Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

DEFF Research Database (Denmark)

Vestentoft, Peter S; Jelnes, Peter; Hopkinson, Branden M

2011-01-01

BACKGROUND: During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic...... in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic...

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin

2017-08-10

The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

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Mesut Sipahi

2015-01-01

Full Text Available Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations.

Molecular Structure of Human-Liver Glycogen.

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Bin Deng

Full Text Available Glycogen is a highly branched glucose polymer which is involved in maintaining blood-sugar homeostasis. Liver glycogen contains large composite α particles made up of linked β particles. Previous studies have shown that the binding which links β particles into α particles is impaired in diabetic mice. The present study reports the first molecular structural characterization of human-liver glycogen from non-diabetic patients, using transmission electron microscopy for morphology and size-exclusion chromatography for the molecular size distribution; the latter is also studied as a function of time during acid hydrolysis in vitro, which is sensitive to certain structural features, particularly glycosidic vs. proteinaceous linkages. The results are compared with those seen in mice and pigs. The molecular structural change during acid hydrolysis is similar in each case, and indicates that the linkage of β into α particles is not glycosidic. This result, and the similar morphology in each case, together imply that human liver glycogen has similar molecular structure to those of mice and pigs. This knowledge will be useful for future diabetes drug targets.

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

Science.gov (United States)

Kitamura, Shigeyuki; Sugihara, Kazumi

2014-01-01

1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

Photosynthetic fuel for heterologous enzymes

DEFF Research Database (Denmark)

Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

2017-01-01

of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells

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Lay Teng Ang

2018-02-01

Full Text Available How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs. Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah−/−Rag2−/−Il2rg−/− mouse model of liver failure.

PVA matches human liver in needle-tissue interaction.

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de Jong, Tonke L; Pluymen, Loes H; van Gerwen, Dennis J; Kleinrensink, Gert-Jan; Dankelman, Jenny; van den Dobbelsteen, John J

2017-05-01

Medical phantoms can be used to study needle-tissue interaction and to train medical residents. The purpose of this research is to study the suitability of polyvinyl alcohol (PVA) as a liver tissue mimicking material in terms of needle-tissue interaction. Insertions into ex-vivo human livers were used for reference. Six PVA samples were created by varying the mass percentage of PVA to water (4m% and 7m%) and the number of freeze-thaw cycles (1, 2 and 3 cycles, 16hours of freezing at -19°C, 8hours of thawing). The inner needle of an 18 Gauge trocar needle with triangular tip was inserted 13 times into each of the samples, using an insertion velocity of 5 mm/s. In addition, 39 insertions were performed in two ex-vivo human livers. Axial forces on the needle were captured during insertion and retraction and characterized by friction along the needle shaft, peak forces, and number of peak forces per unit length. The concentration of PVA and the number of freeze-thaw cycles both influenced the mechanical interaction between needle and specimen. Insertions into 4m% PVA phantoms with 2 freeze-thaw cycles were comparable to human liver in terms of estimated friction along the needle shaft and the number of peak forces. Therefore, these phantoms are considered to be suitable liver mimicking materials for image-guided needle interventions. The mechanical properties of PVA hydrogels can be influenced in a controlled manner by varying the concentration of PVA and the number of freeze-thaw cycles, to mimic liver tissue characteristics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice.

Science.gov (United States)

Shimizu, Makiko; Suemizu, Hiroshi; Mitsui, Marina; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

2017-10-01

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

Cell sources for in vitro human liver cell culture models

Science.gov (United States)

Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-01-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

Molecular Aging of Human Liver: An Epigenetic/Transcriptomic Signature.

Science.gov (United States)

Bacalini, Maria Giulia; Franceschi, Claudio; Gentilini, Davide; Ravaioli, Francesco; Zhou, Xiaoyuan; Remondini, Daniel; Pirazzini, Chiara; Giuliani, Cristina; Marasco, Elena; Gensous, Noémie; Di Blasio, Anna Maria; Ellis, Ewa; Gramignoli, Roberto; Castellani, Gastone; Capri, Miriam; Strom, Stephen; Nardini, Christine; Cescon, Matteo; Grazi, Gian Luca; Garagnani, Paolo

2018-03-15

The feasibility of liver transplantation from old healthy donors suggests that this organ is able to preserve its functionality during aging. To explore the biological basis of this phenomenon, we characterized the epigenetic profile of liver biopsies collected from 45 healthy liver donors ranging from 13 to 90 years old using the Infinium HumanMethylation450 BeadChip. The analysis indicates that a large remodeling in DNA methylation patterns occurs, with 8823 age-associated differentially methylated CpG probes. Notably, these age-associated changes tended to level off after the age of 60, as confirmed by Horvath's clock. Using stringent selection criteria we further identified a DNA methylation signature of aging liver including 75 genomic regions. We demonstrated that this signature is specific for liver compared to other tissues and that it is able to detect biological age-acceleration effects associated with obesity. Finally we combined DNA methylation measurements with available expression data. Although the intersection between the two omic characterizations was low, both approaches suggested a previously unappreciated role of epithelial-mesenchymal transition and Wnt signaling pathways in the aging of human liver.

The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

International Nuclear Information System (INIS)

Parkinson, Andrew; Mudra, Daniel R.; Johnson, Cory; Dwyer, Anne; Carroll, Kathleen M.

2004-01-01

We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6β-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with β-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in

Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

Science.gov (United States)

Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2016-11-01

Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

Assessment of emerging biomarkers of liver injury in human subjects.

Science.gov (United States)

Schomaker, Shelli; Warner, Roscoe; Bock, Jeff; Johnson, Kent; Potter, David; Van Winkle, Joyce; Aubrecht, Jiri

2013-04-01

Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

Cranberry juice suppressed the diclofenac metabolism by human liver microsomes, but not in healthy human subjects

Science.gov (United States)

Ushijima, Kentarou; Tsuruoka, Shu-ichi; Tsuda, Hidetoshi; Hasegawa, Gohki; Obi, Yuri; Kaneda, Tae; Takahashi, Masaki; Maekawa, Tomohiro; Sasaki, Tomohiro; Koshimizu, Taka-aki; Fujimura, Akio

2009-01-01

AIM To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations. PMID:19694738

Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis

NARCIS (Netherlands)

Westra, Inge M.; Mutsaers, Henricus A. M.; Luangmonkong, Theerut; Hadi, Mackenzie; Oosterhuis, Dorenda; de Jong, Koert P.; Groothuis, Geny M. M.; Olinga, Peter

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and

Interaction of rocuronium with human liver cytochromes P450

OpenAIRE

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-01-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells.

NARCIS (Netherlands)

M. Needham; C. Gooding; K. Hudson; M. Antoniou (Michael); F.G. Grosveld (Frank); M. Hollis

1992-01-01

textabstractWe have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

Energy Technology Data Exchange (ETDEWEB)

Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

2011-01-07

Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

International Nuclear Information System (INIS)

Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

2011-01-01

Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Analysis of iron storage proteins in chicken liver and spleen tissues in comparison with human liver ferritin by Moessbauer spectroscopy

International Nuclear Information System (INIS)

Oshtrakh, M.I.; Milder, O.B.; Semionkin, V.A.; Malakheeva, L.I.; Prokopenko, P.G.

2006-01-01

Characterization of iron storage proteins in liver and spleen from normal chicken and chicken with lymphoid leukemia in comparison with human liver ferritin were considered by Moessbauer spectroscopy (preliminary results). Small differences in Moessbauer hyperfine parameters for both normal and lymphoid leukemia chicken liver and spleen were observed. The value of quadrupole splitting for human liver ferritin was higher than those for chicken tissues. A decrease of iron content in lymphoid leukemia chicken tissues was also found, however, the reason of this fact (pathology or feeding) was not clear yet. (author)

AGE WISE HISTOMORPHOLOGICAL CHANGES IN HUMAN LIVER

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Tribeni

2015-11-01

Full Text Available CONTEXT: Hepato cellular carcinoma (HCC results in between 2.5 lakhs to 1million deaths globally per annum. Liver transplantation nowadays is a well accepted treatment option for end-stage liver disease and acute liver failure. AIMS: Keeping this concept in view, a study was conducted in the Guwahati Zone of Northeast India, to compare the histomorphological features of the human liver in different age groups. SETTING AND DESIGN: Apparently healthy livers were obtained from 21 subjects on whom medicolegal post-mortems had been performed. Their ages varied from newborn to 90 years. Subjects were divided into 3 groups. 7 specimens were taken from each group. (1 Pediatric (2 Adult (3 Old age. METHODS AND MATERIALS: In all the above age groups, immediately after removal of the livers, they were washed in normal saline, dried with blotting paper and weighed in an electronic weighing machine. Sections of liver were fixed, processed, cut and stained with Harris Haematoxylin and Eosin stain. RESULTS: The liver loses weight from 50 years onwards. There appears to be racial and environmental differences in the change in liver weight in old age. Autopsy studies show a diminution of nearly 46% in liver weight between the 3rd and 10th decades of life. The liver decreases in size with age. The hepatocytes are radially disposed in the liver lobule. They are piled up, forming a layer one cell thick (except in young children in a fashion similar to the bricks of a wall. These plates are directed from the periphery of the lobule to its centre and anastomose freely forming a complex labyrinthine and sponge-like structure. CONCLUSIONS: From the findings in the present study it can be concluded that: 1. Nowadays, the measurement of liver volume has gained practical use in relation to liver transplantation. 2. We have compared the histomorphology of adult liver with a child. The findings in both the groups are very similar. This feature is important, since in

Past Life and Future Effects—How Heterologous Infections Alter Immunity to Influenza Viruses

Directory of Open Access Journals (Sweden)

Aisha Souquette

2018-05-01

Full Text Available Influenza virus frequently mutates due to its error-prone polymerase. This feature contributes to influenza virus’s ability to evade pre-existing immunity, leading to annual epidemics and periodic pandemics. T cell memory plays a key protective role in the face of an antigenically distinct influenza virus strain because T cell targets are often derived from conserved internal proteins, whereas humoral immunity targets are often sites of increased mutation rates that are tolerated by the virus. Most studies of influenza T cell memory are conducted in naive, specific pathogen free mice and do not account for repetitive influenza infection throughout a lifetime, sequential acute heterologous infections between influenza infections, or heterologous chronic co-infections. By contrast to these mouse models, humans often experience numerous influenza infections, encounter heterologous acute infections between influenza infections, and are infected with at least one chronic virus. In this review, we discuss recent advances in understanding the effects of heterologous infections on the establishment and maintenance of CD8+ T cell immunological memory. Understanding the various factors that affect immune memory can provide insights into the development of more effective vaccines and increase reproducibility of translational studies between animal models and clinical results.

Molecular cloning and nucleotide sequence of cDNA for human liver arginase

International Nuclear Information System (INIS)

Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

1987-01-01

Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

Heterologous expression of plasmodial proteins for structural studies and functional annotation

CSIR Research Space (South Africa)

Birkholtz, LM

2008-01-01

Full Text Available Malaria Journal Open AcceReview Heterologous expression of plasmodial proteins for structural studies and functional annotation Lyn-Marie Birkholtz1, Gregory Blatch2, Theresa L Coetzer3, Heinrich C Hoppe1,4, Esmaré Human1, Elizabeth J Morris1,5, Zoleka Ngcete..., Kwadlangezwa, South Africa Email: Lyn-Marie Birkholtz - lbirkholtz@up.ac.za; Gregory Blatch - G.Blatch@ru.ac.za; Theresa L Coetzer - theresa.coetzer@nhls.ac.za; Heinrich C Hoppe - hhoppe@csir.co.za; Esmaré Human - esmare.human@up.ac.za; Elizabeth J Morris...

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

Science.gov (United States)

Capra, Valérie; Accomazzo, Maria Rosa; Gardoni, Fabrizio; Barbieri, Silvia; Rovati, G. Enrico

2010-01-01

Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage–like U937 cells, extracellular nucleotides heterologously desensitize CysLT1 receptor (CysLT1R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals. PMID:19965602

Heterologous Infection of Pregnant Mice Induces Low Birth Weight and Modifies Offspring Susceptibility to Malaria.

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Ankur Sharma

Full Text Available Pregnancy malaria (PM is associated with poor pregnancy outcomes, and can arise due to relapse, recrudescence or a re-infection with heterologous parasites. We have used the Plasmodium chabaudi model of pregnancy malaria in C57BL/6 mice to examine recrudescence and heterologous infection using CB and AS parasite strains. After an initial course of patent parasitemia and first recrudescence, CB but not AS parasites were observed to recrudesce again in most animals that became pregnant. Pregnancy exacerbated heterologous CB infection of AS-experienced mice, leading to mortality and impaired post-natal growth of pups. Parasites were detected in placental blood without evidence of sequestration, unlike P. falciparum but similar to other malaria species that infect pregnant women. Inflammatory cytokine levels were elevated in pregnant females during malaria, and associated with intensity of infection and with poor outcomes. Pups born to dams during heterologous infection were more resistant to malaria infections at 6-7 weeks of age, compared to pups born to malaria-experienced but uninfected dams or to malaria-naïve dams. In summary, our mouse model reproduces several features of human PM, including recrudescences, heterologous infections, poor pregnancy outcomes associated with inflammatory cytokines, and modulation of offspring susceptibility to malaria. This model should be further studied to explore mechanisms underlying PM pathogenesis.

Expression pattern of thymosin beta 4 in the adult human liver

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S. Nemolato

2011-09-01

Full Text Available Thymosin beta-4 (Tβ4 is a member of beta-thymosins, a family of small peptides involved in polymerization of G-actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. Previous studies in the newborn liver did not reveal any significant reactivity for Tβ4 during the intrauterine life. The aim of the present study was to investigate by immunohistochemistry Tβ4 expression in the adult normal liver. Thirty-five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti-Tβ4 commercial antibody. Tβ4 was detected in the hepatocytes of all adult normal livers examined. A zonation of Tβ4 expression was evident in the vast majority of cases. Immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1. At higher power, Tβ4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. In cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for Tβ4. The current work first evidences a strong diffuse expression of Tβ4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of Tβ4 in adulthood. Moreover, Tβ4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. Identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on Tβ4.

Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

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Matthew J Robertson

Full Text Available Liver-like organoids that recapitulate the complex functions of the whole liver by combining cells, scaffolds, and mechanical or chemical cues are becoming important models for studying liver biology and drug metabolism. The advantages of growing cells in three-dimensional constructs include enhanced cell-cell and cell-extracellular matrix interactions and preserved cellular phenotype including, prevention of de-differentiation. In the current study, biomimetic liver constructs were made via perfusion decellularization of rat liver, with the goal of maintaining the native composition and structure of the extracellular matrix. We optimized our decellularization process to produce liver scaffolds in which immunogenic residual DNA was removed but glycosaminoglycans were maintained. When the constructs were recellularized with rat or human liver cells, the cells remained viable, capable of proliferation, and functional for 28 days. Specifically, the cells continued to express cytochrome P450 genes and maintained their ability to metabolize a model drug, midazolam. Microarray analysis showed an upregulation of genes involved in liver regeneration and fibrosis. In conclusion, these liver constructs have the potential to be used as test beds for studying liver biology and drug metabolism.

Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

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Bateman, Thomas J; Reddy, Vijay G B; Kakuni, Masakazu; Morikawa, Yoshio; Kumar, Sanjeev

2014-06-01

Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

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Alderete JF

2005-03-01

Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

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2010-08-01

or the early liver-stages of the mammalian life cycle . One of these antigens is the cell-traversal protein for ookinetes and sporozoites (CelTOS...Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with... Plasmodium berghei Elke S. Bergmann-Leitner1*, Ryan M. Mease1, Patricia De La Vega1, Tatyana Savranskaya2, Mark Polhemus1, Christian Ockenhouse1, Evelina

Phosphorus-31 spectroscopic imaging of the human liver

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Biran, M.; Raffard, G.; Canioni, P.; Kien, P.

1993-01-01

During the last decade, progresses in the field of nuclear magnetic resonance spectroscopy (M.R.S.), have allowed the metabolic studies of complex biological systems. Since the coming out of whole body magnets, clinical applications are possible; they utilize magnetic field gradients coupled with selective pulse sequences. Study of the phosphorylated metabolism of human liver can be performed with sequences as ISIS, FROGS or 1D-CSI. But they present some disadvantages (for instance contamination by phosphocreatine from muscle). In the present work, we have studied the human liver in vivo by 31 P spectroscopic imaging. Several spectra could be acquired with only one acquisition. This study has needed the building of radiofrequency coils (surface coils), specially designed for liver observation (15 cm diameter 31 P coil and 19 cm diameter proton coil, both transmitter and receiver coils). Preliminary studies have been done on a phantom followed by in vivo measurements on healthy subject livers. We have obtained localized 31 P N.M.R. spectra corresponding to different voxels within the hepatic tissue. The conditions of acquisition of spectra and the problems related to the saturation of phosphorylated metabolite signals (in particular phosphodiesters) are discussed. (author). 5 figs., 15 refs

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells

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Takanori Takebe

2017-12-01

Full Text Available Summary: Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC. By conducting massive “reverse” screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108. Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. : With the goal of clinical translation of liver bud transplant therapy, Takebe et al. established a massive organoid production platform from endoderm, endothelial, and mesenchymal progenitor populations specified entirely from human iPSCs, reproducibly demonstrating functionality both in vitro and in vivo. Keywords: iPSC, liver bud, organoid, transplantation, self-organization, endothelial, mesenchymal, liver failure, clinical grade

Heterologous, Fresh, Human Donor Sclera as Patch Graft Material in Glaucoma Drainage Device Surgery.

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Tsoukanas, Dimitrios; Xanthopoulou, Paraskevi; Charonis, Alexandros C; Theodossiadis, Panagiotis; Kopsinis, Gerasimos; Filippopoulos, Theodoros

2016-07-01

To determine the safety and efficacy of fresh, human sclera allografts as a patch graft material in glaucoma drainage device (GDD) surgery. Retrospective, noncomparative, interventional, consecutive case series. All GDD cases operated between 2008 and 2013 in which fresh human corneoscleral rims were used immediately after the central corneal button was used for penetrating or endothelial keratoplasty. Surgery was performed by 2 surgeons at 2 facilities. The Ahmed Glaucoma Valve (FP-7) was used exclusively in this cohort. Sixty-four eyes of 60 patients were identified; demographic data were recorded along with intraocular pressure (IOP), medication requirements, visual acuity, complications, and subsequent interventions. Incidence of complications. IOP and medication requirements at the last follow-up. Quilified success utilizing Tube Versus Trabeculectomy study criteria. The mean age of the cohort was 66.2±19.1 years; the average preoperative IOP was 33.2±11.1 mm Hg on 4.2±1.3 IOP-lowering agents before GDD surgery. IOP decreased significantly to 14.1±4.7 mm Hg (Pendophthalmitis, and there was 1 case of conjunctival erosion and tube/plate exposure (1.6%) occurring 30 days after surgery. Qualified success was estimated as 90.5% and 81% at 1 and 2 years, respectively, using Tube Versus Trabeculectomy study criteria. Heterologous, fresh, human donor sclera appears to be a safe material for GDD tube coverage. It provides a cost-efficient alternative compared with traditional patch graft materials associated with a low risk of pathogen transmission.

