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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

SU-E-J-240: Development of a Novel 4D MRI Sequence for Real-Time Liver Tumor Tracking During Radiotherapy

Purpose: To develop a Novel 4D MRI Technique that is feasible for realtime liver tumor tracking during radiotherapy. Methods: A volunteer underwent an abdominal 2D fast EPI coronal scan on a 3.0T MRI scanner (Siemens Inc., Germany). An optimal set of parameters was determined based on image quality and scan time. A total of 23 slices were scanned to cover the whole liver in the test scan. For each scan position, the 2D images were retrospectively sorted into multiple phases based on breathing signal extracted from the images. Consequently the 2D slices with same phase numbers were stacked to form one 3D image. Multiple phases of 3D images formed the 4D MRI sequence representing one breathing cycle. Results: The optimal set of scan parameters were: TR= 57ms, TE= 19ms, FOV read= 320mm and flip angle= 30°, which resulted in a total scan time of 14s for 200 frames (FMs) per slice and image resolution of (2.5mm,2.5mm,5.0mm) in three directions. Ten phases of 3D images were generated, each of which had 23 slices. Based on our test scan, only 100FMs were necessary for the phase sorting process which may lower the scan time to 7s/100FMs/slice. For example, only 5 slices/35s are necessary for a 4D MRI scan to cover liver tumor size ≤ 2cm leading to the possibility of tumor trajectory tracking every 35s during treatment. Conclusion: The novel 4D MRI technique we developed can reconstruct a 4D liver MRI sequence representing one breathing cycle (7s/ slice) without an external monitor. This technique can potentially be used for real-time liver tumor tracking during radiotherapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-F-J-103: Assessment of Liver Tumor Contrast for Radiation Therapy: Inter-Patient and Inter-Sequence Variability

Purpose: To determine the variation in tumor contrast between different MRI sequences and between patients for the purpose of MRI-based treatment planning. Methods: Multiple MRI scans of 11 patients with cancer(s) in the liver were included in this IRB-approved study. Imaging sequences consisted of T1W MRI, Contrast-Enhanced T1W MRI, T2W MRI, and T2*/T1W MRI. MRI images were acquired on a 1.5T GE Signa scanner with a four-channel torso coil. We calculated the tumor-to-tissue contrast to noise ratio (CNR) for each MR sequence by contouring the tumor and a region of interest (ROI) in a homogeneous region of the liver using the Eclipse treatment planning software. CNR was calculated (I-Tum-I-ROI)/SD-ROI, where I-Tum and I-ROI are the mean values of the tumor and the ROI respectively, and SD-ROI is the standard deviation of the ROI. The same tumor and ROI structures were used in all measurements for different MR sequences. Inter-patient Coefficient of variation (CV), and inter-sequence CV was determined. In addition, mean and standard deviation of CNR were calculated and compared between different MR sequences. Results: Our preliminary results showed large inter-patient CV (range: 37.7% to 88%) and inter-sequence CV (range 5.3% to 104.9%) of liver tumor CNR, indicating great variations in tumor CNR between MR sequences and between patients. Tumor CNR was found to be largest in CE-T1W (8.5±7.5), followed by T2W (4.2±2.4), T1W (3.4±2.2), and T2*/T1W (1.7±0.6) MR scans. The inter-patient CV of tumor CNR was also the largest in CE-T1W (88%), followed by T1W (64.3%), T1W (56.2%), and T2*/T1W (37.7) MR scans. Conclusion: Large inter-sequence and inter-patient variations were observed in liver tumor CNR. CE-T1W MR images on average provided the best tumor CNR. Efforts are needed to optimize tumor contrast and its consistency for MRI-based treatment planning of cancer in the liver. This project is supported by NIH grant: 1R21CA165384.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-F-J-221: Adjusted Dose and Its Relation to Radiation Induced Liver Disease During Hepatocellular Carcinoma Radiotherapy

