial kidney injury (eg, glomerulus vs tubule) has not been fully explored. Here, we assessed the performance of two algorithms that were constructed using either an empirical or a mathematical model to predict the site of kidney injury using a data set consisting of 22 rat kidney toxicity studies with known urine biomarker and histopathologic outcomes. Two kidney safety biomarkers used in both models, kidney injury molecule 1 (KIM-1) and albumin (ALB), were the best performers to differentiate glomerular injury from tubular injury. The performance of algorithms using these two biomarkers against the gold standard of kidney histopathologic examination showed high sensitivity in differentiating the location of the kidney damage to either the glomerulus or the proximal tubules. These data support the exploration of such an approach for use in clinical settings, leveraging urinary biomarker data to aid in the diagnosis of either glomerular or tubular injury where histopathologic assessments are not conducted.

Sensitization of cholangiocarcinoma cells to chemotherapy through BCRP inhibition with ß-caryophyllene oxide.

Cholangiocarcinomas (CCAs) are cancers originated in the biliary tree, which are characterized by their high mortality and marked chemoresistance, partly due to the activity of ATP-binding cassette (ABC) export pumps, whose inhibition has been proposed as a strategy for enhancing the response to chemotherapy. We have previously shown that ß-caryophyllene oxide (CRYO) acts as a chemosensitizer in hepatocellular carcinoma by inhibiting ABCB1, MRP1, and MRP2. Here, we have evaluated the usefulness of CRYO in inhibiting BCRP and improving the response of CCA to antitumor drugs. The TCGA-CHOL cohort (n = 36) was used for in silico analysis. BCRP expression (mRNA and protein) was assayed in samples from intrahepatic (iCCA) and extrahepatic (eCCA) tumors (n = 50) and CCA-derived cells (EGI-1 and TFK-1). In these cells, BCRP-dependent mitoxantrone transport was determined by flow cytometry. At non-toxic concentrations, CRYO inhibited BCRP function, which enhanced the cytostatic effect of drugs used in the treatment of CCA. The BCRP ability to confer resistance to a panel of antitumor drugs was determined in Chinese hamster ovary (CHO) cells with stable BCRP expression. At non-toxic concentrations, CRYO markedly reduced BCRP-induced resistance to known substrate drugs (mitoxantrone and SN-38) and cisplatin, gemcitabine, sorafenib, and 5-FU but not oxaliplatin. Neither CRYO nor cisplatin alone significantly affected the growth of BCRP-expressing tumors subcutaneously implanted in immunodeficient mice. In contrast, intratumor drug content was enhanced when administered together, and tumor growth was inhibited. In sum, the combined treatment of drugs exported by BCRP with CRYO can improve the response to chemotherapy in CCA patients.

Flucloxacillin Is a Weak Inducer of CYP3A4 in Healthy Adults and 3D Spheroid of Primary Human Hepatocytes.

Flucloxacillin is a widely used antibiotic. It is an agonist to the nuclear receptor PXR that regulates the expression of cytochrome P450 (CYP) enzymes. Treatment with flucloxacillin reduces warfarin efficacy and plasma concentrations of tacrolimus, voriconazole, and repaglinide. We conducted a translational study to investigate if flucloxacillin induces CYP enzymes. We also investigated if flucloxacillin induces its own metabolism as an autoinducer. We performed a randomized, unblinded, two-period, cross-over, clinical pharmacokinetic cocktail study. Twelve healthy adults completed the study. They ingested 1 g flucloxacillin 3 times daily for 31 days, and we assessed the full pharmacokinetics of the Basel cocktail drugs on days 0, 10, and 28, and plasma concentrations of flucloxacillin on days 0, 9, and 27. The 3D spheroid of primary human hepatocytes (PHHs) were exposed to flucloxacillin (concentration range: 0.15-250 µM) for 96 hours. Induction of mRNA expression, protein abundance, and enzyme activity of CYP enzymes were assessed. Flucloxacillin treatment reduced the metabolic ratio of midazolam (CYP3A4), (geometric mean ratio (GMR) 10 days (95% confidence interval (CI)): 0.75 (0.64-0.89)) and (GMR 28 days (95% CI): 0.72 (0.62-0.85)). Plasma concentrations of flucloxacillin did not change during 27 days of treatment. Flucloxacillin caused concentration-dependent induction of CYP3A4 and CYP2B6 (mRNA, protein, and activity), CYP2C9 (mRNA and protein), CYP2C19 (mRNA and activity), and CYP2D6 (activity) in 3D spheroid PHH. In conclusion, flucloxacillin is a weak inducer of CYP3A4, which may lead to clinically relevant drug-drug interactions for some narrow therapeutic range drugs that are substrates of CYP3A4.

Dicloxacillin-warfarin drug-drug interaction-A register-based study and in vitro investigations in 3D spheroid primary human hepatocytes.