Liver Effects of Clinical Drugs Differentiated in Human Liver Slices

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Alison E. M. Vickers

2017-03-01

Full Text Available Drugs with clinical adverse effects are compared in an ex vivo 3-dimensional multi-cellular human liver slice model. Functional markers of oxidative stress and mitochondrial function, glutathione GSH and ATP levels, were affected by acetaminophen (APAP, 1 mM, diclofenac (DCF, 1 mM and etomoxir (ETM, 100 μM. Drugs targeting mitochondria more than GSH were dantrolene (DTL, 10 μM and cyclosporin A (CSA, 10 μM, while GSH was affected more than ATP by methimazole (MMI, 500 μM, terbinafine (TBF, 100 μM, and carbamazepine (CBZ 100 μM. Oxidative stress genes were affected by TBF (18%, CBZ, APAP, and ETM (12%–11%, and mitochondrial genes were altered by CBZ, APAP, MMI, and ETM (8%–6%. Apoptosis genes were affected by DCF (14%, while apoptosis plus necrosis were altered by APAP and ETM (15%. Activation of oxidative stress, mitochondrial energy, heat shock, ER stress, apoptosis, necrosis, DNA damage, immune and inflammation genes ranked CSA (75%, ETM (66%, DCF, TBF, MMI (61%–60%, APAP, CBZ (57%–56%, and DTL (48%. Gene changes in fatty acid metabolism, cholestasis, immune and inflammation were affected by DTL (51%, CBZ and ETM (44%–43%, APAP and DCF (40%–38%, MMI, TBF and CSA (37%–35%. This model advances multiple dosing in a human ex vivo model, plus functional markers and gene profile markers of drug induced human liver side-effects.

Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

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Anne Frentzen

2011-04-01

Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

Dipeptidyl peptidase-4 greatly contributes to the hydrolysis of vildagliptin in human liver.

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Asakura, Mitsutoshi; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2015-04-01

The major metabolic pathway of vildagliptin in mice, rats, dogs, and humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite M20.7 (LAY151), whereas the major metabolic enzyme of vildagliptin has not been identified. In the present study, we determined the contribution rate of dipeptidyl peptidase-4 (DPP-4) to the hydrolysis of vildagliptin in the liver. We performed hydrolysis assay of the cyano group of vildagliptin using mouse, rat, and human liver samples. Additionally, DPP-4 activities in each liver sample were assessed by DPP-4 activity assay using the synthetic substrate H-glycyl-prolyl-7-amino-4-methylcoumarin (Gly-Pro-AMC). M20.7 formation rates in liver microsomes were higher than those in liver cytosol. M20.7 formation rate was significantly positively correlated with the DPP-4 activity using Gly-Pro-AMC in liver samples (r = 0.917, P vildagliptin hydrolysis in the liver. Additionally, we established stable single expression systems of human DPP-4 and its R623Q mutant, which is the nonsynonymous single-nucleotide polymorphism of human DPP-4, in human embryonic kidney 293 (HEK293) cells to investigate the effect of R623Q mutant on vildagliptin-hydrolyzing activity. M20.7 formation rate in HEK293 cells expressing human DPP-4 was significantly higher than that in control HEK293 cells. Interestingly, R623Q mutation resulted in a decrease of the vildagliptin-hydrolyzing activity. Our findings might be useful for the prediction of interindividual variability in vildagliptin pharmacokinetics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

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Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

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Heshof, Ruud; van Schayck, J Paul; Tamayo-Ramos, Juan Antonio; de Graaff, Leo H

2014-01-01

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

Human germline hedgehog pathway mutations predispose to fatty liver.

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Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

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Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

2009-07-01

Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

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Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

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Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

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Nakagawa, Shin-ichiro; Hirata, Yuichi; Kameyama, Takeshi; Tokunaga, Yuko; Nishito, Yasumasa; Hirabayashi, Kazuko; Yano, Junichi; Ochiya, Takahiro; Tateno, Chise; Tanaka, Yasuhito; Mizokami, Masashi; Tsukiyama-Kohara, Kyoko; Inoue, Kazuaki; Yoshiba, Makoto; Takaoka, Akinori; Kohara, Michinori

2013-01-01

The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

A rapid and simple method for cryopreservation of human liver slices

NARCIS (Netherlands)

de Kanter, R; Olinga, Peter; Hof, I.H; de Jager, M.H; Verwillegen, W.A; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Koster, H

1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals. Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

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Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

2015-01-01

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Chip-based human liver-intestine and liver-skin co-cultures--A first step toward systemic repeated dose substance testing in vitro.

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Maschmeyer, Ilka; Hasenberg, Tobias; Jaenicke, Annika; Lindner, Marcus; Lorenz, Alexandra Katharina; Zech, Julie; Garbe, Leif-Alexander; Sonntag, Frank; Hayden, Patrick; Ayehunie, Seyoum; Lauster, Roland; Marx, Uwe; Materne, Eva-Maria

2015-09-01

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures. Copyright © 2015. Published by Elsevier B.V.

Detection of driver metabolites in the human liver metabolic network using structural controllability analysis

Science.gov (United States)

2014-01-01

Background Abnormal states in human liver metabolism are major causes of human liver diseases ranging from hepatitis to hepatic tumor. The accumulation in relevant data makes it feasible to derive a large-scale human liver metabolic network (HLMN) and to discover important biological principles or drug-targets based on network analysis. Some studies have shown that interesting biological phenomenon and drug-targets could be discovered by applying structural controllability analysis (which is a newly prevailed concept in networks) to biological networks. The exploration on the connections between structural controllability theory and the HLMN could be used to uncover valuable information on the human liver metabolism from a fresh perspective. Results We applied structural controllability analysis to the HLMN and detected driver metabolites. The driver metabolites tend to have strong ability to influence the states of other metabolites and weak susceptibility to be influenced by the states of others. In addition, the metabolites were classified into three classes: critical, high-frequency and low-frequency driver metabolites. Among the identified 36 critical driver metabolites, 27 metabolites were found to be essential; the high-frequency driver metabolites tend to participate in different metabolic pathways, which are important in regulating the whole metabolic systems. Moreover, we explored some other possible connections between the structural controllability theory and the HLMN, and find that transport reactions and the environment play important roles in the human liver metabolism. Conclusion There are interesting connections between the structural controllability theory and the human liver metabolism: driver metabolites have essential biological functions; the crucial role of extracellular metabolites and transport reactions in controlling the HLMN highlights the importance of the environment in the health of human liver metabolism. PMID:24885538

Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

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Shin-ichiro Nakagawa

Full Text Available BACKGROUND & AIMS: The interferon (IFN system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV and hepatitis B virus (HBV. METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC. Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1, suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

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Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

2015-01-01

HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

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Irit Meivar-Levy

2011-01-01

Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

Human Precision-Cut Liver Slices as an ex Vivo Model to Study Idiosyncratic Drug-Induced Liver Injury

NARCIS (Netherlands)

Hadi, Mackenzie; Westra, Inge M.; Starokozhko, Viktoriia; Dragovic, Sanja; Merema, M.T.; Groothuis, Geny M. M.

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict

Human precision-cut liver slices as an ex vivo model to study idiosyncratic drug-induced liver injury

NARCIS (Netherlands)

Hadi, Mackenzie; Westra, Inge; Starokozhko, Viktoriia; Dragovic, Sanja; Merema, Maja; Groothuis, Genoveva

2013-01-01

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Due to the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict and to

Mice with humanized liver endothelium

NARCIS (Netherlands)

el Filali, E.

2014-01-01

The only curative treatment option for a large proportion of patients suffering from a liver disorder is liver transplantation. The use of ex vivo genetically modified autologous liver cells instead of whole liver transplantation could overcome the problem of donor scarcity. Even though clinical

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

Science.gov (United States)

Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

2018-02-01

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Proteomic profiling in incubation medium of mouse, rat and human precision-cut liver slices for biomarker detection regarding acute drug-induced liver injury

NARCIS (Netherlands)

van Swelm, Rachel P L; Hadi, Mackenzie; Laarakkers, Coby M M; Masereeuw, R.|info:eu-repo/dai/nl/155644033; Groothuis, Geny M M; Russel, Frans G M

Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker

Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections.

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Zhu, Liangquan; Feng, Yu; Zhang, Ge; Jiang, Hui; Zhang, Zhen; Wang, Nan; Ding, Jiabo; Suo, Xun

2016-01-12

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

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Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

2016-01-01

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models. PMID:27077489

Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

International Nuclear Information System (INIS)

Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi

1991-01-01

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

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Dan Xu

2014-04-01

Full Text Available Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU] developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

Science.gov (United States)

Xu, Dan; Nishimura, Toshi; Nishimura, Sachiko; Zhang, Haili; Zheng, Ming; Guo, Ying-Ying; Masek, Marylin; Michie, Sara A; Glenn, Jeffrey; Peltz, Gary

2014-04-01

Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve

MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

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Erkko Ylösmäki

Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Heterologous expression of a ketohexokinase in potato plants leads to inhibited rates of photosynthesis, severe growth retardation and abnormal leaf development

DEFF Research Database (Denmark)

Geigenberger, P.; Regierer, B.; Lytovchenko, A.

2004-01-01

of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of (14)CO(2) assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis......In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity...

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

Science.gov (United States)

Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

Science.gov (United States)

March, Sandra; Ng, Shengyong; Velmurugan, Soundarapandian; Galstian, Ani; Shan, Jing; Logan, David J; Carpenter, Anne E; Thomas, David; Sim, B Kim Lee; Mota, Maria M; Hoffman, Stephen L; Bhatia, Sangeeta N

2013-07-17

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development. Copyright © 2013 Elsevier Inc. All rights reserved.

Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure

NARCIS (Netherlands)

Nibourg, Geert A. A.; Chamuleau, Robert A. F. M.; van der Hoeven, Tessa V.; Maas, Martinus A. W.; Ruiter, An F. C.; Lamers, Wouter H.; Oude Elferink, Ronald P. J.; van Gulik, Thomas M.; Hoekstra, Ruurdtje

2012-01-01

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into

Heterologous Prime-Boost HIV-1 Vaccination Regimens in Pre-Clinical and Clinical Trials

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Julia L. Hurwitz

2010-02-01

Full Text Available Currently, there are more than 30 million people infected with HIV-1 and thousands more are infected each day. Vaccination is the single most effective mechanism for prevention of viral disease, and after more than 25 years of research, one vaccine has shown somewhat encouraging results in an advanced clinical efficacy trial. A modified intent-to-treat analysis of trial results showed that infection was approximately 30% lower in the vaccine group compared to the placebo group. The vaccine was administered using a heterologous prime-boost regimen in which both target antigens and delivery vehicles were changed during the course of inoculations. Here we examine the complexity of heterologous prime-boost immunizations. We show that the use of different delivery vehicles in prime and boost inoculations can help to avert the inhibitory effects caused by vector-specific immune responses. We also show that the introduction of new antigens into boost inoculations can be advantageous, demonstrating that the effect of ‘original antigenic sin’ is not absolute. Pre-clinical and clinical studies are reviewed, including our own work with a three-vector vaccination regimen using recombinant DNA, virus (Sendai virus or vaccinia virus and protein. Promising preliminary results suggest that the heterologous prime-boost strategy may possibly provide a foundation for the future prevention of HIV-1 infections in humans.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

Science.gov (United States)

Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

2010-01-01

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

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Marina E Fomin

Full Text Available Liver sinusoidal endothelial cells (LSECs form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII. Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene (uPA-NOG mice. Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

2017-01-01

suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis

Science.gov (United States)

2011-01-01

Background Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome. Results We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines. Conclusions This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control. PMID:22023798

Comparative Metabolism Study of Five Protoberberine Alkaloids in Liver Microsomes from Rat, Rhesus Monkey, and Human.

Science.gov (United States)

Li, Yan; Zhou, Yanyan; Si, Nan; Han, Lingyu; Ren, Wei; Xin, Shaokun; Wang, Hongjie; Zuo, Ran; Wei, Xiaolu; Yang, Jian; Zhao, Haiyu; Bian, Baolin

2017-11-01

Protoberberine alkaloids including berberine, palmatine, jatrorrhizine, coptisine, and epiberberine are major components in many medicinal plants. They have been widely used for the treatment of cancer, inflammation, diabetes, depression, hypertension, and various infectious areas. However, the metabolism of five protoberberine alkaloids among different species has not been clarified previously. In order to elaborate on the in vitro metabolism of them, a comparative analysis of their metabolic profile in rat, rhesus monkey, and human liver microsomes was carried out using ultrahigh-performance liquid chromatography coupled with a high-resolution linear trap quadrupole-Orbitrap mass spectrometer (UHPLC-electrospray ionization-Orbitrap MS) for the first time. Each metabolite was identified and semiquantified by its accurate mass data and peak area. Fifteen metabolites were characterized based on accurate MS/MS spectra and the proposed MS/MS fragmentation pathways including demethylation, hydroxylation, and methyl reduction. Among them, the content of berberine metabolites in human liver microsomes was similar with those in rhesus monkey liver microsomes, whereas berberine in rat liver microsomes showed no demethylation metabolites and the content of metabolites showed significant differences with that in human liver microsomes. On the contrary, the metabolism of palmatine in rat liver microsomes resembled that in human liver microsomes. The content of jatrorrhizine metabolites presented obvious differences in all species. The HR-ESI-MS/MS fragmentation behavior of protoberberine alkaloids and their metabolic profile in rat, rhesus monkey, and human liver microsomes were investigated for the first time. The results demonstrated that the biotransformation characteristics of protoberberine alkaloids among different species had similarities as well differences that would be beneficial for us to better understand the pharmacological activities of protoberberine alkaloids

Radionuclide imaging of the liver in human fascioliasis

International Nuclear Information System (INIS)

Rivera, J.V.; Bermudez, R.H.

1984-01-01

The clinical, laboratory, and scintigraphic findings in four cases of human fascioliasis are described. Acute onset of fever, abdominal pain, and weight loss in a person who has ingested watercress constitutes the clinical syndrome often seen. Eosinophilia and alteration in liver function tests, particularly alkaline phosphatase are frequent. Tc-99m sulfur colloid images showed hepatomegaly in four patients, focal defects in two, splenomegaly in three, and increased splenic uptake in two. Gallium citrate (Ga 67) images show increased uptake in the focal lesions in two of two. Sonographic imaging showed focal lucent abnormality in one of three. Liver biopsy findings were nonspecific. The differential diagnosis from other invasive parasitic diseases is discussed. A possible role of hepatic imaging in the evaluation of fascioliasis is suggested

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

Science.gov (United States)

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

Morphology and some biomechanical properties of human liver and spleen

Czech Academy of Sciences Publication Activity Database

Stingl, J.; Bača, V.; Čech, V.; Kovanda, J.; Kovandová, H.; Mandys, Václav; Rejmontová, J.; Sosna, B.

2002-01-01

Roč. 24, - (2002), s. 285-289 ISSN 0930-1038 Institutional research plan: CEZ:AV0Z5039906 Keywords : Human liver and spleen Subject RIV: FE - Other Internal Medicine Disciplines Impact factor: 0.252, year: 2002

Biochemical mechanisms determine the functional compatibility of heterologous genes

DEFF Research Database (Denmark)

Porse, Andreas; Schou, Thea S.; Munck, Christian

2018-01-01

-gene libraries have suggested that sequence composition is a strong barrier for the successful integration of heterologous genes. Here we sample 200 diverse genes, representing >80% of sequenced antibiotic resistance genes, to interrogate the factors governing genetic compatibility in new hosts. In contrast...... factors governing the functionality and fitness of antibiotic resistance genes. These findings emphasize the importance of biochemical mechanism for heterologous gene compatibility, and suggest physiological constraints as a pivotal feature orienting the evolution of antibiotic resistance....

A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

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Yannam, Govardhana Rao [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Han, Bing [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Setoyama, Kentaro [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamamoto, Toshiyuki [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Ito, Ryotaro; Brooks, Jenna M. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Guzman-Lepe, Jorge [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Galambos, Csaba [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Fong, Jason V. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Deutsch, Melvin; Quader, Mubina A. [Department of Radiation Oncology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamanouchi, Kosho [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York (United States); Kabarriti, Rafi; Mehta, Keyur [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Soto-Gutierrez, Alejandro [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); and others

2014-02-01

Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

International Nuclear Information System (INIS)

Yannam, Govardhana Rao; Han, Bing; Setoyama, Kentaro; Yamamoto, Toshiyuki; Ito, Ryotaro; Brooks, Jenna M.; Guzman-Lepe, Jorge; Galambos, Csaba; Fong, Jason V.; Deutsch, Melvin; Quader, Mubina A.; Yamanouchi, Kosho; Kabarriti, Rafi; Mehta, Keyur; Soto-Gutierrez, Alejandro

2014-01-01

Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury

Humanizing π-class glutathione S-transferase regulation in a mouse model alters liver toxicity in response to acetaminophen overdose.

Directory of Open Access Journals (Sweden)

Matthew P Vaughn

Full Text Available Glutathione S-transferases (GSTs metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2-/- mice escape liver injury.To more faithfully model the contribution of π-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2-/- strain, π-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2-/- mice, when hGSTP1+mGstp1/2-/- mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π-class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes.By recapitulating human π-class GST expression, hGSTP1+mGstp1/2-/- mice may better model human drug and xenobiotic toxicology.

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

Directory of Open Access Journals (Sweden)

Ruchi Sharma

2010-01-01

Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

An in silico platform for the design of heterologous pathways in nonnative metabolite production

Directory of Open Access Journals (Sweden)

Chatsurachai Sunisa

2012-05-01

Full Text Available Abstract Background Microorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. Incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. However, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. Hence, metabolic network design could benefit greatly from the availability of an in silico platform for heterologous pathway searching. Results We developed an algorithm for finding feasible heterologous pathways by which nonnative target metabolites are produced by host microorganisms, using Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as templates. Using this algorithm, we screened heterologous pathways for the production of all possible nonnative target metabolites contained within databases. We then assessed the feasibility of the target productions using flux balance analysis, by which we could identify target metabolites associated with maximum cellular growth rate. Conclusions This in silico platform, designed for targeted searching of heterologous metabolic reactions, provides essential information for cell factory improvement.

Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

Science.gov (United States)

Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

2000-10-01

The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

Science.gov (United States)

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Extended normothermic extracorporeal perfusion of isolated human liver after warm ischaemia: a preliminary report.

Science.gov (United States)

Bellomo, Rinaldo; Marino, Bruno; Starkey, Graeme; Fink, Michael; Wang, Bao Zhong; Eastwood, Glenn M; Peck, Leah; Young, Helen; Houston, Shane; Skene, Alison; Opdam, Helen; Jones, Robert

2014-09-01

Donation after circulatory death (DCD) livers are at markedly increased risk of primary graft dysfunction and biliary tract ischaemia. Normothermic extracorporeal liver perfusion (NELP) may increase the ability to transplant DCD livers and may allow their use for artificial extracorporeal liver support of patients with fulminant liver failure. We conducted two proof-of-concept experiments using human livers after DCD to assess the feasibility and functional efficacy of NELP over an extended period. We applied extracorporeal membrane oxygenation, parenteral nutrition, separate hepatic artery and portal vein perfusion and physiological perfusion pressures to two livers obtained after DCD. We achieved NELP and evidence of liver function (bile production, paracetamol removal and maintenance of normal lactate levels) in both livers; one for 24 hours and the other for 43 hours. Histological examination showed areas of patchy ischaemia but preserved biliary ducts and canaliculi. Our experiments justify further investigations of the feasibility and efficacy of extended DCD liver preservation by ex-vivo perfusion.