Purpose: Many patients with hepatocellular carcinoma (HCC) had hepatic anatomy variations as a result of inter-fraction deformation during fractionated radiotherapy, which may result in difference from the planned dose. This study aimed to investigate the relationship between adjusted dose and radiation induced liver disease (RILD) in HCC patients receiving three dimensional conformal radiotherapy (3DCRT). Methods: Twenty-three HCC patients received conventional fractionated 3DCRT were enrolled in this retrospective investigation. Among them, seven patients had been diagnosed of RILD post-radiotherapy, including 4 cases of grade 2, 3 cases of grade 3 according to the CTCAE Version 3.0. Daily cone-beam CT (CBCT) scans were acquired throughout the whole treatment course for each patient. To reconstruct the daily dose to a patient considering the interfraction anatomy variations, the planned beams from each patient’s treatment plan were firstly applied to each daily modified CBCT (mCBCT). The daily doses were then summed together with the help of deformable image registration (DIR) to obtain the adjusted dose (Dadjusted) of the patient. Finally, the dose changes in normal liver between planned dose (Dplan) and Dadjusted were evaluated by V20, V30, V40 and the mean dose to normal liver (MDTNL). Univariate analysis was performed to identify the significant dose changes. Results: Among the twenty-three patients, the adjusted liver V20, V30, V40 and MDTNL showed significant changes from the planned ones (p<0.05) and averagely increased by 4.1%, 4.7%, 4.5% and 3.9Gy, respectively. And the adjusted liver dose in twenty-one patients (91%) were higher than planned value, the adjusted dose of patients with RILD (6/7) exceeds to the hepatic radiation tolerance. Conclusion: The adjusted dose of all the studied patients significantly differs from planned dose, and mCBCT-based dose reconstruction can aid in evaluating the robustness of the planning solutions, and adjusted dose has the potential to reduce the risk of RILD. The author would like to express great thanks to Lei Xing, Daniel S Kapp and Yong Yang in the Stanford University School of Medicine for their valuable suggestions to this work.This work is supported by NSFC(61471226),China Postdoctoral Science Foundation (2015T80739, 2014M551949) and research funding from Shandong Province(JQ201516).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MO-FG-CAMPUS-JeP3-05: Evaluation of 4D CT-On-Rails Target Localization Methods for Free Breathing Liver Stereotactic Body Radiotherapy (SBRT)

Purpose: Liver SBRT patients unable to tolerate breath-hold for radiotherapy are treated free-breathing with image guidance. Target localization using 3D CBCT requires extra margins to accommodate the respiratory motion. The purpose of this study is to evaluate the accuracy and reproducibility of 4D CT-on-rails in target localization for free-breathing liver SBRT. Methods: A Siemens SOMATOM CT-on-Rails 4D with Anzai Pressure Belt system was used both as the simulation and the localization CT. Fiducial marker was placed close to the center of the target prior to the simulation. Amplitude based sorting was used in the scan. Eight or sixteen phases of reconstructed CT sets (depends on breathing pattern) can be sent to Velocity to create the maximum intensity projection (MIP) image set. Target ITV and fiducial ITV were drawn based on the MIP image. In patient localization, a 4D scan was taken with the same settings as the sim scan. Images were registered to match fiducial ITVs. Results: Ten liver cancer patients treated for 50Gy over 5 fractions, with amplitudes of breathing motion ranging from 4.3–14.5 mm, were analyzed in this study. Results show that the Intra & inter fraction variability in liver motion amplitude significantly less than the baseline inter-fraction shifts in liver position. 90% of amplitude change is less than 3 mm. The differences in the D99 and D95 GTV dose coverage between the 4D CT-on-Rails and the CBCT plan were small (within 5%) for all the selected cases. However, the average PTV volume by using the 4D CT-on-Rails is 37% less than the CBCT PTV volume. Conclusion: Simulation and Registration using 4D CT-on-Rails provides accurate target localization and is unaffected by larger breathing amplitudes as seen with 3D CBCT image registration. Localization with 4D CT-on-Rails can significantly reduce the PTV volume with sufficient tumor.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

TU-H-BRC-02: Biological Dose Escalation for Liver SBRT Through Spatiotemporal Fractionation