AIMS: Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. METHODS: We conducted a register-based study and analysed international normalized ratio (INR) levels in chronic warfarin users before and after short- and long-term use of dicloxacillin (n = 1023) and flucloxacillin (n = 123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes at the level of mRNA, and protein and enzyme activity. RESULTS: Short- and long-term dicloxacillin treatments decreased INR levels by -0.65 (95% confidence interval [CI]: -0.57 to -0.74) and -0.76 (95% CI: -0.50 to -1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long-term dicloxacillin treatment. Flucloxacillin decreased INR levels by -0.37 (95% CI: -0.14 to -0.60). In 3D spheroid primary human hepatocytes, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9-, 2.9- and 2.4-fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7-fold. CONCLUSION: Dicloxacillin induces CYPs and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long-term treatment with dicloxacillin. The in vitro results corroborated this drug-drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long-term treatment of endocarditis.

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction.

Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

Characterization of hepatic zonation in mice by mass-spectrometric and antibody-based proteomics approaches.

Periportal and perivenous hepatocytes show zonal heterogeneity in metabolism and signaling. Here, hepatic zonation in mouse liver was analyzed by non-targeted mass spectrometry (MS) and by the antibody-based DigiWest technique, yielding a comprehensive overview of protein expression in periportal and perivenous hepatocytes. Targeted immunoaffinity-based proteomics were used to substantiate findings related to drug metabolism. 165 (MS) and 82 (DigiWest) zonated proteins were identified based on the selected criteria for statistical significance, including 7 (MS) and 43 (DigiWest) proteins not identified as zonated before. New zonated proteins especially comprised kinases and phosphatases related to growth factor-dependent signaling, with mainly periportal localization. Moreover, the mainly perivenous zonation of a large panel of cytochrome P450 enzymes was characterized. DigiWest data were shown to complement the MS results, substantially improving possibilities to bioinformatically identify zonated biological processes. Data mining revealed key regulators and pathways preferentially active in either periportal or perivenous hepatocytes, with ß-catenin signaling and nuclear xeno-sensing receptors as the most prominent perivenous regulators, and several kinase- and G-protein-dependent signaling cascades active mainly in periportal hepatocytes. In summary, the present data substantially broaden our knowledge of hepatic zonation in mouse liver at the protein level.

Organic Cation Transporter I and Na+ /taurocholate Co-Transporting Polypeptide are Involved in Retrorsine- and Senecionine-Induced Hepatotoxicity in HepaRG cells.

SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Pesticide mixture effects on liver protein abundance in HepaRG cells.

Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.

Cross-species analysis of hepatic cytochrome P450 and transport protein expression.

Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models.

Induction and repression effects on CYP and transporter protein abundance by azole mixture uptake in rat liver.

Detection of mixture effects is a major challenge in current experimental and regulatory toxicology. Robust markers are needed that are easy to quantify and responsive to chemical stressors in a broad dose range. Several hepatic enzymes and proteins related to drug metabolism like cytochrome-P-450 (CYP) enzymes and transporters have been shown to be responsive to pesticide active substances in a broad dose range and are therefore good candidates to be used as markers for mixture toxicity. Even though they can be well quantified at the mRNA level, quantification on the protein level is challenging because most of these proteins are membrane bound. Here we report the development of mass spectrometry-based assays using triple-x-proteomics (TXP) antibodies in combination with targeted selected ion monitoring (tSIM) to quantify changes of protein levels due to exposure to mixtures of pesticide active substances. Our results indicate that changes on the protein level of CYP1A1, ABCB2, ABCC3 are in line with observations on the mRNA and enzyme activity level and are indicative of mixture effects. Therefore, the tests are promising to reveal effects by chemical mixture effects in toxicological studies in rats.

Impact of iron oxide nanoparticles on xenobiotic metabolism in HepaRG cells.

Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach.

Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

Representing dynamic biological networks with multi-scale probabilistic models.

Dynamic models analyzing gene regulation and metabolism face challenges when adapted to modeling signal transduction networks. During signal transduction, molecular reactions and mechanisms occur in different spatial and temporal frames and involve feedbacks. This impedes the straight-forward use of methods based on Boolean networks, Bayesian approaches, and differential equations. We propose a new approach, ProbRules, that combines probabilities and logical rules to represent the dynamics of a system across multiple scales. We demonstrate that ProbRules models can represent various network motifs of biological systems. As an example of a comprehensive model of signal transduction, we provide a Wnt network that shows remarkable robustness under a range of phenotypical and pathological conditions. Its simulation allows the clarification of controversially discussed molecular mechanisms of Wnt signaling by predicting wet-lab measurements. ProbRules provides an avenue in current computational modeling by enabling systems biologists to integrate vast amounts of available data on different scales.

Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice.

The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500 ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells.

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that ß-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that ß-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, ß-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of ß-catenin in melanoma progression and therapy resistance. We further demonstrate that ß-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.