Pentose pathway in human liver

International Nuclear Information System (INIS)

Magnusson, I.; Chandramouli, V.; Schumann, W.C.; Kumaran, K.; Wahren, J.; Landau, B.R.

1988-01-01

[1- 14 C]Ribose and [1- 14 C]glucose were given to normal subjects along with glucose loads (1 g per kg of body weight) after administration of diflunisal and acetaminophen, drugs that are excreted in urine as glucuronides. Distributions of 14 C were determined in the carbons of the excreted glucoronides and in the glucose from blood samples drawn from hepatic veins before and after glucagon administration. Eighty percent or more of the 14 C from [1- 14 C]ribose incorporated into the glucuronic acid moiety of the glucuronides was in carbons 1 and 3, with less than 8% in carbon 2. In glucuronic acid from glucuronide excreted when [2- 14 C]glucose was given, 3.5-8.1% of the 14 C was in carbon 1, 2.5-4.3% in carbon 3, and more than 70% in carbon 2. These distributions are in accord with the glucuronides sampling the glucose unit of the glucose 6-phosphate pool that is a component of the pentose pathway and is intermediate in glycogen formation. It is concluded that the glucuronic acid conjugates of the drugs can serve as a noninvasive means of sampling hepatic glucose 6-phosphate. In human liver, as in animal liver, the classical pentose pathway functions, not the L-type pathway, and only a small percentage of the glucose is metabolized via the pathway

Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

Directory of Open Access Journals (Sweden)

Donna N Douglas

2010-02-01

Full Text Available Severe Combined Immune Deficient (SCID/Urokinase-type Plasminogen Activator (uPA mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk/ganciclovir (GCV system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK/GCV system of hepatic failure in SCID/uPA mice.In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%. Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

NARCIS (Netherlands)

Geuken, Erwin; Visser, Dorien; Kuipers, Folkert; Blokzijl, Hans; Leuvenink, Henri G. D.; de Jong, Koert P.; Peeters, Paul M. J. G.; Jansen, Peter L. M.; Slooff, Maarten J. H.; Gouw, Annette S. H.; Porte, Robert J.

2004-01-01

BACKGROUND/AIMS: Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. METHODS: In 28 liver transplant recipients, bile samples were collected daily

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

NARCIS (Netherlands)

Geuken, E; Visser, D; Kuipers, F; Blokzijl, H; Leuvenink, HGD; de Jong, KP; Peeters, PMJG; Jansen, PLM; Slooff, MJH; Gouw, ASH; Porte, RJ

2004-01-01

Background/Aims: Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. Methods: In 28 liver transplant recipients, bile samples were collected daily

In vivo time-harmonic multifrequency elastography of the human liver

International Nuclear Information System (INIS)

Tzschätzsch, Heiko; Guo, Jing; Streitberger, Kaspar-Josche; Fischer, Thomas; Sack, Ingolf; Ipek-Ugay, Selcan; Braun, Jürgen; Gentz, Enno; Klaua, Robert; Schultz, Michael

2014-01-01

Elastography is capable of noninvasively detecting hepatic fibrosis by imposing mechanical stress and measuring the viscoelastic response in the liver. Magnetic resonance elastography (MRE) relies on time-harmonic vibrations, while most dynamic ultrasound elastography methods employ transient stimulation methods. This study attempts to benefit from the advantages of time-harmonic tissue stimulation, i.e. relative insensitivity to obesity and ascites and mechanical approachability of the entire liver, and the advantages of ultrasound, i.e. time efficiency, low costs, and wide availability, by introducing in vivo time-harmonic elastography (THE) of the human liver using ultrasound and a broad range of harmonic stimulation frequencies. THE employs continuous harmonic shear vibrations at 7 frequencies from 30 to 60 Hz in a single examination and determines the elasticity and the viscosity of the liver from the dispersion of the shear wave speed within the applied frequency range. The feasibility of the method is demonstrated in the livers of eight healthy volunteers and a patient with cirrhosis. Multifrequency MRE at the same drive frequencies was used as elastographic reference method. Similar values of shear modulus and shear viscosity according the Kelvin–Voigt model were obtained by MRE and THE, indicating that the new method is suitable for in vivo quantification of the shear viscoelastic properties of the liver, however, in real-time and at a fraction of the costs of MRE. In conclusion, THE may provide a useful tool for fast assessment of the viscoelastic properties of the liver at low costs and without limitations in obesity, ascites or hemochromatosis. (paper)

Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity

NARCIS (Netherlands)

Wenker, M. A.; Kezić, S.; Monster, A. C.; de Wolff, F. A.

2001-01-01

1. The interindividual variation and enantioselectivity of the in vitro styrene oxidation by cytochrome P450 have been investigated in 20 human microsomal liver samples. Liver samples were genotyped for the CYP2E1*6 and CYP2E1*5B alleles. 2. Kinetic analysis indicated the presence of at least two

Potential genotoxic and cytotoxicity of emamectin benzoate in human normal liver cells.

Science.gov (United States)

Zhang, Zhijie; Zhao, Xinyu; Qin, Xiaosong

2017-10-10

Pesticide residue inducing cancer-related health problems draw people more attention recently. Emamectin benzoate (EMB) has been widely used in agriculture around the world based on its specificity targets. Although potential risk and the molecular mechanism of EMB toxicity to human liver has not been well-characterized. Unlike well-reported toxicity upon central nervous system, potential genotoxic and cytotoxicity of EMB in human liver cell was ignored and very limited. In this study, we identify genotoxicity and cytotoxicity of EMB to human normal liver cells (QSG7701 cell line) in vitro . We demonstrate that EMB inhibited the viability of QSG7701 cells and induced the DNA damage. Established assays of cytotoxicity were performed to characterize the mechanism of EMB toxicity on QSG7701 cells. Typical chromatin condensation and DNA fragmentation indicated the apoptosis of QSG7701 cells induced by EMB. And the intracellular biochemical results demonstrated that EMB-enhanced apoptosis of QSG7701 cells concurrent with generated ROS, a loss of mitochondrial membrane potential, the cytochrome-c release, up regulate the Bax/Bcl-2 and the activation of caspase-9/-3. Our results of EMB induces the death of QSG7701 cells maybe via mitochondrial-mediated intrinsic apoptotic pathways would contribute to promote the awareness of EMB as an extensive used pesticide to human being effects and reveal the underlying mechanisms of potential genotoxic.

Interaction of rocuronium with human liver cytochromes P450.

Science.gov (United States)

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-02-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

International Nuclear Information System (INIS)

Lake, April D.; Novak, Petr; Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D.; Lu, Zhenqiang; Lehman-McKeeman, Lois D.; Cherrington, Nathan J.

2013-01-01

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

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Lake, April D. [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States); Novak, Petr [Biology Centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice 37001 (Czech Republic); Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Lu, Zhenqiang [The Arizona Statistical Consulting Laboratory, University of Arizona, Tucson, AZ 85721 (United States); Lehman-McKeeman, Lois D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Cherrington, Nathan J., E-mail: cherrington@pharmacy.arizona.edu [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States)

2013-04-15

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

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Kazuya Shiogama

2013-01-01

Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

Production of a heterologous proteinase A by Saccharomyces kluyveri

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Møller, Kasper; Tidemand, L.D.; Winther, J.R.

2001-01-01

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a refer......In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter...

Methods of measuring metabolism during surgery in humans: focus on the liver-brain relationship.

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Battezzati, Alberto; Bertoli, Simona

2004-09-01

The purpose of this work is to review recent advances in setting methods and models for measuring metabolism during surgery in humans. Surgery, especially solid organ transplantation, may offer unique experimental models in which it is ethically acceptable to gain information on difficult problems of amino acid and protein metabolism. Two areas are reviewed: the metabolic study of the anhepatic phase during liver transplantation and brain microdialysis during cerebral surgery. The first model offers an innovative approach to understand the relative role of liver and extrahepatic organs in gluconeogenesis, and to evaluate whether other organs can perform functions believed to be exclusively or almost exclusively performed by the liver. The second model offers an insight to intracerebral metabolism that is closely bound to that of the liver. The recent advances in metabolic research during surgery provide knowledge immediately useful for perioperative patient management and for a better control of surgical stress. The studies during the anhepatic phase of liver transplantation have showed that gluconeogenesis and glutamine metabolism are very active processes outside the liver. One of the critical organs for extrahepatic glutamine metabolism is the brain. Microdialysis studies helped to prove that in humans there is an intense trafficking of glutamine, glutamate and alanine among neurons and astrocytes. This delicate network is influenced by systemic amino acid metabolism. The metabolic dialogue between the liver and the brain is beginning to be understood in this light in order to explain the metabolic events of brain damage during liver failure.

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease.

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Strom, Stephen C; Gramignoli, Roberto

2016-09-01

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations. Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient. Copyright © 2016. Published by Elsevier Inc.

Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

1987-01-01

A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

Bioartificial liver and liver transplantation: new modalities for the treatment of liver failure

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DING Yitao

2017-09-01

Full Text Available The main features of liver failure are extensive necrosis of hepatocytes, rapid disease progression, and poor prognosis, and at present, there are no effective drugs and methods for the treatment of liver failure. This article summarizes four treatment methods for liver failure, i.e., medical treatment, cell transplantation, liver transplantation, and artificial liver support therapy, and elaborates on the existing treatment methods. The current medical treatment regimen should be optimized; cell transplantation has not been used in clinical practice; liver transplantation is the most effective method, but it is limited by donor liver shortage and high costs; artificial liver can effectively remove toxic substances in human body. Therefore, this article puts forward artificial liver as a transition for liver transplantation; artificial liver can buy time for liver regeneration or liver transplantation and prolong patients′ survival time and thus has a promising future. The new treatment modality of bioartificial liver combined with liver transplantation may bring good news to patients with liver failure.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

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Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

Heterologous Protein Expression by Lactococcus lactis

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Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

2012-01-01

This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Hou, Jin; Österlund, Tobias; Liu, Zihe

2013-01-01

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration

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Adam, Aziza A. A.; van Wenum, Martien; van der Mark, Vincent A.; Jongejan, Aldo; Moerland, Perry D.; Houtkooper, Riekelt H.; Wanders, Ronald J. A.; Oude Elferink, Ronald P.; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

2017-01-01

Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we

Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

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Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

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Gorden, D Lee; Ivanova, Pavlina T; Myers, David S; McIntyre, J Oliver; VanSaun, Michael N; Wright, J Kelly; Matrisian, Lynn M; Brown, H Alex

2011-01-01

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

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Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Erratico, Claudio [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Exarchou, Vassiliki [Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Maho, Walid; Neels, Hugo [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Covaci, Adrian, E-mail: adrian.covaci@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium)

2015-04-15

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum

Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae.

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Zhu, Lin; Nemoto, Takeshi; Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2012-01-01

Proteolytic degradation is one of the serious bottlenecks limiting the yields of heterologous protein production by Aspergillus oryzae. In this study, we selected a tripeptidyl peptidase gene AosedD (AO090166000084) as a candidate potentially degrading the heterologous protein, and performed localization analysis of the fusion protein AoSedD-EGFP in A. oryzae. As a result, the AoSedD-EGFP was observed in the septa and cell walls as well as in the culture medium, suggesting that AoSedD is a secretory enzyme. An AosedD disruptant was constructed to investigate an effect of AoSedD on the production level of heterologous proteins and protease activity. Both of the total protease and tripeptidyl peptidase activities in the culture medium of the AosedD disruptant were decreased as compared to those of the control strain. The maximum yields of recombinant bovine chymosin (CHY) and human lysozyme (HLY) produced by the AosedD disruptants showed approximately 2.9- and 1.7-fold increases, respectively, as compared to their control strains. These results suggest that AoSedD is one of the major proteases involved in the proteolytic degradation of recombinant proteins in A. oryzae.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

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Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

2016-07-01

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

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Marta Moya

Full Text Available Triglyceride accumulation in nonalcoholic fatty liver (NAFL results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB. Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

Microsomal UDP-glucuronyltransferase-catalyzed bilirubin diglucuronide formation in human liver

NARCIS (Netherlands)

Peters, W. H.; Jansen, P. L.

1986-01-01

Human liver microsomal bilirubin UDP-glucuronyltransferase catalyzes formation of bilirubin mono- and diglucuronide. KmUDPGA and Vmax of the enzyme are 0.6 mM and 1.69 nmol/mg protein X min. In vitro, bilirubin readily dissolves in the microsomal lipid phase. Taking this into account a Kmbilirubin

Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

International Nuclear Information System (INIS)

Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

1988-01-01

Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

Proton MR spectroscopic features of the human liver: in-vivo application to the normal condition

International Nuclear Information System (INIS)

Cho, Soon Gu; Kim, Mi Young; Kim, Young Soo; Choi, Won; Shin, Seok Hwan; Ok, Chul Soo; Suh, Chang Hae

1999-01-01

To determine the feasibility of MR spectroscopy in the living human liver, and to evaluate the corresponding proton MR spectroscopic features. In fifteen normal volunteers with neither previous nor present liver disease, the proton MR spectroscopic findings were reviewed. Twelve subjects were male and three were female ; they were aged between 28 and 32 (mean, 30) years. MR spectroscopy involved the use of a 1.5T GE Signa Horizon system with body coil(GE Medical System, Milwaukee, U.S.A). We used STEAM (Stimulated Echo-Acquisition Mode) with 3000/30 msec of TR/TE for signal acquisition, and the prone position without respiratory interruption. Mean and standard deviation of the ratios of glutamate+glutamine/lipids, phosphomonoesters/lipids, and glycogen+glucose/lipids were calculated from the area of their peaks. The proton MR spectroscopic findings of normal human livers showed four distinctive peaks, i.e. lipids, glutamate and glutamine complex, phosphomonoesters, and glycogen and glucose complex. The mean and standard deviation of the ratios of glutamate+glutamine/lipids, phosphomonoesters/lipids, and glycogen+glucose/lipids were 0.02±0.01, 0.01±0.01, and 0.04±0.03, respectively. In living normal human livers, MR spectroscopy can be successfully applied. When applied to a liver whose condition is pathologic, the findings can be used as a standard

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

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Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

2017-01-02

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations

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Murphy, Sean C.; Kas, Arnold; Stone, Brad C.; Bevan, Michael J.

2013-01-01

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins. PMID:23530242

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

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Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2011-02-01

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

Heterologous production of human papillomavirus type-16 L1 protein by a lactic acid bacterium

Directory of Open Access Journals (Sweden)

Bermúdez-Humarán Luis G

2009-08-01

Full Text Available Abstract Background The expression of vaccine antigens in lactic acid bacteria (LAB is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i the expression of Human papillomavirus type 16 (HPV-16 L1 major capsid protein in the model LAB Lactococcus lactis and ii the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs. Results and conclusion HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

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Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

1989-02-28

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

Dynamic PET of human liver inflammation: impact of kinetic modeling with optimization-derived dual-blood input function.

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Wang, Guobao; Corwin, Michael T; Olson, Kristin A; Badawi, Ramsey D; Sarkar, Souvik

2018-05-30

The hallmark of nonalcoholic steatohepatitis is hepatocellular inflammation and injury in the setting of hepatic steatosis. Recent work has indicated that dynamic 18F-FDG PET with kinetic modeling has the potential to assess hepatic inflammation noninvasively, while static FDG-PET did not show a promise. Because the liver has dual blood supplies, kinetic modeling of dynamic liver PET data is challenging in human studies. The objective of this study is to evaluate and identify a dual-input kinetic modeling approach for dynamic FDG-PET of human liver inflammation. Fourteen human patients with nonalcoholic fatty liver disease were included in the study. Each patient underwent one-hour dynamic FDG-PET/CT scan and had liver biopsy within six weeks. Three models were tested for kinetic analysis: traditional two-tissue compartmental model with an image-derived single-blood input function (SBIF), model with population-based dual-blood input function (DBIF), and modified model with optimization-derived DBIF through a joint estimation framework. The three models were compared using Akaike information criterion (AIC), F test and histopathologic inflammation reference. The results showed that the optimization-derived DBIF model improved the fitting of liver time activity curves and achieved lower AIC values and higher F values than the SBIF and population-based DBIF models in all patients. The optimization-derived model significantly increased FDG K1 estimates by 101% and 27% as compared with traditional SBIF and population-based DBIF. K1 by the optimization-derived model was significantly associated with histopathologic grades of liver inflammation while the other two models did not provide a statistical significance. In conclusion, modeling of DBIF is critical for kinetic analysis of dynamic liver FDG-PET data in human studies. The optimization-derived DBIF model is more appropriate than SBIF and population-based DBIF for dynamic FDG-PET of liver inflammation. © 2018

Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

International Nuclear Information System (INIS)

Lenich, A.C.

1985-01-01

Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5- 14 C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively

[Detection of human parvovirus B19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in Nanjing by nested PCR].

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Tong, Rui; Zhou, Wei-Min; Liu, Xi-Jun; Wang, Yue; Lou, Yong-Liang; Tan, Wen-Jie

2013-04-01

To analyze the infection of human parvovirus B19, human bocavirus (HBoV) and human parvovirus 4 (PARV4) in blood samples among patients with liver disease in Nanjing by molecular detection. Nested PCR assays were designed and validated to detect B19, HBoV and PARV4, respectively. The assays were used to screen three parvoviruses in blood samples from 95 patients with different liver disease in Nanjing. The parvovirus infection was analyzed statistically. The detection limits were 10 copies of genomic DNA equivalents per reaction for each assays and the good specificity were observed. The frequency of B19 and HBoV were 2/95 (2.1%) and 9/95 (9.5%) in blood samples respectively. No PARV4 was detected. HBoV was detected in 3/5 patients with drug-induced hepatitis. Both B19 and HBoV infection were detected in blood from patients with liver disease.

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

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Nakada, Naoyuki; Oda, Kazuo

2015-01-01

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

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Lee-Montiel, Felipe T; George, Subin M; Gough, Albert H; Sharma, Anup D; Wu, Juanfang; DeBiasio, Richard; Vernetti, Lawrence A; Taylor, D Lansing

2017-10-01

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we

Human Adipose Tissue Derived Stem Cells Promote Liver Regeneration in a Rat Model of Toxic Injury

Directory of Open Access Journals (Sweden)

Eva Koellensperger

2013-01-01

Full Text Available In the light of the persisting lack of donor organs and the risks of allotransplantations, the possibility of liver regeneration with autologous stem cells from adipose tissue (ADSC is an intriguing alternative. Using a model of a toxic liver damage in Sprague Dawley rats, generated by repetitive intraperitoneal application of retrorsine and allyl alcohol, the ability of human ADSC to support the restoration of liver function was investigated. A two-thirds hepatectomy was performed, and human ADSC were injected into one remaining liver lobe in group 1 (n = 20. Injection of cell culture medium performed in group 2 (n = 20 served as control. Cyclosporine was applied to achieve immunotolerance. Blood samples were drawn weekly after surgery to determine liver-correlated blood values. Six and twelve weeks after surgery, animals were sacrificed and histological sections were analyzed. ADSC significantly raised postoperative albumin (P < 0.017, total protein (P < 0.031, glutamic oxaloacetic transaminase (P < 0.001, and lactate dehydrogenase (P < 0.04 levels compared to injection of cell culture medium alone. Transplanted cells could be found up to twelve weeks after surgery in histological sections. This study points towards ADSC being a promising alternative to hepatocyte or liver organ transplantation in patients with severe liver failure.