Purpose: Stereotactic body radiotherapy (SBRT) has become an established treatment option for liver cancer. For patients with large tumors, the prescription dose is often limited by constraints on the mean liver dose, leading to tumor recurrence. In this work, we demonstrate that spatiotemporal fractionation schemes, ie delivering distinct dose distributions in different fractions, may allow for a 10% increase in biologically effective dose (BED) in the tumor compared to current practice where each fraction delivers the same dose distribution. Methods: We consider rotation therapy delivered with x-ray beams. Treatment plan optimization is performed using objective functions evaluated for the cumulative BED delivered at the end of treatment. This allows for simultaneously optimizing multiple distinct treatment plans for different fractions. Results: The treatment that optimally exploits fractionation effects is designed such that each fraction delivers a similar dose bath to the uninvolved liver while delivering high single fraction doses to complementary parts of the target volume. Thereby, partial hypofractionation in the tumor is achieved along with near uniform fractionation in the surrounding liver - leading to an improvement in the therapeutic ratio. The benefit of such spatiotemporal fractionation schemes depends on tumor geometry and location as well as the number of fractions. For 5-fraction treatments (allowing for 5 distinct dose distributions) an improvement in the order of 10% is observed. Conclusion: Delivering distinct dose distributions in different fractions, purely motivated by fractionation effects rather than geometric changes, may improve the therapeutic ratio. For treatment sites where the prescriptions dose is limited by mean dose constraints in the surrounding organ, such as liver cancer, this approach may facilitate biological dose escalation and improved cure rates.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T Cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4 + and CD8 + T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8 + tissue-resident memory T (T RM ) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4 + and CD8 + T cells, including CD8 + T RM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4 + T helper (T H ) cell and cytotoxic CD8 + T cell epitopes of NS5B in vivo , which were subsequently used to design a K d NS5B 451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo . The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting T H and cytotoxic CD8 + T cell responses and intrahepatic CD8 + T RM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal. There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis

Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3) -/- mice are completely protected from ethanol-induced liver injury, Mlkl -/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. Bone marrow transplants between Mlkl -/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl -/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→Mlkl -/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1–mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl -/- mice phagocytosed fewer bioparticles than KCs of WT mice. Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1990

Experiment K-6-12. Morphometric studies of atrial or granules and hepatocytes. Part 1: Morphometric study of the liver; Part 2: The atrial granular accumulations

The livers of flight, F, rats from the Cosmos 1887 mission were markedly paler and heavier than those of the synchronous, S, and vivarium, V, controls. In the F group, microscopic study revealed extensive hepatocytic intracytoplasmic vacuolization which was moderate in the S and minimal in the V groups. The vacuoles were not sudanophilic and therefore were regarded as glycogenic in origin. To obtain objective data concerning the extent of the vacuolization, livers were examined by computer assisted morphometry. Measurements of profile area and perimeter of the hepatocyte nuclei and vacuoles were evaluated according to stereological principles. Results indicated that the volume density of the nuclei was less in the F group than in the S(p equal less than 0.0002) and V(p equal less than 0.001) groups. Mean volume of individual nuclei did not differ. Volume density of the vacuoles was greater in the F than in the V group (p equal less than 0.02) while their mean diameter was less (p equal less than 0.05). To ascertain the relationship between increase in liver weight of the flight animals and the results of this study, an assumption was made that the specific gravity of the vacuolar contents was similar to the other extranuclear components of the hepatocyte. On that basis, calculations showed that the elevated vacuolar volume density in the flight group did not cause the increased liver weight in those animals, but that the non-nuclear, non-vacuolar parenchymal compartment did contribute significantly. Factors that may have played a causal role in liver weight and vacuolar compartment increases are discussed.

Kraft, L. M.↗
NASA NTRS1999

Induction of three-dimensional assembly of human liver cells by simulated microgravity

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

NASA Center JSC↗
NASA NTRS2023

Exploring the ARNTL and RORc Genes' Impact on Lipid Accumulation in the Liver via the Circadian Rhythm

