21 research outputs found
Recommended from our members
Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans
Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform
Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis
Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFÎČ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFÎČ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH
Recommended from our members
The effect of cyclosporine A on cytokeratin intermediate filament phosphorylation
MCF7 cell cultures exposed to cyclosporine A (CsA) from 1-5 ÎŒM and from 0-48 hr have decreased viability and clonogenic ability. When these cells are examined by indirect immunofluorescence for cytoskeletal filaments, there is altered cell shape, but no alteration in microfilaments or microtubules. However, there is a collapse in cytokeratin intermediate filament arrays resulting in a peri-nuclear reorganization at 24-48 hr incubation (5 ÎŒM). When MCF7 cells are pre-treated in 5 ÎŒM CsA for 24 hr, and then placed in non-drug media for 12-24 hr, there is recovery of cytokeratin arrays and cell shape. Analysis of cytokeratin proteins by 2-D electrophoresis depict decreased phosphorylated proteins at 2-5 ÎŒM CsA (24 hr). CsA (1-5 ÎŒM CsA, 1-48 hr) inhibits phosphate content on cytokeratin protein to 35% levels on cytokeratin #8, and to 50% levels on cytokeratins #18, and #19. CsA decreases phosphate content on cytokeratin protein during incorporation, at steady state, and during turnover. Determinations of phosphate loss indicate that CsA increases turnover at greater levels than it inhibits incorporation, suggesting the activation of a phosphatase enzyme. CsA-induced decreased phosphorylation levels can be rescued by addition of 100 nM 12-o-tetradecanoylphorbol-13-acetate (TPA), but not upon addition of 50 ÎŒM forskolin. This indicates a CsA-sensitive protein kinase C site on cytokeratin protein. An in vitro urea assay was utilized to examine assembly and disassembly or cytokeratin protofilaments. CsA (5 ÎŒM, 48-72 hr) does not alter assembly of cytokeratin protofilaments from monomeric proteins. Analysis of disassembly of protofilaments into monomeric units resulted in CsA-induced (5 ÎŒM, 48-72 hr) inhibition of disassembly. Regulation of phosphorylation may therefore be important in the assembly and disassembly of cytokeratin filaments, and a resistance to disassembly may indicate a CsA interaction that is toxic to the cell
Recommended from our members
Effects of cyclosporin A on cytokeratin intermediate filaments in potaroo kangaroo rat renal cell cultures
Cyclosporin A (CsA) was incubated at concentrations of 5.0 x 10â»â¶ M for 72 hours, and at concentrations of 1.0 and 0.5 x 10â»â¶ M for 30 days with kangaroo rat proximal tubular epithelial cells (PtKâ) in order to evaluate its effects on the cytoskeleton. Alterations in the cytoskeleton were assessed by indirect immunofluorescence of viable cells, and by two dimensional electrophoresis of a high salt extract from the cells. There is a selective alteration of the cytokeratin intermediate filament organization in both the short term (5 x 10â»â¶ M, 72 hr) and long term (1 and 0.5 x 10â»â¶ M, 30 days) exposures. There are either peri-nuclear rings formed or the formation of a single aggregate clump of the cytokeratins within the cytoplasm. Other components of the cytoskeleton, the microtubules and the microfilaments remain unaffected at both short term and long term exposures. Along with this cytokeratin alteration in CsA exposed cells is the decrease or elimination of an acidic triplet of cytokeratin protein monomers, human equivalent K15 (50 kd), K16 (48 kd), K17 (46 kd). This may be related to CsA-associated nephrotoxicity
Identifying and quantifying heterogeneity in high content analysis: application of heterogeneity indices to drug discovery.
One of the greatest challenges in biomedical research, drug discovery and diagnostics is understanding how seemingly identical cells can respond differently to perturbagens including drugs for disease treatment. Although heterogeneity has become an accepted characteristic of a population of cells, in drug discovery it is not routinely evaluated or reported. The standard practice for cell-based, high content assays has been to assume a normal distribution and to report a well-to-well average value with a standard deviation. To address this important issue we sought to define a method that could be readily implemented to identify, quantify and characterize heterogeneity in cellular and small organism assays to guide decisions during drug discovery and experimental cell/tissue profiling. Our study revealed that heterogeneity can be effectively identified and quantified with three indices that indicate diversity, non-normality and percent outliers. The indices were evaluated using the induction and inhibition of STAT3 activation in five cell lines where the systems response including sample preparation and instrument performance were well characterized and controlled. These heterogeneity indices provide a standardized method that can easily be integrated into small and large scale screening or profiling projects to guide interpretation of the biology, as well as the development of therapeutics and diagnostics. Understanding the heterogeneity in the response to perturbagens will become a critical factor in designing strategies for the development of therapeutics including targeted polypharmacology
A Quantitative Systems Pharmacology Platform Reveals NAFLD Pathophysiological States and Targeting Strategies
Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD. Analysis of a 182 patient-derived hepatic RNA-sequencing dataset generated 12 gene signatures mirroring these states. Screening against the LINCS L1000 database led to the identification of drugs predicted to revert these signatures and corresponding disease states. A proof-of-concept study in LAMPS demonstrated mitigation of steatosis, inflammation, and fibrosis, especially with drug combinations. Mechanistically, several structurally diverse drugs were predicted to interact with a subnetwork of nuclear receptors, including pregnane X receptor (PXR; NR1I2), that has evolved to respond to both xenobiotic and endogenous ligands and is intrinsic to NAFLD-associated transcription dysregulation. In conjunction with iPSC-derived cells, this platform has the potential for developing personalized NAFLD therapeutic strategies, informing disease mechanisms, and defining optimal cohorts of patients for clinical trials
Heterogeneity in the activation STAT3 in Cal33 cells.
<p>Cal33 cells were treated with IL-6 (50 ng/ml) for 15 min. then fixed and labeled with an antibody to phospho-STAT3-Y705. A) Pseudocolor image of STAT3 activation shows a high degree of heterogeneity in the intensity of the Cy5-labeled secondary antibody (color scale at lower right indicates mapping of relative fluorescent intensities to colors). Scale bar is 100 um (lower left). B) The standard deviation of the well average STAT3 activity in replicate wells (EC50â=â3.3 ng/ml, error bars are ±1Ï, Nâ=â8) indicates the assay is highly reproducible despite the observed cellular heterogeneity (Zââ=â0.54) C) The standard deviation of the cellular STAT3 activity (error bars are ±1Ï) indicates the high variability in the cell-to-cell STAT3 Activity consistent with the appearance of the image (A).</p
Comparison of the activation of STAT3 across 5 cell lines.
<p>Application of the HI's to the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102678#pone-0102678-g002" target="_blank">Figure 2</a>. Left Panel) Activation of pSTAT3 by exposure to IL-6 for 15 min at the indicated concentrations. Right Panel) Activation of pSTAT3 by exposure to Oncostatin M for 15 min at the indicated concentrations. Red Bars) Diversity index (DIV) indicating the relative heterogeneity associated with the activation of pSTAT3. The horizontal red line indicates the selected threshold for classifying populations a heterogeneous. Green Bars) The non-Normality index (nNRM) indicating the extent of deviation from a single, normally distributed population. The green horizontal line indicates the selected threshold for classifying a population as having macro-heterogeneity. Blue Bars) The percent outliers (%OL) indicates the percentage of cells with an activity level that is above the upper inner fence or below the lower inner fence. The horizontal blue line indicates the selected threshold that is used to classify a population as having more than the expected number of outliers.</p