Alloxan-Induced Diabetes Causes Morphological and Ultrastructural Changes in Rat Liver that Resemble the Natural History of Chronic Fatty Liver Disease in Humans

Directory of Open Access Journals (Sweden)

Amanda Natália Lucchesi

2015-01-01

Full Text Available Purpose. This study evaluated the long-term effects of alloxan-induced diabetes in rat liver. Methods. Thirty nondiabetic control rats (NC and 30 untreated diabetic (UD rats were divided into three subgroups sacrificed after 6, 14, or 26 weeks. Clinical and laboratory parameters were assessed. Fresh liver weight and its relationship with body weight were obtained, and liver tissue was analyzed. Results. UD rats showed sustained hyperglycemia, high glycosylated hemoglobin, and low plasma insulin. High serum levels of AST and ALT were observed in UD rats after 2 weeks, but only ALT remained elevated throughout the experiment. Fresh liver weight was equal between NC and UD rats, but the fresh liver weight/body weight ratio was significantly higher in UD rats after 14 and 26 weeks. UD rats showed liver morphological changes characterized by hepatic sinusoidal enlargement and micro- and macrovesicular hepatocyte fatty degeneration with progressive liver structure loss, steatohepatitis, and periportal fibrosis. Ultrastructural changes of hepatocytes, such as a decrease in the number of intracytoplasmic organelles and degeneration of mitochondria, rough endoplasmic reticulum, and nuclei, were also observed. Conclusion. Alloxan-induced diabetes triggered liver morphological and ultrastructural changes that closely resembled human disease, ranging from steatosis to steatohepatitis and liver fibrosis.

A computer-simulated liver phantom (virtual liver phantom) for multidetector computed tomography evaluation

Energy Technology Data Exchange (ETDEWEB)

Funama, Yoshinori [Kumamoto University, Department of Radiological Sciences, School of Health Sciences, Kumamoto (Japan); Awai, Kazuo; Nakayama, Yoshiharu; Liu, Da; Yamashita, Yasuyuki [Kumamoto University, Department of Diagnostic Radiology, Graduate School of Medical Sciences, Kumamoto (Japan); Miyazaki, Osamu; Goto, Taiga [Hitachi Medical Corporation, Tokyo (Japan); Hori, Shinichi [Gate Tower Institute of Image Guided Therapy, Osaka (Japan)

2006-04-15

The purpose of study was to develop a computer-simulated liver phantom for hepatic CT studies. A computer-simulated liver phantom was mathematically constructed on a computer workstation. The computer-simulated phantom was calibrated using real CT images acquired by an actual four-detector CT. We added an inhomogeneous texture to the simulated liver by referring to CT images of chronically damaged human livers. The mean CT number of the simulated liver was 60 HU and we added numerous 5-to 10-mm structures with 60{+-}10 HU/mm. To mimic liver tumors we added nodules measuring 8, 10, and 12 mm in diameter with CT numbers of 60{+-}10, 60{+-}15, and 60{+-}20 HU. Five radiologists visually evaluated similarity of the texture of the computer-simulated liver phantom and a real human liver to confirm the appropriateness of the virtual liver images using a five-point scale. The total score was 44 in two radiologists, and 42, 41, and 39 in one radiologist each. They evaluated that the textures of virtual liver were comparable to those of human liver. Our computer-simulated liver phantom is a promising tool for the evaluation of the image quality and diagnostic performance of hepatic CT imaging. (orig.)

Determination of cobalt in human liver by atomic absorption spectrometry with electrothermal atomization

International Nuclear Information System (INIS)

Caldas, E.D.; Gine-Rosias, M.F.; Dorea, J.G.

1991-01-01

A detailed study of the use of electrothermal atomic absorption spectrometry for the determination of cobalt in human liver is described. Comparisons of sample digestion using nitric acid or nitric acid plus perchloric acid, atomization procedures and the application of palladium and magnesium nitrate chemical modifiers were studied using NBS SRM 1577a Bovine Liver. The best results were achieved with sample decomposition in nitric acid, atomization from the tube wall and no chemical modifier. Cobalt was determined in 90 samples of livers from foetuses and deceased newborns using the standard addition method with an average recovery of 99.8%. (author). 30 refs.; 4 figs.; 2 tabs

Liver-related deaths in persons infected with the human immunodeficiency virus: the D:A:D study

NARCIS (Netherlands)

Weber, Rainer; Sabin, Caroline A.; Friis-Møller, Nina; Reiss, Peter; El-Sadr, Wafaa M.; Kirk, Ole; Dabis, Francois; Law, Matthew G.; Pradier, Christian; de Wit, Stephane; Akerlund, Börje; Calvo, Gonzalo; D'Arminio Monforte, Antonella; Rickenbach, Martin; Ledergerber, Bruno; Phillips, Andrew N.; Lundgren, Jens D.

2006-01-01

BACKGROUND: An increasing proportion of deaths among human immunodeficiency virus (HIV)-infected persons with access to combination antiretroviral therapy (cART) are due to complications of liver diseases. METHODS: We investigated the frequency of and risk factors associated with liver-related

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b.

Science.gov (United States)

Gasmi, Najla; Ayed, Atef; Nicaud, Jean-Marc; Kallel, Héla

2011-05-20

The non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host. Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity. Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃ and MnSO₄ had the most inhibitory effect. We have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no

Sinusoidal obstruction syndrome (SOS): A light and electron microscopy study in human liver.

Science.gov (United States)

Vreuls, C P H; Driessen, A; Olde Damink, S W M; Koek, G H; Duimel, H; van den Broek, M A J; Dejong, C H C; Braet, F; Wisse, E

2016-05-01

Oxaliplatin is an important chemotherapeutic agent, used in the treatment of hepatic colorectal metastases, and known to induce the sinusoidal obstruction syndrome (SOS). Pathophysiological knowledge concerning SOS is based on a rat model. Therefore, the aim was to perform a comprehensive study of the features of human SOS, using both light microscopy (LM) and electron microscopy (EM). Included were all patients of whom wedge liver biopsies were collected during a partial hepatectomy for colorectal liver metastases, in a 4-year period. The wedge biopsy were perfusion fixated and processed for LM and EM. The SOS lesions were selected by LM and details were studied using EM. Material was available of 30 patients, of whom 28 patients received neo-adjuvant oxaliplatin. Eighteen (64%) of the 28 patients showed SOS lesions, based on microscopy. The lesions consisted of sinusoidal endothelial cell detachment from the space of Disse on EM. In the enlarged space of Disse a variable amount of erythrocytes were located. Sinusoidal endothelial cell detachment was present in human SOS, accompanied by enlargement of the space of Disse and erythrocytes in this area. These findings, originally described in a rat model, were now for the first time confirmed in human livers under clinically relevant settings. Copyright © 2016 Elsevier Ltd. All rights reserved.

CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

NARCIS (Netherlands)

LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

Science.gov (United States)

Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

2005-05-01

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion.

Science.gov (United States)

Laing, Richard W; Bhogal, Ricky H; Wallace, Lorraine; Boteon, Yuri; Neil, Desley A H; Smith, Amanda; Stephenson, Barney T F; Schlegel, Andrea; Hübscher, Stefan G; Mirza, Darius F; Afford, Simon C; Mergental, Hynek

2017-11-01

Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

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Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

2016-08-11

Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

Science.gov (United States)

Gates, Leah A; Lu, Ding; Peterson, Lisa A

2012-03-01

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

Preoperative assessment of congestive liver dysfunction using technetium-99m galactosyl human Serum albumin liver scintigraphy in patients with severe valvular heart disease

International Nuclear Information System (INIS)

Nishi, Hiroyuki; Matsumiya, Goro; Takano, Hiroshi; Ichikawa, Hajime; Miyagawa, Shigeru; Sawa, Yoshiki; Takahashi, Toshiki

2007-01-01

Severe valvular heart disease is often complicated by congestive liver dysfunction, which greatly compromises the operative results. We evaluated congestive liver dysfunction by a novel approach using technetium-99m galactosyl human serum albumin ( 99m Tc-GSA) with liver scintigraphy. Between 1998 and 2004, we performed scintigraphy accompanied by 99m Tc-GSA in 28 patients who had valvular heart disease with moderate-to-severe tricuspid regurgitation and who showed symptoms of right heart failure. Based on the results, we calculated a receptor index (LHL15) and an index of blood clearance (HH15) and assessed the correlation between these factors and postoperative liver dysfunction, defined as the maximum serum total bilirubin level (max T-bil) as >2.0 mg/dl. Nineteen patients, including four who died in hospital, had postoperative liver dysfunction. The level of HH15 was significantly higher and the level of cholinesterase was significantly lower (P 99m Tc-GSA is a clinically useful predictor of postoperative liver dysfunction in patients with severe valvular disease. (author)

PEDIATRIC LIVER TRANSPLANTATION WITH EX-SITU LIVER TRANSECTION AND THE APPLICATION OF THE HUMAN FIBRINOGEN AND THROMBIN SPONGE IN THE WOUND AREA.

Science.gov (United States)

Vicentine, Fernando Pompeu Piza; Gonzalez, Adriano Miziara; Azevedo, Ramiro Anthero de; Benini, Barbara Burza; Linhares, Marcelo Moura; Lopes-Filho, Gaspar de Jesus; Martins, Jose Luiz; Salzedas-Netto, Alcides Augusto

2016-01-01

Surgical strategy to increase the number of liver transplants in the pediatric population is the ex-situ liver transection (reduction or split). However, it is associated with complications such as hemorrhage and leaks. The human fibrinogen and thrombin sponge is useful for improving hemostasis in liver surgery. Compare pediatric liver transplants with ex-situ liver transection (reduction or split) with or without the human fibrinogen and thrombin sponge. Was performed a prospective analysis of 21 patients submitted to liver transplantation with ex-situ liver transection with the application of the human fibrinogen and thrombin sponge in the wound area (group A) and retrospective analysis of 59 patients without the sponge (group B). The characteristics of recipients and donors were similar. There were fewer reoperations due to bleeding in the wound area in group A (14.2%) compared to group B (41.7%, p=0.029). There was no difference in relation to the biliary leak (group A: 17.6%, group B: 5.1%, p=0.14). There was a lower number of reoperations due to bleeding of the wound area of ​​the hepatic graft when the human fibrinogen and thrombin sponge were used. Estratégia cirúrgica para aumentar o número de transplantes hepáticos na população pediátrica é a transecção hepática ex-situ (redução ou split). No entanto, ela está associada com complicações, tais como hemorragia e fístulas. A esponja de fibrinogênio e trombina humana é útil para melhorar a hemostasia nas operações hepáticas. Comparar transplantes hepáticos pediátricos com transecção hepática ex-situ (redução ou split) com ou sem a esponja de fibrinogênio e trombina humana. Foi realizada análise prospectiva de 21 pacientes submetidos ao transplante de fígado com transecção hepática ex-situ com a aplicação da esponja de fibrinogênio e trombina humana na área cruenta (grupo A) e análise retrospectiva de 59 pacientes sem a esponja (grupo B). As características dos

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABAA Receptor

International Nuclear Information System (INIS)

Chen, Zhi-ao; Bao, Mei-yan; Xu, Yong-fen; Zha, Ruo-peng; Shi, Hai-bing; Chen, Tao-yang; He, Xiang-huo

2012-01-01

To investigate the roles of the γ-aminobutyric acid (GABA) in the metastasis of hepatocellular carcinoma (HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. The expression levels of GABA receptor subunit genes in various HCC cell lines and patients‘ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis. Transwell cell migration and invasion assays were carried out for functional analysis. The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue. GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABA A receptor as a result of the induction of liver cancer cell cytoskeletal reorganization. Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

NARCIS (Netherlands)

Mazza, G. (Giuseppe); Al-Akkad, W. (Walid); Telese, A. (Andrea); Longato, L. (Lisa); Urbani, L. (Luca); Robinson, B. (Benjamin); Hall, A. (Andrew); Kong, K. (Kenny); Frenguelli, L. (Luca); Marrone, G. (Giusi); Willacy, O. (Oliver); Shaeri, M. (Mohsen); A.J. Burns (Alan); Malago, M. (Massimo); Gilbertson, J. (Janet); Rendell, N. (Nigel); Moore, K. (Kevin); Hughes, D. (David); Notingher, I. (Ioan); Jell, G. (Gavin); Del Rio Hernandez, A. (Armando); P. de Coppi (Paolo); Rombouts, K. (Krista); Pinzani, M. (Massimo)

2017-01-01

textabstractThe development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of

METHOD FOR THE PRODUCTION OF HETEROLOGOUS POLYPEPTIDES IN TRANSFORMED YEAST CELLS

DEFF Research Database (Denmark)

2000-01-01

The invention describes industrial fermentation of a $i(Saccharomyces) yeast species for production of a heterologous product encoded by a plasmid or DNA contained in said $i(Saccharomyces) yeast species with method utilizes the substrate more efficiently and without fermentative metabolism...... resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained. The $i(Saccharomyces) yeast species is preferably a Crabtree negative $i(Saccharomyces species) in particular $i(Saccharomyces kluyveri)....

Spatial organization of heterologous metabolic system in vivo based on TALE.

Science.gov (United States)

Zhu, Lv-yun; Qiu, Xin-Yuan; Zhu, Ling-Yun; Wu, Xiao-Min; Zhang, Yuan; Zhu, Qian-Hui; Fan, Dong-Yu; Zhu, Chu-Shu; Zhang, Dong-Yi

2016-05-17

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

An Experimental Study to Measure the Mechanical Properties of the Human Liver.

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Karimi, Alireza; Shojaei, Ahmad

2018-01-01

Since the liver is one of the most important organs of the body that can be injured during trauma, that is, during accidents like car crashes, understanding its mechanical properties is of great interest. Experimental data is needed to address the mechanical properties of the liver to be used for a variety of applications, such as the numerical simulations for medical purposes, including the virtual reality simulators, trauma research, diagnosis objectives, as well as injury biomechanics. However, the data on the mechanical properties of the liver capsule is limited to the animal models or confined to the tensile/compressive loading under single direction. Therefore, this study was aimed at experimentally measuring the axial and transversal mechanical properties of the human liver capsule under both the tensile and compressive loadings. To do that, 20 human cadavers were autopsied and their liver capsules were excised and histologically analyzed to extract the mean angle of a large fibers population (bundle of the fine collagen fibers). Thereafter, the samples were cut and subjected to a series of axial and transversal tensile/compressive loadings. The results revealed the tensile elastic modulus of 12.16 ± 1.20 (mean ± SD) and 7.17 ± 0.85 kPa under the axial and transversal loadings respectively. Correspondingly, the compressive elastic modulus of 196.54 ± 13.15 and 112.41 ± 8.98 kPa were observed under the axial and transversal loadings respectively. The compressive axial and transversal maximum/failure stress of the capsule were 32.54 and 37.30 times higher than that of the tensile ones respectively. The capsule showed a stiffer behavior under the compressive load compared to the tensile one. In addition, the axial elastic modulus of the capsule was found to be higher than that of the transversal one. The findings of the current study have implications not only for understanding the mechanical properties of the human capsule tissue under tensile

Volumetric Growth of the Liver in the Human Fetus: An Anatomical, Hydrostatic, and Statistical Study

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Michał Szpinda

2015-01-01

Full Text Available Using anatomical, hydrostatic, and statistical methods, liver volumes were assessed in 69 human fetuses of both sexes aged 18–30 weeks. No sex differences were found. The median of liver volume achieved by hydrostatic measurements increased from 6.57 cm3 at 18–21 weeks through 14.36 cm3 at 22–25 weeks to 20.77 cm3 at 26–30 weeks, according to the following regression: y = −26.95 + 1.74 × age ± Z  × (−3.15 + 0.27 × age. The median of liver volume calculated indirectly according to the formula liver volume = 0.55 × liver length × liver transverse diameter × liver sagittal diameter increased from 12.41 cm3 at 18–21 weeks through 28.21 cm3 at 22–25 weeks to 49.69 cm3 at 26–30 weeks. There was a strong relationship (r=0.91, p<0.001 between the liver volumes achieved by hydrostatic (x and indirect (y methods, expressed by y = −0.05 + 2.16x  ± 7.26. The liver volume should be calculated as follows liver volume = 0.26 × liver length × liver transverse diameter × liver sagittal diameter. The age-specific liver volumes are of great relevance in the evaluation of the normal hepatic growth and the early diagnosis of fetal micro- and macrosomias.

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression.

Science.gov (United States)

Kim, Geon A; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Oh, Hyun Ju; Hwang, Joing-Ik; Ahn, Curie; Saadeldin, Islam M; Lee, Byeong Chun

2017-01-01

Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets ( P hHO-1 piglets ( P hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

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Rigo, Federica; De Stefano, Nicola; Navarro-Tableros, Victor; David, Ezio; Rizza, Giorgia; Catalano, Giorgia; Gilbo, Nicholas; Maione, Francesca; Gonella, Federica; Roggio, Dorotea; Martini, Silvia; Patrono, Damiano; Salizzoni, Mauro; Camussi, Giovanni; Romagnoli, Renato

2018-05-01

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029.

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Bian, Xiaoying; Tang, Biao; Yu, Yucong; Tu, Qiang; Gross, Frank; Wang, Hailong; Li, Aiying; Fu, Jun; Shen, Yuemao; Li, Yue-Zhong; Stewart, A Francis; Zhao, Guoping; Ding, Xiaoming; Müller, Rolf; Zhang, Youming

2017-07-21

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L -1 . These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

Science.gov (United States)

Ellis, Ewa C S; Naugler, Willscott Edward; Nauglers, Scott; Parini, Paolo; Mörk, Lisa-Mari; Jorns, Carl; Zemack, Helen; Sandblom, Anita Lövgren; Björkhem, Ingemar; Ericzon, Bo-Göran; Wilson, Elizabeth M; Strom, Stephen C; Grompe, Markus

2013-01-01

Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

Science.gov (United States)

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

The L polymerase protein of parainfluenza virus 3 forms an oligomer and can interact with the heterologous Sendai virus L, P and C proteins

International Nuclear Information System (INIS)

Smallwood, Sherin; Moyer, Sue A.