The objective of our proposed experiment is to prove the significance of the upregulation of the ARNTL gene and down regulation of the RORc gene in space, highlighting their crucial roles in causing circadian rhythm misalignment during spaceflight, ultimately resulting in liver lipid accumulation. This experiment was created by thoroughly analyzing the genetic data (UseGalaxy) in OSD-245, Transcriptional analysis of liver from mice flown on the RR-6 mission (NASA GeneLab), and making a comparison between the Flight ISS Terminal and the Ground Control ISS Terminal groups. In our study, we intend to establish experimental groups of Mus musculus (lab mice), with 4 of these groups designated for space travel, while the remaining groups will serve as ground control samples on Earth. Both the space and ground conditions are categorized into four groups according to the genetic makeup of the mice. Within each condition, there will be a control group representing the wild type genes, and the experimental groups will involve knockout mice for ARNTL and RORc. We will then compare the lipid accumulation between the ground control mice and FLT mice to understand whether or not spaceflight causes lipid accumulation, due to a misaligned circadian rhythm. The experiment is anticipated to yield two main outcomes. Firstly, the upregulation of ARNTL is expected to rectify the circadian rhythm misalignment in space, consequently heightening lipid accumulation within the liver. Secondly, the downregulation of RORc is predicted to result in a diminished immune response in the liver, thereby facilitating lipid accumulation. Our space research is expected to aid in lipid buildup countermeasures, informing scientists regarding astronaut protection and providing insights into obesity and non-alcoholic fatty liver disease.

GL4HS↗
DOE OSTI2013

Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Regulation of retinoid X receptor gamma expression by fed state in mouse liver

Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting–feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting–feeding cycle. - Highlights: • Refeeding increases the RXRγ expression level in mouse liver. • RXRγ expression is induced by high glucose condition in primary hepatocytes. • RXRγ and LXRα have synergistic effect on SREBP-1c promoter activity. • RXRγ binds to LXRE(-299/-280) located within SREBP-1c promoter region and interacts with LXRα.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers

Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 μM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R. - Highlights: • A mechanism of carbamazepine (CBZ)-induced cytoprotection in livers is proposed. • Impaired autophagy is a key event contributing to lethal reperfusion injury. • The importance of autophagy is extended and confirmed in an in vivo model. • CBZ is a potential agent to improve liver function after liver surgery.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Promoter-region hypermethylation and expression downregulation of Yy1 (Yin yang 1) in preneoplastic liver lesions in a thioacetamide rat hepatocarcinogenesis model

Thioacetamide (TAA) has been used to develop a rodent model for hepatocarcinogenesis. To determine the genes with epigenetic modifications in early hepatocarcinogenesis, we did a genome-wide scan for hypermethylated promoter regions using CpG island microarrays in TAA-promoted rat liver tissue. Eight genes were selected based on the microarray profile; of these, Yy1 and Wdr45b were confirmed to be hypermethylated by methylation-specific polymerase chain reaction (PCR) and pyrosequencing and downregulated by real-time reverse transcription PCR. Non-neoplastic liver cells had nuclear Yy1 immunoreactivity, while preneoplastic foci with glutathione S-transferase placental form (GST-P) immunoreactivity had decreased Yy1 immunoreactivity. The incidence of these foci was proportional to the dose of TAA administered. Co-expression analysis of gene products downstream of Yy1 revealed increased nuclear phospho-c-Myc{sup +} foci as well as nuclear and cytoplasmic p21{sup Cip1+} foci in Yy1{sup −} or GST-P{sup +} foci in response to TAA-promotion dose. Although the absolute number of cells was low, the incidence of death receptor 5{sup −} foci was increased in Yy1{sup −} foci in proportion to the TAA dose. Yy1{sup −}/GST-P{sup +} foci revealed a higher number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells than Yy1{sup +}/GST-P{sup +} foci, while cleaved caspase-3{sup +} cells were unchanged between Yy1{sup –}/GST-P{sup +} and Yy1{sup +}/GST-P{sup +} foci. In the case of Wdr45b, most GST-P{sup +} foci were Wdr45b{sup –} and were not increased by TAA promotion. These results suggest involvement of Yy1 in the epigenetic gene regulation at the early stages of TAA promoted cell proliferation and concomitant cell cycle arrest in preneoplastic lesions. - Highlights: • Epigenetically downregulated genes were searched in TAA-promnoted rat livers. • Yy1 and Wdr45b showed promoter-region hypermethylation and mRNA downregulation. • TAA promoted increase of preneoplastic Yy1{sup –}/GST-P{sup +} foci showing high proliferation. • TAA promoted activation of c-Myc or p21{sup Cip1} within Yy1{sup −}/GST-P{sup +} foci. • Epigenetic Yy1 downregulation plays a role in rat liver tumor promotion by TAA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Di-(2-ethylhexyl) phthalate could disrupt the insulin signaling pathway in liver of SD rats and L02 cells via PPARγ