2004-01-01

We recently showed that the L protein of Sendai virus is present as an oligomer in the active P-L polymerase complex [Smallwood et al., Virology 304 (2002) 235]. We now demonstrate using two different epitope tags that the L protein of a second respirovirus, human parainfluenza type 3 virus (PIV3), also forms an L-L complex. L oligomerization requires the coexpression of the differentially epitope tagged L proteins. By exploiting a series of C-terminal truncations the L-L binding site maps to the N-terminal half of L. There is some complex formation between the heterologous PIV3 and Sendai L and P proteins; however, the heterologous L protein does not function in transcription of either the PIV3 or Sendai template. The PIV3 C protein binds PIV3 L and inhibits RNA synthesis in vitro and in vivo. Significant homology exists between the C proteins of PIV3 and Sendai and complex formation occurs between the PIV3 and Sendai heterologous C and L proteins. In addition, the heterologous C proteins can inhibit transcription at ∼50% of the level of the homologous protein. These data suggest that while the C proteins may be functionally somewhat interchangeable, the L and P proteins are specific for each virus

Volumetric Growth of the Liver in the Human Fetus: An Anatomical, Hydrostatic, and Statistical Study

Science.gov (United States)

Szpinda, Michał; Paruszewska-Achtel, Monika; Mila-Kierzenkowska, Celestyna; Elminowska-Wenda, Gabriela; Dombek, Małgorzata; Szpinda, Anna; Badura, Mateusz

2015-01-01

Using anatomical, hydrostatic, and statistical methods, liver volumes were assessed in 69 human fetuses of both sexes aged 18–30 weeks. No sex differences were found. The median of liver volume achieved by hydrostatic measurements increased from 6.57 cm3 at 18–21 weeks through 14.36 cm3 at 22–25 weeks to 20.77 cm3 at 26–30 weeks, according to the following regression: y = −26.95 + 1.74 × age ± Z  × (−3.15 + 0.27 × age). The median of liver volume calculated indirectly according to the formula liver volume = 0.55 × liver length × liver transverse diameter × liver sagittal diameter increased from 12.41 cm3 at 18–21 weeks through 28.21 cm3 at 22–25 weeks to 49.69 cm3 at 26–30 weeks. There was a strong relationship (r = 0.91, p hydrostatic (x) and indirect (y) methods, expressed by y = −0.05 + 2.16x  ± 7.26. The liver volume should be calculated as follows liver volume = 0.26 × liver length × liver transverse diameter × liver sagittal diameter. The age-specific liver volumes are of great relevance in the evaluation of the normal hepatic growth and the early diagnosis of fetal micro- and macrosomias. PMID:26413551

Adaption of Saccharomyces cerevisiae expressing a heterologous protein

DEFF Research Database (Denmark)

Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

2008-01-01

Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos

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Rachel E. Jennings

2017-11-01

Full Text Available To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

Science.gov (United States)

Lhuaire, Martin; Tonnelet, Romain; Renard, Yohann; Piardi, Tullio; Sommacale, Daniele; Duparc, Fabrice; Braun, Marc; Labrousse, Marc

2015-07-01

Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23. Copyright © 2015 Elsevier GmbH. All rights reserved.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

Science.gov (United States)

Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

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Melanie Werner

Full Text Available Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7 hepatocytes, 1.8 ± 0.5 × 10(6 Kupffer cells, 4.3 ± 1.9 × 10(5 liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5 stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7% and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2% and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+ (97.8 ± 1.1% and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%. These cells further exhibited retinol (vitamin A-mediated autofluorescence.Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

DEFF Research Database (Denmark)

Starokozhko, Viktoriia; Hemmingsen, Mette; Larsen, Layla

2018-01-01

Hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions...... and to perform functional comparisons with fresh human precision-cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis...... in the hiPSC-derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted...

In vitro Inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea, and Curcuma xanthorrhiza Extracts and Constituents on Human Liver Glucuronidation Activity.

Science.gov (United States)

Husni, Zulhilmi; Ismail, Sabariah; Zulkiffli, Mohd Halimhilmi; Afandi, Atiqah; Haron, Munirah

2017-07-01

Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza are commonly consumed as herbal medicines. However their effects on human liver glucuronidation activity are not yet evaluated. In this study, we evaluate the inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza extracts and their constituents on human liver glucuronidation activity. Herbal extracts (aqueous, methanolic and ethanolic extracts) and their constituents were incubated with human liver microsomes with the addition of UDPGA to initiate the reaction. Working concentrations of herbal extracts and their constituents ranged from 10 μg/mL to 1000 μg/mL and 10 μM to 300 μM respectively. IC50 was determined by monitoring the decrement of glucuronidation activity with the increment of herbal extracts or phytochemical constituent's concentrations. All herbal extracts inhibited human liver glucuronidation activity in range of 34.69 μg/mL to 398.10 μg/mL whereas for the constituents, only xanthorrhizol and curcumin (constituents of Curcuma xanthorrhiza ) inhibited human liver glucuronidation activity with IC50 of 538.50 and 32.26 μM respectively. In the present study, we have proved the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to interfere with in vitro glucuronidation process in human liver microsomes. This study documented the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to inhibit human liver glucuronidation activity which may affect the metabolism of therapeutic drugs or hazardous toxicants that follow the same glucuronidation pathway. Abbreviations used: UGT: Uridine 5'-diphospho-glucuronosyltransferase; 4-MU: 4-methylumbelliferone; IC50: Half Maximal Inhibitory Concentration; Km: Michaelis constant; Vmax: Maximum velocity.

Radiation-induced liver damage

International Nuclear Information System (INIS)

Marcial, V.A.; Santiago-Delpin, E.A.; Lanaro, A.E.; Castro-Vita, H.; Arroyo, G.; Moscol, J.A.; Gomez, C.; Velazquez, J.; Prado, K.

1977-01-01

Due to the recent increase in the use of radiation therapy in the treatment of cancer with or without chemotherapy, the risk of liver radiation damage has become a significant concern for the radiotherapist when the treated tumour is located in the upper abdomen or lower thorax. Clinically evident radiation liver damage may result in significant mortality, but at times patients recover without sequelae. The dose of 3000 rads in 3 weeks to the entire liver with 5 fractions per week of 200 rads each, seems to be tolerated well clinically by adult humans. Lower doses may lead to damage when used in children, when chemotherapy is added, as in recent hepatectomy cases, and in the presence of pre-existent liver damage. Reduced fractionation may lead to increased damage. Increased fractionation, limitation of the dose delivered to the entire liver, and restriction of the high dose irradiation volume may afford protection. With the aim of studying the problems of hepatic radiation injury in humans, a project of liver irradiation in the dog is being conducted. Mongrel dogs are being conditioned, submitted to pre-irradiation studies (haemogram, blood chemistry, liver scan and biopsy), irradiated under conditions resembling human cancer therapy, and submitted to post-irradiation evaluation of the liver. Twenty-two dogs have been entered in the study but only four qualify for the evaluation of all the study parameters. It has been found that dogs are susceptible to liver irradiation damage similar to humans. The initial mortality has been high mainly due to non-radiation factors which are being kept under control at the present phase of the study. After the initial experiences, the study will involve variations in total dose and fractionation, and the addition of anticoagulant therapy for possible prevention of radiation liver injury. (author)

Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

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Leavell Sarah

2010-05-01

Full Text Available Abstract Background Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control and then cats were vaginally challenged with cell-associated or cell-free FIV. Results Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls. Conclusions The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus can be modulated by mucosal exposure to uninfected heterologous cells.

Effect of ultrasound frequency on the Nakagami statistics of human liver tissues.

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Po-Hsiang Tsui

Full Text Available The analysis of the backscattered statistics using the Nakagami parameter is an emerging ultrasound technique for assessing hepatic steatosis and fibrosis. Previous studies indicated that the echo amplitude distribution of a normal liver follows the Rayleigh distribution (the Nakagami parameter m is close to 1. However, using different frequencies may change the backscattered statistics of normal livers. This study explored the frequency dependence of the backscattered statistics in human livers and then discussed the sources of ultrasound scattering in the liver. A total of 30 healthy participants were enrolled to undergo a standard care ultrasound examination on the liver, which is a natural model containing diffuse and coherent scatterers. The liver of each volunteer was scanned from the right intercostal view to obtain image raw data at different central frequencies ranging from 2 to 3.5 MHz. Phantoms with diffuse scatterers only were also made to perform ultrasound scanning using the same protocol for comparisons with clinical data. The Nakagami parameter-frequency correlation was evaluated using Pearson correlation analysis. The median and interquartile range of the Nakagami parameter obtained from livers was 1.00 (0.98-1.05 for 2 MHz, 0.93 (0.89-0.98 for 2.3 MHz, 0.87 (0.84-0.92 for 2.5 MHz, 0.82 (0.77-0.88 for 3.3 MHz, and 0.81 (0.76-0.88 for 3.5 MHz. The Nakagami parameter decreased with the increasing central frequency (r = -0.67, p < 0.0001. However, the effect of ultrasound frequency on the statistical distribution of the backscattered envelopes was not found in the phantom results (r = -0.147, p = 0.0727. The current results demonstrated that the backscattered statistics of normal livers is frequency-dependent. Moreover, the coherent scatterers may be the primary factor to dominate the frequency dependence of the backscattered statistics in a liver.

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

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Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

International Nuclear Information System (INIS)

Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

2012-01-01

Highlights: ► Cigarette smoke may induce liver fibrosis via nicotine receptors. ► Nicotine induces proliferation of hepatic stellate cells (HSCs). ► Nicotine activates hepatic fibrogenic pathways. ► Nicotine receptor antagonists attenuate HSC proliferation. ► Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine – which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed – RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type (α1, β1, delta and epsilon) and neuronal type (α3, α6, α7, β2 and β4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, β1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-α2 and TGF-β1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers’ blood is pro-fibrogenic, through

Human intrahepatic ILC2 are IL-13positive amphiregulinpositive and their frequency correlates with model of end stage liver disease score.

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Hannah C Jeffery

Full Text Available Innate lymphoid cells (ILC have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue.Human intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function.All three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+ and NCRneg ILC3 (CRTH2negCD117pos subsets were also detected. ILC2 (CRTH2pos frequency correlated with disease severity measured by model of end stage liver disease (MELD scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin.Intrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease.

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

International Nuclear Information System (INIS)

Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C.; Grzeschik, K.H.

1988-01-01

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration

The sinusoidal lining cells in "normal" human liver. A scanning electron microscopic investigation

DEFF Research Database (Denmark)

Horn, T; Henriksen, Jens Henrik Sahl; Christoffersen, P

1986-01-01

The scanning electron microscopic was used to study the fenestrations of human liver sinusoids. Thirteen biopsies, where light microscopy and transmission electron microscopy revealed normal sinusoidal architecture, were investigated. The number of fenestrae was calculated in acinar zone 3...

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

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Hoang, Huy-Dung; Maruyama, Jun-ichi

2014-01-01

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

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Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2013-07-01

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

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Egel-Mitani; Andersen; Diers; Hach; Thim; Hastrup; Vad

2000-06-01

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

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Silvia Berardis

Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

Gene expression analysis of precision-cut human liver slices indicates stable expression of ADME-Tox related genes

NARCIS (Netherlands)

Elferink, M. G. L.; Olinga, P.; van Leeuwen, E. M.; Bauerschmidt, S.; Polman, J.; Schoonen, W. G.; Heisterkamp, S. H.; Groothuis, G. M. M.

2011-01-01

In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

Science.gov (United States)

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling

DEFF Research Database (Denmark)

Le Pabic, Hélène; Bonnier, Dominique; Wewer, Ulla M

2003-01-01

"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT...... carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12......-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression...

Tc-99 m-GSA liver scintigraphy in alcoholic liver cirrhosis

International Nuclear Information System (INIS)

Itano, Satoshi; Harada, Masaru; Nagamatsu, Hiroaki

2003-01-01

We compared 15 alcoholic liver cirrhosis patients with 10 viral liver cirrhosis patients using technetium-99 m-galactosyl human serum albumin (Tc-99 m-GSA) liver scintigraphy and could clinically reveal the disorder of metabolism of asialoglycoprotein in alcoholic liver cirrhosis. Receptor index (LHL 15 = liver count divided by the sum of liver and heart counts at 15 minutes) was significantly (p <0.01) lower in patients with alcoholic cirrhosis (median: 0.821), compared with patients with viral cirrhosis (0.915). Grading score, which was an index showed by the difference in the isotope uptake patterns between liver and heart, was significantly (p <0.01) worse in patients with alcoholic cirrhosis, compared with patients with viral cirrhosis. These results suggested that alcoholic liver cirrhosis had a specific disorder of a metabolic function for asialoglycoprotein. (author)

Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

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Li, Runqin; Zhang, Yinglin

2016-01-01

Background and Objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. Materials and Methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells. PMID:27402632

Heterologous transporter expression for improved fatty alcohol secretion in yeast

DEFF Research Database (Denmark)

Hu, Yating; Zhu, Zhiwei; Nielsen, Jens

2017-01-01

The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production...... transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl......-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known...

Hordeum vulgare cysteine protease heterologous expressed in yeast

DEFF Research Database (Denmark)

Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

, (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression...

Regulation of coagulation factor XI expression by microRNAs in the human liver.

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Salam Salloum-Asfar

Full Text Available High levels of factor XI (FXI increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.

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Henning W Zimmermann

Full Text Available BACKGROUND: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C(+ monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14(+CD16(- and non-classical CD14(+CD16(+ cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that 'non-classical' monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14(+CD16(+ subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14(+CD16(+ macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC in vitro. CD14(+CD16(+ monocytes released abundant proinflammatory cytokines. Furthermore, CD14(+CD16(+, but not CD14(+CD16(- monocytes could directly activate collagen-producing HSC. CONCLUSIONS/SIGNIFICANCE: Our data

Priming T-cell responses with recombinant measles vaccine vector in a heterologous prime-boost setting in non-human primates.

Science.gov (United States)

Bolton, Diane L; Santra, Sampa; Swett-Tapia, Cindy; Custers, Jerome; Song, Kaimei; Balachandran, Harikrishnan; Mach, Linh; Naim, Hussein; Kozlowski, Pamela A; Lifton, Michelle; Goudsmit, Jaap; Letvin, Norman; Roederer, Mario; Radošević, Katarina

2012-09-07

Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.

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Ijssennagger, Noortje; Janssen, Aafke W F; Milona, Alexandra; Ramos Pittol, José M; Hollman, Danielle A A; Mokry, Michal; Betzel, Bark; Berends, Frits J; Janssen, Ignace M; van Mil, Saskia W C; Kersten, Sander

2016-05-01

The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

Science.gov (United States)

Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

2014-06-01

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

The liver taxis of receptor mediated lactosaminated human growth hormone

International Nuclear Information System (INIS)

Chen Zelian; Shi Lin; Li Tongling; Pang Qijie; He Juying; Guan Changtian

2002-01-01

Radiography imaging is used to assess liver taxis mechanism of anti-dwarfism drug lactosaminated human growth hormone (L-rhGH). Both L-rhGH and rhGH labelled with 131 I are used to study their biodistribution in animals (including rabbits, cocks and rats). The results show that L-rhGH is of specific hepatic targeting property, and the maximum hepatic concentration rate is 76.8%, which is two times of rhGH. Its hepatic binding is receptor mediated

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

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Selma Majerowicz

1994-09-01

Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology.

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Wen Liang

Full Text Available The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD by the NASH Clinical Research Network (NASH-CRN has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system. For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively. The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively and very high for the analysis of inflammation (ICC = 0.931, p

Upper respiratory tract infection, heterologous immunisation and meningococcal disease

NARCIS (Netherlands)

Scholten, R. J.; Bijlmer, H. A.; Tobi, H.; Dankert, J.; Bouter, L. M.

1999-01-01

To test the hypothesis that an episode of upper respiratory tract infection or heterologous immunisation is a predisposing factor for the occurrence of meningococcal disease, data from 377 cases of meningococcal disease and their household contacts (n = 1124) were analysed by conditional logistic

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

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Oberlin, Estelle; Fleury, Maud; Clay, Denis; Petit-Cocault, Laurence; Candelier, Jean-Jacques; Mennesson, Benoît; Jaffredo, Thierry; Souyri, Michèle

2010-11-25

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

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Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

International Nuclear Information System (INIS)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

2013-01-01

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker

Science.gov (United States)

Bolamba, D.; Patino, R.; Yoshizaki, G.; Thomas, P.

2003-01-01

Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-??m segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P<0.05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. ?? 2003 Elsevier Science (USA). All rights reserved.

Uptake and Persistence of Homologous and Heterologous Zooxanthellae in the Temperate Sea Anemone Cereus pedunculatus (Pennant).

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Davy, S K; Lucas, I A N; Turner, J R

1997-04-01

The uptake and persistence of symbiotic dinoflagellates (zooxanthellae) were measured in the temperate sea anemone Cereus pedunculatus (Pennant). Aposymbiotic specimens of C. pedunculatus were inoculated with zooxanthellae freshly isolated from a range of temperate and subtropical Anthozoa. Each inoculate consisted of zooxanthellae from a single host species and was either homologous (zooxanthellae from a host of the same species as the one being inoculated) or heterologous (from a host of a different species than the one being inoculated). The densities of zooxanthellae in host tissues were determined at regular intervals. C. pedunculatus took up homologous and heterologous zooxanthellae to similar degrees, except for zooxanthellae from the temperate Anthopleura ballii, which were taken up to a lesser extent. The densities of all zooxanthellae declined between 4 hours and 4 days after uptake, indicating that zooxanthellae were expelled, digested, or both during this period. The densities of all zooxanthellae increased between 2 and 8 weeks after inoculation, indicating zooxanthella growth. Over the entire 8-week period after uptake, densities of homologous zooxanthellae were always greater than those of heterologous zooxanthellae. Between 8 and 36 weeks after infection, densities of homologous zooxanthellae declined markedly and densities of some heterologous zooxanthellae increased further, resulting in homologous and heterologous zooxanthella densities being the same at 36 weeks. These densities were the same as those in naturally infected C. pedunculatus of similar size. The results suggest that zooxanthellae from a range of host species and environments can establish symbioses with C. pedunculatus and that, over long periods under laboratory conditions, heterologous zooxanthellae may populate C. pedunculatus to the same extent as homologous zooxanthellae.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

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Qinghua Cao

2016-01-01

Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Long-term outcomes of liver transplant patients with human immunodeficiency virus infection and end-stage-liver-disease: single center experience

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Vernadakis S

2011-08-01

Full Text Available Abstract Objective Orthotopic-liver-transplantation (OLT in patients with Human-Immunodeficiency-Virus infection (HIV and end-stage-liver-disease (ESDL is rarely reported. The purpose of this study is to describe our institutional experience on OLT for HIV positive patients. Material and methods This is a retrospective study of all HIV-infected patients who underwent OLT at the University Hospital of Essen, from January 1996 to December 2009. Age, sex, HIV transmission-way, CDC-stage, etiology of ESDL, concomitant liver disease, last CD4cell count and HIV-viral load prior to OLT were collected and analysed. Standard calcineurin-inhibitors-based immunosuppression was applied. All patients received anti-fungal and anti-pneumocystis carinii pneumonia prophylaxis post-OLT. Results Eight transplanted HIV-infected patients with a median age of 46 years (range 35-61 years were included. OLT indications were HCV (n = 5, HBV (n = 2, HCV/HBV/HDV-related cirrhosis (n = 1 and acute liver-failure (n = 1. At OLT, CD4 cell-counts ranged from 113-621 cells/μl, and HIV viral-loads from Conclusions OLT in HIV-infected patients and ESLD is an acceptable therapeutic option in selected patients. Long-term survival can be achieved without HIV disease-progression under antiretroviral therapy and management of the viral hepatitis co-infection.

CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

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Yu. В. Basok

2018-01-01

Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

The nutritional geometry of liver disease including non-alcoholic fatty liver disease.

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Simpson, Stephen J; Raubenheimer, David; Cogger, Victoria C; Macia, Laurence; Solon-Biet, Samantha M; Le Couteur, David G; George, Jacob

2018-02-01

Nutrition has a profound effect on chronic liver disease, especially non-alcoholic fatty liver disease (NAFLD). Most observational studies and clinical trials have focussed on the effects of total energy intake, or the intake of individual macronutrients and certain micronutrients, such as vitamin D, on liver disease. Although these studies have shown the importance of nutrition on hepatic outcomes, there is not yet any unifying framework for understanding the relationship between diet and liver disease. The Geometric Framework for Nutrition (GFN) is an innovative model for designing nutritional experiments or interpreting nutritional data that can determine the effects of nutrients and their interactions on animal behaviour and phenotypes. Recently the GFN has provided insights into the relationship between dietary energy and macronutrients on obesity and ageing in mammals including humans. Mouse studies using the GFN have disentangled the effects of macronutrients on fatty liver and the gut microbiome. The GFN is likely to play a significant role in disentangling the effects of nutrients on liver disease, especially NAFLD, in humans. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Liver Immunology

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Bogdanos, Dimitrios P.; Gao, Bin; Gershwin, M. Eric

2014-01-01

The liver is the largest organ in the body and is generally regarded by non-immunologists as not having lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates a tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and is also instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena which if are not controlled by regulatory lymphoid populations may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events which lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discus select, but not all, immune mediated liver disease and attempt to place these data in the context of human autoimmunity. PMID:23720323

Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts

International Nuclear Information System (INIS)

Massou, S.; Puech, V.; Talmont, F.; Demange, P.; Lindley, N.D.; Tropis, M.; Milon, A.

1999-01-01

Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human μ-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%-90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water

Detection of titanium particles in human liver and spleen and possible health implications

NARCIS (Netherlands)

Heringa, M.B.; Peters, R.J.B.; Bleys, R.L.A.W.; Lee, van der M.K.; Tromp, P.C.; Kesteren, van P.C.E.; Eijkeren, van J.C.H.; Undas, A.K.; Oomen, A.G.; Bouwmeester, H.

2018-01-01

Background: Titanium dioxide (TiO2) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal

Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples

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Robim M. Rodrigues

2016-06-01

Full Text Available This data set is composed of transcriptomics analyses of (i liver samples from patients suffering from acetaminophen-induced acute liver failure (ALF and (ii hepatic cell systems exposed to acetaminophen and their respective controls. The in vitro systems include widely employed cell lines i.e. HepaRG and HepG2 cells as well as a novel stem cell-derived model i.e. human skin-precursors-derived hepatocyte-like cells (hSKP-HPC. Data from primary human hepatocytes was also added to the data set “Open TG-GATEs: a large-scale toxicogenomics database” (Igarashi et al., 2015 [1]. Changes in gene expression due to acetaminophen intoxication as well as comparative information between human in vivo and in vitro samples are provided. The microarray data have been deposited in NCBI׳s Gene Expression Omnibus and are accessible through GEO Series accession number GEO: GSE74000. The provided data is used to evaluate the predictive capacity of each hepatic in vitro system and can be directly compared with large-scale publically available toxicogenomics databases. Further interpretation and discussion of these data feature in the corresponding research article “Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems” (Rodrigues et al., 2016 [2].

Heterologous human/rat HER2-specific exosome-targeted T cell vaccine stimulates potent humoral and CTL responses leading to enhanced circumvention of HER2 tolerance in double transgenic HLA-A2/HER2 mice.

Science.gov (United States)

Xie, Yufeng; Wu, Jie; Xu, Aizhang; Ahmeqd, Shahid; Sami, Amer; Chibbar, Rajni; Freywald, Andrew; Zheng, Changyu; Xiang, Jim

2018-03-07

DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-T EXO capable of stimulating HER2-specific CD8 + T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdV HuRt expressing HuRt fusion protein composed of NH 2 -HER2 1-407 (Hu) and COOH-neu 408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-T EXO using polyclonal CD4 + T-cells uptaking exosomes released by AdV HuRt -transfected dendritic cells. We found that the HuRt-T EXO vaccine stimulates enhanced CD4 + T-cell responses leading to increased induction of HER2-specific antibody (∼70 µg/ml) compared to that (∼40 µg/ml) triggered by the homologous HER2-T EXO vaccine. By using PE-H-2K d /HER2 23-71 tetramer, we determined that HuRt-T EXO stimulates stronger HER2-specific CD8 + T-cell responses eradicating 90% of HER2-specific target cells, while HER2-T EXO -induced CD8 + T-cell responses only eliminating 53% targets. Furthermore, HuRt-T EXO , but not HER2-T EXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1 HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10 A2/HER2 melanoma. HuRt-T EXO -stimulated HER2-specific CD8 + T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-T EXO, circumventing HER2 tolerance, may provide a new

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression

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Geon A. Kim

2017-01-01

Full Text Available Soluble human tumor necrosis factor (shTNFRI-Fc and human heme oxygenase 1 (hHO-1 are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

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Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

2009-10-26

The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

DEFF Research Database (Denmark)

Starokozhko, Viktoriia; Hemmingsen, Mette; Larsen, Layla

2018-01-01

and to perform functional comparisons with fresh human precision-cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis...... in the hiPSC-derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted...... in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels...

Heterologous prime-boost strategy in non-human primates combining the infective dengue virus and a recombinant protein in a formulation suitable for human use.

Science.gov (United States)

Valdés, Iris; Hermida, Lisset; Gil, Lázaro; Lazo, Laura; Castro, Jorge; Martín, Jorge; Bernardo, Lídice; López, Carlos; Niebla, Olivia; Menéndez, Tamara; Romero, Yaremis; Sánchez, Jorge; Guzmán, María G; Guillén, Gerardo

2010-05-01

The aim of the present work was to test the concept of the heterologous prime-boost strategy combining an infective dengue virus with a recombinant chimeric protein carrying domain III of the envelope protein. Two studies in monkeys, combining recombinant protein PD5 (domain III of the envelope protein from dengue-2 virus, fused to the protein carrier P64k) and the infective dengue virus in the same immunization schedules were carried out. Humoral and cell-mediated immunity were evaluated. In the first study, monkeys received four doses of the protein PD5 and were subsequently infected with one dose of dengue virus. Antibody response measured after virus inoculation was significantly higher compared to that in non-primed monkeys and comparable to that elicited after two doses of infective virus. In a second study, monkeys were infected with one dose of the virus and subsequently boosted with one dose of the recombinant protein, reaching high levels of neutralizing antibodies, which were still detectable 14 months after the last immunization. In addition, the cellular immune response was also recalled. The results obtained in the present work support the approach of heterologous prime-boosting, in either order prime or boost, combining the chimeric protein PD5 (formulated in alum-CPS-A) and an infective dengue virus. The latter could potentially be replaced by an attenuated vaccine candidate. Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

Science.gov (United States)

Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

2001-01-01

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

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Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

2013-09-05

The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Aryl hydrocarbon receptor nuclear translocator in human liver is regulated by miR-24

Energy Technology Data Exchange (ETDEWEB)

Oda, Yuki; Nakajima, Miki; Mohri, Takuya [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan); Takamiya, Masataka; Aoki, Yasuhiro [Department of Legal Medicine, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka 020-8505 (Japan); Fukami, Tatsuki [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan); Yokoi, Tsuyoshi, E-mail: tyokoi@kenroku.kanazawa-u.ac.jp [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan)

2012-05-01

Aryl hydrocarbon receptor nuclear translocator (ARNT) forms a heterodimer with aryl hydrocarbon receptor or hypoxia inducible factor 1α to mediate biological responses to xenobiotic exposure and hypoxia. Although the regulation mechanism of the ARNT expression is largely unknown, earlier studies reported that the human ARNT protein level was decreased by hydrogen peroxide or reactive oxygen species. These stimuli increase the miR-24 level in various human cell lines. In silico analysis predicts that some microRNAs including miR-16 and miR-23b may bind to ARNT mRNA. This background prompted us to investigate whether human ARNT is regulated by microRNAs. Overexpression of miR-24 into HuH-7 and HepG2 cells significantly decreased the ARNT protein level, but not the ARNT mRNA level, indicating translational repression. However, overexpression of miR-16 or miR-23b caused no change in the ARNT expression. The miR-24-dependent down-regulation of ARNT decreased the expression of its downstream genes such as CYP1A1 and carbonic anhydrase IX. Luciferase assay was performed to determine the element on the ARNT mRNA to which miR-24 binds. Finally, it was demonstrated that the miR-24 levels in a panel of 26 human livers were inversely correlated with the protein levels or the translational efficiency of ARNT. Taken together, we found that miR-24 negatively regulates ARNT expression in human liver, affecting the expression of its downstream genes. miR-24 would be one of the factors underlying the mechanisms by which ARNT protein is decreased by reactive oxygen species. -- Highlights: ► Overexpression of miR-24 into human cell lines decreased the ARNT protein level. ► miR-24-dependent down-regulation of ARNT affected the expression of CYP1A1 and CA IX. ► Luciferase assay was performed to identify functional MREs for miR-24 in ARNT mRNA. ► The miR-24 levels inversely correlated with the ARNT protein levels in human liver.

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

Science.gov (United States)

Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

2015-03-01

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

International Nuclear Information System (INIS)

Vickers, Alison E.M.; Sinclair, John R.; Fisher, Robyn L.; Morris, Stephen R.; Way, William

2010-01-01

A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (≥ 1000 μM at 48 h) and human tissues (≥ 1000 μM at 48 h, ≥ 750 μM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

Directory of Open Access Journals (Sweden)

Thomas Gronemeyer

Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis

OpenAIRE

Zhang, Guo-Zun; Sun, Hui-Cong; Zheng, Li-Bo; Guo, Jin-Bo; Zhang, Xiao-Lan

2017-01-01

AIM To investigate the hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and to evaluate their therapeutic effect on liver fibrosis/cirrhosis. METHODS A CCl4-induced liver fibrotic/cirrhotic rat model was used to assess the effect of hUC-MSCs. Histopathology was assessed by hematoxylin and eosin (H&E), Masson trichrome and Sirius red staining. The liver biochemical profile was measured using a Beckman Coulter analyzer. Expression analysis was ...

Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

Science.gov (United States)

Yamazaki, H; Mimura, M; Oda, Y; Inui, Y; Shiraga, T; Iwasaki, K; Guengerich, F P; Shimada, T

1993-07-01

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the

Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

DEFF Research Database (Denmark)

2016-01-01

A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

Science.gov (United States)

Herrera Sanchez, Maria Beatriz; Previdi, Sara; Bruno, Stefania; Fonsato, Valentina; Deregibus, Maria Chiara; Kholia, Sharad; Petrillo, Sara; Tolosano, Emanuela; Critelli, Rossana; Spada, Marco; Romagnoli, Renato; Salizzoni, Mauro; Tetta, Ciro; Camussi, Giovanni

2017-07-27

Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme

Bisphenol A sulfonation is impaired in metabolic and liver disease

International Nuclear Information System (INIS)

Yalcin, Emine B.; Kulkarni, Supriya R.; Slitt, Angela L.; King, Roberta

2016-01-01

Background: Bisphenol A (BPA) is a widely used industrial chemical and suspected endocrine disruptor to which humans are ubiquitously exposed. The liver metabolizes and facilitates BPA excretion through glucuronidation and sulfonation. The sulfotransferase enzymes contributing to BPA sulfonation (detected in human and rodents) is poorly understood. Objectives: To determine the impact of metabolic and liver disease on BPA sulfonation in human and mouse livers. Methods: The capacity for BPA sulfonation was determined in human liver samples that were categorized into different stages of metabolic and liver disease (including obesity, diabetes, steatosis, and cirrhosis) and in livers from ob/ob mice. Results: In human liver tissues, BPA sulfonation was substantially lower in livers from subjects with steatosis (23%), diabetes cirrhosis (16%), and cirrhosis (18%), relative to healthy individuals with non-fatty livers (100%). In livers of obese mice (ob/ob), BPA sulfonation was lower (23%) than in livers from lean wild-type controls (100%). In addition to BPA sulfonation activity, Sult1a1 protein expression decreased by 97% in obese mouse livers. Conclusion: Taken together these findings establish a profoundly reduced capacity of BPA elimination via sulfonation in obese or diabetic individuals and in those with fatty or cirrhotic livers versus individuals with healthy livers. - Highlights: • Present study demonstrates that hepatic SULT 1A1/1A3 are primarily sulfonate BPA in mouse and human. • Hepatic BPA sulfonation is profoundly reduced steatosis, diabetes and cirrhosis. • With BPA-S detectable in urine under low or common exposures, these findings are novel and important.

Bisphenol A sulfonation is impaired in metabolic and liver disease

Energy Technology Data Exchange (ETDEWEB)

Yalcin, Emine B.; Kulkarni, Supriya R.; Slitt, Angela L., E-mail: angela_slitt@uri.edu; King, Roberta, E-mail: rking@uri.edu

2016-02-01

Background: Bisphenol A (BPA) is a widely used industrial chemical and suspected endocrine disruptor to which humans are ubiquitously exposed. The liver metabolizes and facilitates BPA excretion through glucuronidation and sulfonation. The sulfotransferase enzymes contributing to BPA sulfonation (detected in human and rodents) is poorly understood. Objectives: To determine the impact of metabolic and liver disease on BPA sulfonation in human and mouse livers. Methods: The capacity for BPA sulfonation was determined in human liver samples that were categorized into different stages of metabolic and liver disease (including obesity, diabetes, steatosis, and cirrhosis) and in livers from ob/ob mice. Results: In human liver tissues, BPA sulfonation was substantially lower in livers from subjects with steatosis (23%), diabetes cirrhosis (16%), and cirrhosis (18%), relative to healthy individuals with non-fatty livers (100%). In livers of obese mice (ob/ob), BPA sulfonation was lower (23%) than in livers from lean wild-type controls (100%). In addition to BPA sulfonation activity, Sult1a1 protein expression decreased by 97% in obese mouse livers. Conclusion: Taken together these findings establish a profoundly reduced capacity of BPA elimination via sulfonation in obese or diabetic individuals and in those with fatty or cirrhotic livers versus individuals with healthy livers. - Highlights: • Present study demonstrates that hepatic SULT 1A1/1A3 are primarily sulfonate BPA in mouse and human. • Hepatic BPA sulfonation is profoundly reduced steatosis, diabetes and cirrhosis. • With BPA-S detectable in urine under low or common exposures, these findings are novel and important.

Expression and Regulation of the Bile Acid Transporter, OST alpha-OST beta in Rat and Human Intestine and Liver

NARCIS (Netherlands)

Khan, Ansar A.; Chow, Edwin C. Y.; Porte, Robert J.; Pang, K. Sandy; Groothuis, Geny M. M.

The regulation of the OST alpha and OST beta expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue

Longitudinal study of experimental induction of AA amyloidosis in mice seeded with homologous and heterologous AA fibrils.

Science.gov (United States)

Muhammad, Naeem; Murakami, Tomoaki; Inoshima, Yasuo; Ishiguro, Naotaka

2016-09-01

To investigate pathogenesis and kinetics of experimentally induced murine AA amyloidosis seeded with homologous (murine) and heterologous (bovine) AA fibrils. Experimental AA amyloidosis was induced by administration of inflammatory stimulus and preformed AA fibrils to a total of 111 female C57/Black mice. In this longitudinal study, heterologous (bovine) as well as homologous (murine) AA fibrils were injected intraperitoneally to mice in various combinations. Re-stimulation was done at 120 or 300 days post first inoculation. To analyze the intensity of amyloid depositions in mice organs, immunohistochemical techniques and image J software were used. Assessment of cytokines level in sera was done using a Mouse Th1/Th2/Th17 Cytokine CBA Kit. Incidence and severity of AA amyloidosis were quite low in mice inoculated with heterologous bovine AA fibrils than homologous murine one. Homologous AA fibrils administration at first and second inoculation caused maximum amount of amyloid depositions and severe systemic form of amyloidosis. Increase in the level of pro-inflammatory cytokine IL-6 was observed after first inoculation, while second inoculation caused a further increase in the level of anti-inflammatory cytokine IL-10. AA amyloidosis can be induced by heterologous as well as homologous AA fibrils. Severity of AA amyloidosis induced with homologous AA fibrils is higher compared to heterologous AA fibrils.

Metabolism of 5-fluorouracil in human liver: an in vivo 19F NMR study

International Nuclear Information System (INIS)

Mohankrishnan, P.; Sprigg, J.; Cardwell, D.; Komoroski, R.A.; Hutchins, L.; Nauke, S.; Williamson, M.R.; Jagannathan, N.R.

1999-01-01

In vivo fluorine-19 nuclear magnetic resonance ( 19 F NMR) spectroscopy was used to study the metabolism and pharmacokinetics of 5-fluorouracil (5-FU) in human liver. Nine patients received 5-FU, and additional chemotherapeutic agents (methotrexate, leucovorin, or levamisole) either prophylactically after breast cancer surgery or for colorectal cancer. The time constant for the disappearance of 5-FU from the liver in vivo varied from 5 to 17 min, while the time constant for the appearance of α-fluoro-β-alanine (the major catabolite of 5 FU) varied from 7 to 86 min. The modulators of 5-FU metabolism did not appear to affect the time constant for the disappearance of 5-FU from the liver or for the appearance of α-fluoro-β-alanine. Results obtained indicate that the pharmacokinetics of 5-FU and α-fluoro-β-alanine may vary substantially at different times in a given individual. (author)

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes

Directory of Open Access Journals (Sweden)

Moayad Saad

2018-02-01

Full Text Available This article represents data regarding a study published in Toxicology in vitro entitled “ in vitro CYP-mediated drug metabolism in the zebrafish (embryo using human reference compounds” (Saad et al., 2017 [1]. Data were acquired with ultra-performance liquid chromatography – accurate mass mass spectrometry (UPLC-amMS. A full spectrum scan was conducted for the testosterone (TST metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM and female (FLM livers, whole body homogenates of 96 h post fertilization larvae (EM and a pool of human liver microsomes from 50 donors (HLM. Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

Heterologous surface display on lactic acid bacteria: non-GMO alternative?

Science.gov (United States)

Zadravec, Petra; Štrukelj, Borut; Berlec, Aleš

2015-01-01

Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

Initial prevalence of anal human papilloma virus infection in liver transplant recipients.

Science.gov (United States)

Grąt, Michał; Grąt, Karolina; Hołówko, Wacław; Malejczyk, Magdalena; Walter de Walthoffen, Szymon; Lewandowski, Zbigniew; Kobryń, Konrad; Patkowski, Waldemar; Majewski, Sławomir; Młynarczyk, Grażyna; Krawczyk, Marek

2014-08-01

Although liver transplant recipients are at increased risk of human papilloma virus (HPV)-related anal cancer, limited data are available regarding the initial prevalence of anal HPV infection in this population. Anal swabs collected from 50 liver transplant recipients within the first three postoperative weeks were subjected to real-time polymerase chain reaction for detection of the four HPV genotypes: 6, 11, 16, and 18. Predictors of any, low-risk, and high-risk anal HPV infection were evaluated. Overall, the prevalence of any anal HPV infection was 18.0%, with the corresponding rates for high- and low-risk HPV genotypes being 8.0% and 10.0%, respectively. Infection with any type of anal HPV was higher in patients with hepatitis B virus (HBV) infection (P = 0.027), ≥3 sexual partners (P = 0.031), and alcoholic liver disease (P = 0.063). HBV infection was the only factor significantly associated with high-risk HPV infection (P = 0.038). Male sex (P = 0.050), age ≥52 years (P = 0.016), ≥30 sexual partners (P = 0.003), age at first intercourse ≤18 years (P = 0.045), and time since first intercourse ≥38 years (P = 0.012) were identified as predictors of low-risk HPV infection. These results indicate that HPV vaccination of liver transplant candidates and screening for anal HPV infection in high-risk groups should be considered. © 2014 Steunstichting ESOT.