Di-(2-ethylhexyl)-phthalate (DEHP), a ubiquitous industrial pollutant in our daily life, has been reported to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies previously. Recently, it has been reported to be an endocrine disrupter and ligand to peroxisome proliferator activated receptor, which could influence the homeostasis of liver metabolic systems and contribute to the development of type-2 diabetes. However, the potential mechanisms are not known yet. This study was designed to solve these problems with male SD rats and normal human hepatocyte line, L02 cells, exposed to DEHP for toxicological experiments. Adult male SD rats were divided into four groups, normal group fed with regular diets and three DEHP-treated groups (dissolved in olive oil at doses of 0.05, 5 and 500 mg/kg body weight, respectively, once daily through gastric intubations for 15 weeks). L02 cells were divided into 6 groups, normal group with 5, 10, 25, 50, and 100 μmol/l DEHP groups. DEHP-exposed rats exhibited significant liver damage, glucose tolerance, and insulin tolerance along with reduced expression of insulin receptor and GLUT4 proteins in the liver tissues. The results of in vitro experiments could determine that the DEHP-induced activation of peroxisome proliferator activated receptor γ (PPARγ) played a key role in the production of oxidative stress and down-regulated expression of insulin receptor and GLUT4 proteins in L02 cells. This conclusion could be supported by the results of in vitro experiments, in which the cells were exposed to DEHP with GW9662 (PPARγ inhibitor). In general, these results highlight the key role of PPARγ in the process of insulin resistance induced by DEHP. - Highlights: • DEHP exacerbates insulin resistance both in liver tissues and cells. • Expression of insulin receptor and GLUT4 were altered with PPARγ. • DEHP can induce oxidative stress to disrupt the metabolic homeostasis. • The dose of exposed DEHP is closed to daily exposure by human. • Determine the key role of PPARγ to insulin resistance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

An optimized method for mouse liver sinusoidal endothelial cell isolation

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic –activated cell sorting step.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions

The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p < 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p < 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p < 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.

42 ENGINEERING↗
DOE OSTI2019

Diagnostic accuracy of acoustic radiation force impulse elastography (ARFI) in comparison to other non-invasive modalities in staging of liver fibrosis in chronic HCV patients: single-center experience

Purpose: To evaluate the reliability of ARFI elastography for liver fibrosis staging and compare it to other non-invasive assessment of hepatic fibrosis (FIB-4 and APRI) in chronic HCV (CHC) patients. Methods: A single-center, prospective study included 2103 CHC patients. Liver stiffness (LS) was evaluated by TE and ARFI elastography. FIB-4 and APRI were calculated. The area under the receiver-operating characteristic curve (AUROCs) was used to assess the diagnostic performance of ARFI elastography for staging of liver fibrosis using TE as a reference standard. Results: The best cut off values of ARFI elastography for diagnosis of ≥ F2, ≥ F3and F4 were 1.36 m/s, 1.45 m/s, and 1.7 m/s with AUROCs of 0.89, 0.94 and 0.95, respectively. ARFI elastography cut offs are lower in patients with normal ALT level compared to those with ALT level (1.1–< 3 ULN) and those with ALT level ≥ 3ULN (1.35 m/s vs 1.39 m/s vs 1.54 for F ≥ 2, 1.44 m/s vs 1.58 m/s vs 1.6 m/s for F3, 1.69 m/s, 1.84 m/s, 1.86 m/s for F4). FIB-4 (0.82–0.86) and APRI (0.78–0.82) yielded lower AUC in prediction of significant fibrosis and cirrhosis than ARFI elastography (0.89–0.95). Conclusion: ARFI elastography is a reliable method for non-invasive staging of liver fibrosis in CHC patients when compared to TE with a good diagnostic performance comparable to FIB-4 and APRI scores for the prediction of significant fibrosis and cirrhosis.

62 RADIOLOGY AND NUCLEAR MEDICINE↗