A high level of liver-specific expression of oncogenic KrasV12 drives robust liver tumorigenesis in transgenic zebrafish

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Anh Tuan Nguyen

2011-11-01

Human liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC being the most common type. Aberrant Ras signaling has been implicated in the development and progression of human HCC, but a complete understanding of the molecular mechanisms of this protein in hepatocarcinogenesis remains elusive. In this study, a stable in vivo liver cancer model using transgenic zebrafish was generated to elucidate Ras-driven tumorigenesis in HCC. Using the liver-specific fabp10 (fatty acid binding protein 10 promoter, we overexpressed oncogenic krasV12 specifically in the transgenic zebrafish liver. Only a high level of krasV12 expression initiated liver tumorigenesis, which progressed from hyperplasia to benign and malignant tumors with activation of the Ras-Raf-MEK-ERK and Wnt–β-catenin pathways. Histological diagnosis of zebrafish tumors identified HCC as the main lesion. The tumors were invasive and transplantable, indicating malignancy of these HCC cells. Oncogenic krasV12 was also found to trigger p53-dependent senescence as a tumor suppressive barrier in the pre-neoplastic stage. Microarray analysis of zebrafish liver hyperplasia and HCC uncovered the deregulation of several stage-specific and common biological processes and signaling pathways responsible for krasV12-driven liver tumorigenesis that recapitulated the molecular hallmarks of human liver cancer. Cross-species comparisons of cancer transcriptomes further defined a HCC-specific gene signature as well as a liver cancer progression gene signature that are evolutionarily conserved between human and zebrafish. Collectively, our study presents a comprehensive portrait of molecular mechanisms during progressive Ras-induced HCC. These observations indicate the validity of our transgenic zebrafish to model human liver cancer, and this model might act as a useful platform for drug screening and identifying new therapeutic targets.

Test systems in drug discovery for hazard identification and risk assessment of human drug-induced liver injury.

Science.gov (United States)

Weaver, Richard J; Betts, Catherine; Blomme, Eric A G; Gerets, Helga H J; Gjervig Jensen, Klaus; Hewitt, Philip G; Juhila, Satu; Labbe, Gilles; Liguori, Michael J; Mesens, Natalie; Ogese, Monday O; Persson, Mikael; Snoeys, Jan; Stevens, James L; Walker, Tracy; Park, B Kevin

2017-07-01

The liver is an important target for drug-induced toxicities. Early detection of hepatotoxic drugs requires use of well-characterized test systems, yet current knowledge, gaps and limitations of tests employed remains an important issue for drug development. Areas Covered: The current state of the science, understanding and application of test systems in use for the detection of drug-induced cytotoxicity, mitochondrial toxicity, cholestasis and inflammation is summarized. The test systems highlighted herein cover mostly in vitro and some in vivo models and endpoint measurements used in the assessment of small molecule toxic liabilities. Opportunities for research efforts in areas necessitating the development of specific tests and improved mechanistic understanding are highlighted. Expert Opinion: Use of in vitro test systems for safety optimization will remain a core activity in drug discovery. Substantial inroads have been made with a number of assays established for human Drug-induced Liver Injury. There nevertheless remain significant gaps with a need for improved in vitro tools and novel tests to address specific mechanisms of human Drug-Induced Liver Injury. Progress in these areas will necessitate not only models fit for application, but also mechanistic understanding of how chemical insult on the liver occurs in order to identify translational and quantifiable readouts for decision-making.

Influence of nanoparticles accumulation on optical properties of human normal and cancerous liver tissue in vitro estimated by OCT

International Nuclear Information System (INIS)

Zhou, Fang; Wei, Huajiang; Guo, Zhouyi; Ye, Xiangping; Hu, Kun; Wu, Guoyong; Yang, Hongqin; Xie, Shusen; He, Yonghong

2015-01-01

In this work, the potential use of nanoparticles as contrast agents by using spectral domain optical coherence tomography (SD-OCT) in liver tissue was demonstrated. Gold nanoparticles (average size of 25 and 70 nm), were studied in human normal and cancerous liver tissues in vitro, respectively. Each sample was monitored with SD-OCT functional imaging for 240 min. Continuous OCT monitoring showed that, after application of gold nanoparticles, the OCT signal intensities of normal liver and cancerous liver tissue both increase with time, and the larger nanoparticles tend to produce a greater signal enhancement in the same type of tissue. The results show that the values of attenuation coefficients have significant differences between normal liver tissue and cancerous liver tissue. In addition, 25 nm gold nanoparticles allow higher penetration depth than 70 nm gold nanoparticles in liver tissues. (paper)

Peroxisome proliferator-activated receptor α (PPARα mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue

Directory of Open Access Journals (Sweden)

Kurokawa Tsuyoshi

2011-12-01

Full Text Available Abstract Background Peroxisome proliferator-activated receptor α (PPARα regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer. Methods The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system. Results The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections. Conclusion The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

A novel “humanized mouse” model for autoimmune hepatitis and the association of gut microbiota with liver inflammation

Science.gov (United States)

Yuksel, Muhammed; Wang, Yipeng; Tai, Ningwen; Peng, Jian; Guo, Junhua; Beland, Kathie; Lapierre, Pascal; David, Chella; Alvarez, Fernando; Colle, Isabelle; Yan, Huiping; Mieli-Vergani, Giorgina; Vergani, Diego; Ma, Yun; Wen, Li

2016-01-01

Background Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease, characterized by interface hepatitis, the presence of circulating autoantibodies and hyper-gammaglobulinemia. There are two types of AIH, type-1 (AIH-1) and type-2 (AIH-2) characterized by distinct autoimmune serology. Patients with AIH-1 are positive for anti-smooth muscle and/or anti-nuclear (SMA/ANA) autoantibodies whereas patients with AIH-2 have anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) autoantibodies. Cytochrome P4502D6 (CYP2D6) is the antigenic target of anti-LKM1 and formiminotransferase cyclodeaminase (FTCD) is the antigenic target of anti-LC1. It is known that AIH, both type-1 and type-2, is strongly linked to the Human Leukocyte Antigen (HLA) alleles -DR3, -DR4 and -DR7. However, the direct evidence of the association of HLA with AIH is lacking. Methods We developed a novel mouse model of AIH using the HLA-DR3 transgenic mouse on the non-obese diabetic (NOD) background (HLA-DR3 NOD) by immunization of HLA-DR3− and HLA-DR3+ NOD mice with a DNA plasmid, coding for human CYP2D6/FTCD fusion protein. Results Immunization with CYP2D6/FTCD leads to a sustained elevation of alanine aminotransferase (ALT), development of ANA and anti-LKM1/anti-LC1 autoantibodies, chronic immune cell infiltration and parenchymal fibrosis on liver histology in HLA-DR3+ mice. Immunized mice also showed an enhanced Th1 immune response and paucity of the frequency of regulatory T-cell (Treg) in the liver. Moreover, HLA-DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. PMID:26185095

Liver afferents contribute to water drinking-induced sympathetic activation in human subjects: a clinical trial.

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Marcus May

Full Text Available Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (p<0.05 between groups after 30-40 minutes of water drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects.ClinicalTrials.gov NCT01237431.

Liver Afferents Contribute to Water Drinking-Induced Sympathetic Activation in Human Subjects: A Clinical Trial

Science.gov (United States)

May, Marcus; Gueler, Faikah; Barg-Hock, Hannelore; Heiringhoff, Karl-Heinz; Engeli, Stefan; Heusser, Karsten; Diedrich, André; Brandt, André; Strassburg, Christian P.; Tank, Jens; Sweep, Fred C. G. J.; Jordan, Jens

2011-01-01

Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant) as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant) as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (pwater drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects. Trial Registration ClinicalTrials.gov NCT01237431 PMID:22016786

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

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Jiafei Xi

Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

Influence of starvation, triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

International Nuclear Information System (INIS)

Harikumar, P.; Ninjoor, V.

1986-01-01

The response of rat liver lysosomes to starvation and administration of lysosomotropic agents viz. Triton WR-1339 and [ 131 I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [ 131 I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment. (author)

Immune mediated liver failure.

Science.gov (United States)

Wang, Xiaojing; Ning, Qin

2014-01-01

Liver failure is a clinical syndrome of various etiologies, manifesting as jaundice, encephalopathy, coagulopathy and circulatory dysfunction, which result in subsequent multiorgan failure. Clinically, liver failure is classified into four categories: acute, subacute, acute-on-chronic and chronic liver failure. Massive hepatocyte death is considered to be the core event in the development of liver failure, which occurs when the extent of hepatocyte death is beyond the liver regenerative capacity. Direct damage and immune-mediated liver injury are two major factors involved in this process. Increasing evidence has suggested the essential role of immune-mediated liver injury in the pathogenesis of liver failure. Here, we review the evolved concepts concerning the mechanisms of immune-mediated liver injury in liver failure from human and animal studies. Both innate and adaptive immunity, especially the interaction of various immune cells and molecules as well as death receptor signaling system are discussed. In addition, we highlight the concept of "immune coagulation", which has been shown to be related to the disease progression and liver injury exacerbation in HBV related acute-on-chronic liver failure.

Processing highly porous calcium phosphate ceramics for use in bioreactor cores for culturing human liver cells in-vitro

Science.gov (United States)

Finoli, Anthony

Chronic liver disease is the 11th highest cause of death in the United States claiming over 30,000 lives in 2009. The current treatment for chronic liver failure is liver transplantation but the availability of tissue is far less than the number of patients in need. To develop human liver tissue in the lab a 3D culturing environment must be created to support the growth of a complex tissue. Hydroxyapatite (HAp) has been chosen as a scaffold material because of its biocompatibility in the body and the ability to create a bioresorbable scaffold. By using a ceramic material, it is possible to create a three dimensional, protective environment in which tissue can grow. The first part of this study is to examine the behavior of adult human liver cells grown on composites of HAp and different biocompatible hydrogels. Porous HAp has been created using an emulsion foaming technique and cells are injected into the structure after being suspended in a hydrogel and are kept in culture for up to 28 days. Functional assays, gene expression and fluorescent microscopy will be used to examine these cultures. The second part of this study will be to develop a processing technique to create a resorbable scaffold that incorporates a vascular system template. Previous experiments have shown the high temperature decomposition of HAp into resorbable calcium phosphates will be used to create a multiphase material. By controlling the amount of transformation product formed, it is proposed that the resorption of the scaffold can be tailored. To introduce a pore network to guide the growth of a vascular system, a positive-negative casting technique has also been developed. A positive polymer copy can be made of a natural vascular system and ceramic is foamed around the copy. During sintering, the polymer is pyrolyzed leaving a multiscale pore network in the ceramic. By combining these techniques, it is proposed that a calcium phosphate bioreactor core can be processed that is suitable for

Heterologous production of peptides in plants: fusion proteins and beyond.

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Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

2013-11-01

Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

Science.gov (United States)

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

[Molecular detection of hepatitis E virus in pig livers destined for human consumption in the state of Nuevo Leon, Mexico].

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Cantú-Martínez, Marco Antonio; Roig-Sagués, Artur Xavier; Cedillo-Rosales, Sibilina; Zamora-Ávila, Diana Elisa; Avalos-Ramírez, Ramiro

2013-04-01

Molecular detection of HEV in pig livers destined for human consumption in Nuevo Leon, Mexico. 87 livers were collected from pigs slaughtered in TIF and 40 livers from butchers. A 212 pb fragment of HEV ORF2 gene was amplified by semi-nested RT-PCR. 19.54% (17) of tif's and 22.5% (9) of butcher's livers were positive for HEV. Sequencing of the amplified products showed a 94%-95% homology with the sequences reported for genotype 3. Our results indicate that HEV is circulating in swine herds in the state, constituting a probable source of contamination of pig meat products.

Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

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Warfield, Kelly L.; Dye, John M.; Wells, Jay B.; Unfer, Robert C.; Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Swenson, Dana L.; Bavari, Sina; Aman, M. Javad

2015-01-01

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components. PMID:25793502

Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles.

Directory of Open Access Journals (Sweden)

Kelly L Warfield

Full Text Available Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV and Marburg virus (MARV following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system and then demonstrate protection against Sudan virus (SUDV and Taï Forest virus (TAFV. Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

Quantification of human hepatic binding protein (HBP) via 99mTc-galactosyl-neoglycoalbumin (NGA) liver scintigraphy

International Nuclear Information System (INIS)

Virgolini, I.; Hoebart, J.; Bergmann, H.; Sinzinger, H.; Mueller, C.; Angelberger, P.

1991-01-01

99m Tc-galactosyl-neoglycoalbumin ( 99m Tc-NGA) was synthesized by covalent coupling of 2-imino-2-methoxyethyl-1-thio-β-D-galactopyranoside to the primary amino groups of human serum albumin. Injections of 99m Tc-NGA (150 MBq; 3.5 mg (=50 nmol)/ml) demonstrated the liver to be the exclusive site of tracer-uptake. Simulation of 99m Tc-NGA-kinetics allowed quantification of binding to the hepatic binding protein (HBP). Using this model we studied 250 patients with various liver disease. In alcoholic liver cirrhosis such patients with Child B and Child C stage cirrhosis had a lower HBP-concentration in the liver compared to control individuals (0.85-1.2 μmol/l). The group with the most advanced cirrhosis (Child C stage) had a significantly lower HBP-concentration (0.20-0.45 μmol/l) than Child A patients (0.60-0.85 μmol/l; p 99m Tc-galactosyl-neoglycoalbumin (NGA) scanning of the liver during the course of the disease. Return of liver function tests to normal values was associated with an increased hepatic imaging size as well as increase in HBP-concentration (up to a 3-fold of initial concentration). In patients exhibiting a prolonged course of the disease changes in NGA-kinetic data were borderline and the hepatic image size unchanged. The values obtained for HBP-concentration in the liver amounted to 0.30-0.50 μmol/l liver for patients with hepatoma, to 0.40-0.60 μmol/l in patients with liver metastasis and to 0.90-1.20 μmol/l in cancer patients without liver malignancy. It is concluded that scintigraphic evaluation of functional hepatic cell mass using the new receptor-tracer 99m Tc-NGA provides an in vivo diagnostic mean allowing quantitative data on liver function beside assessment of liver morphology. (Authors)

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

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Burra Patrizia

2012-07-01

Full Text Available Abstract Background Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs, a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. Methods Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion. To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification. Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons. Results We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However

Bioengineered Liver Models for Drug Testing and Cell Differentiation Studies

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Gregory H. Underhill

2018-01-01

Full Text Available In vitro models of the human liver are important for the following: (1 mitigating the risk of drug-induced liver injury to human beings, (2 modeling human liver diseases, (3 elucidating the role of single and combinatorial microenvironmental cues on liver cell function, and (4 enabling cell-based therapies in the clinic. Methods to isolate and culture primary human hepatocytes (PHHs, the gold standard for building human liver models, were developed several decades ago; however, PHHs show a precipitous decline in phenotypic functions in 2-dimensional extracellular matrix–coated conventional culture formats, which does not allow chronic treatment with drugs and other stimuli. The development of several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment for enhancing the function of both PHHs and induced pluripotent stem cell–derived human hepatocyte-like cells; long-term (4+ weeks stabilization of hepatocellular function typically requires co-cultivation with liver-derived or non–liver-derived nonparenchymal cell types. In addition, the recent development of liver organoid culture systems can provide a strategy for the enhanced expansion of therapeutically relevant cell types. Here, we discuss advances in engineering approaches for constructing in vitro human liver models that have utility in drug screening and for determining microenvironmental determinants of liver cell differentiation/function. Design features and validation data of representative models are presented to highlight major trends followed by the discussion of pending issues that need to be addressed. Overall, bioengineered liver models have significantly advanced our understanding of liver function and injury, which will prove useful for drug development and ultimately cell-based therapies.

Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

NARCIS (Netherlands)

Amerongen, A. van; Helms, J.B.; Krift, T.P. van der; Schutgens, R.B.H.; Wirtz, K.W.A.

1987-01-01

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Science.gov (United States)

Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

2015-04-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Directory of Open Access Journals (Sweden)

Wenjing Law

2015-04-01

Full Text Available Monoamines, such as 5-HT and tyramine (TA, paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for

Heterologous gene expression in filamentous fungi.

Science.gov (United States)

Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

2012-01-01

Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

DEFF Research Database (Denmark)

Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

Science.gov (United States)

Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

2015-01-01

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice.

Science.gov (United States)

Soulard, Valérie; Bosson-Vanga, Henriette; Lorthiois, Audrey; Roucher, Clémentine; Franetich, Jean-François; Zanghi, Gigliola; Bordessoulles, Mallaury; Tefit, Maurel; Thellier, Marc; Morosan, Serban; Le Naour, Gilles; Capron, Frédérique; Suemizu, Hiroshi; Snounou, Georges; Moreno-Sabater, Alicia; Mazier, Dominique

2015-07-24

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.

Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China

Institute of Scientific and Technical Information of China (English)

Xue-Qin Meng; Xuan Zhang; Jun Fan; Lin Zhou; Bing Hao; Xiao-Ming Chen; Wei-Hang Ma; Shu-Sen Zheng

2009-01-01

BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05).CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Speciifc investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers.

Science.gov (United States)

Furuta, Mayuko; Tanaka, Hiroko; Shiraishi, Yuichi; Unida, Takuro; Imamura, Michio; Fujimoto, Akihiro; Fujita, Masahi; Sasaki-Oku, Aya; Maejima, Kazuhiro; Nakano, Kaoru; Kawakami, Yoshiiku; Arihiro, Koji; Aikata, Hiroshi; Ueno, Masaki; Hayami, Shinya; Ariizumi, Shun-Ichi; Yamamoto, Masakazu; Gotoh, Kunihito; Ohdan, Hideki; Yamaue, Hiroki; Miyano, Satoru; Chayama, Kazuaki; Nakagawa, Hidewaki

2018-05-18

Integration of Hepatitis B virus (HBV) into the human genome can cause genetic instability, leading to selective advantages for HBV-induced liver cancer. Despite the large number of studies for HBV integration into liver cancer, little is known about the mechanism of initial HBV integration events owing to the limitations of materials and detection methods. We conducted an HBV sequence capture, followed by ultra-deep sequencing, to screen for HBV integrations in 111 liver samples from human-hepatocyte chimeric mice with HBV infection and human clinical samples containing 42 paired samples from non-tumorous and tumorous liver tissues. The HBV infection model using chimeric mice verified the efficiency of our HBV-capture analysis and demonstrated that HBV integration could occur 23 to 49 days after HBV infection via microhomology-mediated end joining and predominantly in mitochondrial DNA. Overall HBV integration sites in clinical samples were significantly enriched in regions annotated as exhibiting open chromatin, a high level of gene expression, and early replication timing in liver cells. These data indicate that HBV integration in liver tissue was biased according to chromatin accessibility, with additional selection pressures in the gene promoters of tumor samples. Moreover, an integrative analysis using paired non-tumorous and tumorous samples and HBV-related transcriptional change revealed the involvement of TERT and MLL4 in clonal selection. We also found frequent and non-tumorous liver-specific HBV integrations in FN1 and HBV-FN1 fusion transcript. Extensive survey of HBV integrations facilitates and improves the understanding of the timing and biology of HBV integration during infection and HBV-related hepatocarcinogenesis.

(99m) Tc-labelled human serum albumin cannot replace (125) I-labelled human serum albumin to determine plasma volume in patients with liver disease

DEFF Research Database (Denmark)

Henriksen, Ulrik Lütken; Henriksen, Jens H; Bendtsen, Flemming

2013-01-01

Summary Background and aims Determination of plasma volume (PV) is important in several clinical situations. Thus, patients with liver disease often have augmented PV as part of their sodium–water retention. This study was undertaken to compare PV determination by two indicators: technetium......-labelled human serum albumin (99mTc-HSA) and iodine-labelled human serum albumin (125I-HSA), as the former may have advantages at repeated measurements and the latter is the classical gold standard. Study population and methods In 88 patients, (64 with liver disease, mainly cirrhosis, and 24 patients without...... In all patients, a close correlation was present between PV determined by the two indicators (r = 0·89, Pdetermined with 99mTc-HSA exceeded PV determined with 125I-HSA by 367 ml (5·2 ml kg...

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

Science.gov (United States)

Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

2005-10-01

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Experimental autoimmune glomerulonephritis induced by anti-glomerular basement membrane antibody. II. Effects of injecting heterologous, homologous, or autologous glomerular basement membranes and complete Freund's adjuvant into sheep.

OpenAIRE

Steblay, R. W.; Rudofsky, U. H.

1983-01-01

The effects of injecting human, rabbit, rat, or single-kidney homologous glomerular basement membrane (GBM) or autologous GBM, each in complete Freund's adjuvant (CFA), into 15- to 18-month-old sheep are compared. All sheep receiving heterologous GBM and 3 of 6 sheep receiving homologous GBM had anti-GBM nephritis, but such sheep did not bind autoantibodies or have Goodpasturelike lesions in their lungs. Sheep given injections of human GBM had autoantibodies to antigenic determinants shared b...

A heterologous prime-boost Ebola virus vaccine regimen induces durable neutralizing antibody response and prevents Ebola virus-like particle entry in mice.

Science.gov (United States)

Chen, Tan; Li, Dapeng; Song, Yufeng; Yang, Xi; Liu, Qingwei; Jin, Xia; Zhou, Dongming; Huang, Zhong

2017-09-01

Ebola virus (EBOV) is one of the most virulent pathogens known to humans. Neutralizing antibodies play a major role in the protection against EBOV infections. Thus, an EBOV vaccine capable of inducing a long-lasting neutralizing antibody response is highly desirable. We report here that a heterologous prime-boost vaccine regimen can elicit durable EBOV-neutralizing antibody response in mice. A chimpanzee serotype 7 adenovirus expressing EBOV GP (denoted AdC7-GP) was generated and used for priming. A truncated version of EBOV GP1 protein (denoted GP1t) was produced at high levels in Drosophila S2 cells and used for boosting. Mouse immunization studies showed that the AdC7-GP prime/GP1t boost vaccine regimen was more potent in eliciting neutralizing antibodies than either the AdC7-GP or GP1t alone. Neutralizing antibodies induced by the heterologous prime-boost regimen sustained at high titers for at least 18 weeks after immunization. Significantly, in vivo challenge studies revealed that the entry of reporter EBOV-like particles was efficiently blocked in mice receiving the heterologous prime-boost regimen even at 18 weeks after the final dose of immunization. These results suggest that this novel AdC7-GP prime/GP1t boost regimen represents an EBOV vaccine approach capable of establishing long-term protection, and therefore warrants further development. Copyright © 2017 Elsevier B.V. All rights reserved.

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

Science.gov (United States)

Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

Transcriptional profiling of human liver identifies sex-biased genes associated with polygenic dyslipidemia and coronary artery disease.

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Yijing Zhang

Full Text Available Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease. Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5 and LDLR; reduced expression of LIPC that is consistent with the lower female risk of coronary artery disease. Female-biased expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of 879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally, the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human hepatic sex differences are widespread and affect diverse cell

Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

Energy Technology Data Exchange (ETDEWEB)

Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

2016-08-05

Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

Science.gov (United States)

Kruse, Robert L; Shum, Thomas; Tashiro, Haruko; Barzi, Mercedes; Yi, Zhongzhen; Whitten-Bauer, Christina; Legras, Xavier; Bissig-Choisat, Beatrice; Garaigorta, Urtzi; Gottschalk, Stephen; Bissig, Karl-Dimiter

2018-04-06

Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

International Nuclear Information System (INIS)

Zhang, Hongxia; Cui, Ruina; Guo, Xuejiang; Hu, Jiayue; Dai, Jiayin

2016-01-01

Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

Malignant phyllodes tumor of the breast with heterologous high-grade angiosarcoma

Directory of Open Access Journals (Sweden)

Ghassan Tranesh

2017-03-01

Full Text Available Phyllodes tumors (PTs account for <3% of fibroepithelial breast lesions and for 0.3% to 1.0% of primary breast tumors. They occur predominantly in middle-aged women (mean age range, 40–50 years. PTs can be categorized into benign, borderline, and malignant; the first 2 categories are distinguished only by degree of cellular atypia and mitotic activity. Malignant PTs are more frequent among persons of Hispanic ethnicity, especially those born in Central America or South America. Heterologous sarcomatous elements may be present in malignant PTs, predominantly liposarcoma and rarely fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma, and chondrosarcoma. Breast angiosarcoma (BA is a rare heterologous, sarcomatous element that may arise secondary to malignant PT. We report a 47-year-old woman with no history of previous surgery or radiation therapy who presented to the emergency department with a painful right breast mass. She admittedly noticed the right breast mass for many years; however, recently it increased in size. Mammography and ultrasonography identified a partially cystic mass. Core needle biopsy showed dense hyalinized fibrous tissue with old blood clots, suggestive of infarcted fibroadenoma. The patient received antibiotics and analgesics; however, she reported intractable pain and a worsening skin rash of her right breast. Chest computed tomography and magnetic resonance imaging showed a doubling in mass size, with pectoralis major muscle involvement. Incisional biopsy showed malignant PT with heterologous high-grade angiosarcoma. The diagnosis of angiosarcoma was confirmed through immunoreactivity for CD31, FLI1, and ERG immunostains.

Merlin, the product of NF2 gene, is associated with aromatase expression and estrogen formation in human liver tissues and liver cancer cells.

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Cocciadiferro, Letizia; Miceli, Vitale; Granata, Orazia M; Carruba, Giuseppe

2017-09-01

The product of neurofibromatosis type 2 (NF2) gene, also known as Merlin/neurofibromin 2, homeostatically regulates liver stem cells by controlling abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Hippo pathway. We have reported that locally elevated estrogen formation, driven by abnormally high expression and function of aromatase, may be implicated in development and progression of human hepatocellular carcinoma (HCC) through activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR. We have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development of major human tumors (liver, breast, prostate) and other chronic diseases (diabetes, obesity, Alzheimer's and heart disease). In this study, we investigated NF2 expression in liver cancer cells and tissues in relation to aromatase expression/function, estrogen receptor (ER) status and amphiregulin. Our data indicate that NF2 expression is associated with aromatase and AREG expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro. Furthermore, incubation with estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in HCC cell lines. Based on the above combined evidence, we hypothesize that NF2 behaves as a protein sensing tissue damage and aromatase-driven local estrogen formation

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

Science.gov (United States)

Katoch, Meenu; Mazmouz, Rabia; Chau, Rocky; Pearson, Leanne A.; Pickford, Russell

2016-01-01

ABSTRACT Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens. PMID:27520810

Lack of cilia and differentiation defects in the liver of human foetuses with the Meckel syndrome.

Science.gov (United States)

Clotman, Frédéric; Libbrecht, Louis; Killingsworth, Murray C; Loo, Christine C K; Roskams, Tania; Lemaigre, Frédéric P

2008-03-01

Meckel syndrome is an autosomal-recessive disease characterized by a combination of renal cysts, anomalies of the central nervous system, polydactyly and ductal plate malformations (DPM), which are hepatic anomalies consisting of excessive and abnormal foetal biliary structures. Among the genomic loci associated with Meckel syndrome, mutations in four genes were recently identified. These genes code for proteins associated with primary cilia and are possibly involved in cell differentiation. The aim of the present work was to investigate the formation of the primary cilia and the differentiation of the hepatic cells in foetuses with Meckel syndrome. Sections of livers from human foetuses with Meckel syndrome were analysed by immunofluorescence, immunohistochemistry and electron microscopy. The primary cilia of the biliary cells were absent in some Meckel foetuses, but were present in others. In addition, defects in hepatic differentiation were observed in Meckel livers, as evidenced by the presence of hybrid cells co-expressing hepatocytic and biliary markers. Defects in cilia formation occur in some Meckel livers, and most cases show DPM associated with abnormal hepatic cell differentiation. Because differentiation precedes the formation of the cilia during liver development, we propose that defective differentiation may constitute the initial defect in the liver of Meckel syndrome foetuses.

The role of heterologous chloroplast sequence elements in transgene integration and expression.

Science.gov (United States)

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-04-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

Determination of trace elements in human liver biopsy samples by ICP-MS and TXRF: hepatic steatosis and nickel accumulation

Energy Technology Data Exchange (ETDEWEB)

Varga, Imre; Szoboszlai, Norbert [L. Eoetvoes University, Department of Inorganic and Analytical Chemistry, P.O. Box 32, Budapest (Hungary); Szebeni, Agnes [MI Central Hospital, Ultrasound Laboratory, Budapest (Hungary); Kovacs, Bela [Debrecen University - Centre of Agricultural Sciences, Department of Food Science and Quality Assurance, P.O. Box 36, Debrecen (Hungary)

2005-10-01

Human liver biopsy samples, collected from 52 individuals, were analysed by inductively coupled plasma-mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TXRF) spectrometry in a retrospective study (i.e. patient selection and liver biopsy were not for the purpose of element analysis). The freeze-dried samples (typically 0.5-2 mg dry weight) were digested in a laboratory microwave digestion system and solutions with a final volume of 1 mL were prepared. The concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Pb were determined by use of a Thermo Elemental X7 ICP-MS spectrometer. TXRF measurements were performed with an Atomika Extra IIA spectrometer. Yttrium was employed as an internal standard, prepared by dissolution of 5N-purity yttria (Y{sub 2}O{sub 3}) in our laboratory. The accuracy was tested by analysis of NIST 1577a Bovine Liver certified reference material. The concentrations of Fe, Cu, Zn, and Rb determined in human liver biopsy samples were in good agreement with data published by other authors. The distribution of nickel in the samples was surprisingly uneven - nickel concentrations ranged from 0.7 to 12 {mu}g g{sup -1} (dry weight) in 38 samples and in several samples were extremely high, 36-693 {mu}g g{sup -1}. Analysis of replicate procedural blanks and control measurements were performed to prevent misinterpretation of the data. For patients with steatosis (n=14) Ni concentrations were consistently high except for two who had levels close to those measured for the normal group. As far as we are aware no previous literature data are available on the association of steatosis with high concentration of nickel in human liver biopsies taken from living patients. (orig.)

Human hepatocytes loaded in 3D bioprinting generate mini-liver.

Science.gov (United States)

Zhong, Cheng; Xie, Hai-Yang; Zhou, Lin; Xu, Xiao; Zheng, Shu-Sen

2016-10-01

Because of an increasing discrepancy between the number of potential liver graft recipients and the number of organs available, scientists are trying to create artificial liver to mimic normal liver function and therefore, to support the patient's liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydrogel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. Hematoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evaluated. The survival time of the mice was also compared among the four groups. 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CYP1A2, CYP2C9, a-GST and UGT-2 were significantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the

Case study: an evaluation of the human relevance of the synthetic pyrethroid metofluthrin-induced liver tumors in rats based on mode of action.

Science.gov (United States)

Yamada, Tomoya; Uwagawa, Satoshi; Okuno, Yasuyoshi; Cohen, Samuel M; Kaneko, Hideo

2009-03-01

In recent years, mode of action (MOA) frameworks have been developed through the International Life Sciences Institute Risk Science Institute and the International Programme on Chemical Safety, including an evaluation of the human relevance of the animal MOA data. In the present paper, the MOA for rat liver tumors induced by Metofluthrin is first analyzed through this framework based on data from studies on Metofluthrin and information on related chemicals from the literature. The human relevance of the rat liver carcinogenic response is then discussed based upon the human relevance framework. Two-year treatment with high dose of Metofluthrin produced hepatocellular tumors in both sexes of the Wistar rats. Metofluthrin induced CYP2B (increased smooth endoplasmic reticulum), resulted in increased liver weights which were associated with centrilobular hepatocyte hypertrophy, and induction of increased hepatocellular DNA replications. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. Furthermore, CYP2B induction by Metofluthrin was shown to involve activation of the constitutive androstane receptor in rat hepatocytes. Based on the evidence, including a comparison with the results with another chemical, phenobarbital, acting by a similar MOA, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production

Directory of Open Access Journals (Sweden)

Paul N. Schwarz

2018-03-01

Full Text Available The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Phytanic acid alpha-oxidation: decarboxylation of 2-hydroxyphytanoyl-CoA to pristanic acid in human liver

NARCIS (Netherlands)

Verhoeven, N. M.; Wanders, R. J.; Schor, D. S.; Jansen, G. A.; Jakobs, C.

1997-01-01

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

Science.gov (United States)

Sarkar, Ujjal; Ravindra, Kodihalli C; Large, Emma; Young, Carissa L; Rivera-Burgos, Dinelia; Yu, Jiajie; Cirit, Murat; Hughes, David J; Wishnok, John S; Lauffenburger, Douglas A; Griffith, Linda G; Tannenbaum, Steven R

2017-07-01

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1 β , IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor- α , RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1 β , and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug

Long-term prognosis of fatty liver: risk of chronic liver disease and death

DEFF Research Database (Denmark)

Dam-Larsen, S.; Franzmann, M.; Andersen, I.B.

2004-01-01

BACKGROUND AND AIMS: Fatty liver is a common histological finding in human liver biopsy specimens. It affects 10-24% of the general population and is believed to be a marker of risk of later chronic liver disease. The present study examined the risk of development of cirrhotic liver disease...... and the risk of death in a cohort diagnosed with pure fatty liver without inflammation. METHODS: A total of 215 patients who had a liver biopsy performed during the period 1976-1987 were included in the study. The population consisted of 109 non-alcoholic and 106 alcoholic fatty liver patients. Median follow...... up time was 16.7 (0.2-21.9) years in the non-alcoholic and 9.2 (0.6-23.1) years in the alcoholic group. Systematic data collection was carried out by review of all medical records. All members of the study cohort were linked through their unique personal identification number to the National Registry...

Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

Science.gov (United States)

Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

2015-03-27

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

Quantification of human hepatic binding protein (HBP) via sup 99m Tc-galactosyl-neoglycoalbumin (NGA) liver scintigraphy

Energy Technology Data Exchange (ETDEWEB)

Virgolini, I; Hoebart, J; Bergmann, H; Sinzinger, H [Vienna Univ. (Austria). Abt. fuer Nuklearmedizin; Mueller, C [Vienna Univ. (Austria). 2. Klinik fuer Gastroenterologie und Hepatologie; Angelberger, P [Oesterreichisches Forschungszentrum Seibersdorf GmbH (Austria). Inst. fuer Chemie

1991-01-01

{sup 99m}Tc-galactosyl-neoglycoalbumin ({sup 99m}Tc-NGA) was synthesized by covalent coupling of 2-imino-2-methoxyethyl-1-thio-{beta}-D-galactopyranoside to the primary amino groups of human serum albumin. Injections of {sup 99m}Tc-NGA (150 MBq; 3.5 mg (=50 nmol)/ml) demonstrated the liver to be the exclusive site of tracer-uptake. Simulation of {sup 99m}Tc-NGA-kinetics allowed quantification of binding to the hepatic binding protein (HBP). Using this model we studied 250 patients with various liver disease. In alcoholic liver cirrhosis such patients with Child B and Child C stage cirrhosis had a lower HBP-concentration in the liver compared to control individuals. The group with the most advanced cirrhosis had a significantly lower HBP-concentration (0.20-0.45 {mu}mol/l) than Child A patients (0.60-0.85 {mu}mol/l; p<0.01) and Child B patients (0.45-0.60 {mu}mol/l; p<0.05). In patients with biopsy proven liver fibrosis (0.80-1.22 {mu}mol/l) no difference in receptor concentration to normal individuals was estimated. Patients with recently diagnosed acute viral hepatitis underwent repeated {sup 99m}Tc-galactosyl-neoglycoalbumin (NGA) scanning of the liver during the course of the disease. Return of liver function tests to normal values was associated with an increased hepatic imaging size as well as increase in HBP-concentration. In patients exhibiting a prolonged course of the disease changes in NGA-kinetic data were borderline and the hepatic image size unchanged. The values obtained for HBP-concentration in the liver amounted to 0.30-0.50 {mu}mol/l liver for patients with hepatoma, to 0.40-0.60 {mu}mol/l in patients with liver metastasis and to 0.90-1.20 {mu}mol/l in cancer patients without liver malignancy. It is concluded that scintigraphic evaluation of functional hepatic cell mass using the new receptor-tracer {sup 99m}Tc-NGA provides an in vivo diagnostic mean allowing quantitative data on liver function beside assessment of liver morphology.

Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

Science.gov (United States)

Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

2011-01-01

Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

Heterologous Prime-Boost Immunizations with a Virosomal and an Alphavirus Replicon Vaccine

NARCIS (Netherlands)

Walczak, Mateusz; de Mare, Arjan; Riezebos-Brilman, Annelies; Regts, Joke; Hoogeboom, Baukje-Nynke; Visser, Jeroen T.; Fiedler, Marc; Jansen-Duerr, Pidder; van der Zee, Ate G. J.; Nijman, Hans W.; Wilschut, Jan; Daemen, Toos

2011-01-01

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of

Exacerbating effects of human parvovirus B19 NS1 on liver fibrosis in NZB/W F1 mice.

Directory of Open Access Journals (Sweden)

Tsai-Ching Hsu

Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19 is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling.

Exacerbating Effects of Human Parvovirus B19 NS1 on Liver Fibrosis in NZB/W F1 Mice

Science.gov (United States)

Hsu, Tsai-Ching; Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Tzang, Bor-Show

2013-01-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling. PMID:23840852

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity.

Science.gov (United States)

Caira, Simonetta; Iannelli, Antonio; Sciarrillo, Rosaria; Picariello, Gianluca; Renzone, Giovanni; Scaloni, Andrea; Addeo, Pietro

2017-12-01

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

Heterogeneity of ductular reactions in adult rat and human liver revealed by novel expression of deleted in malignant brain tumor 1

DEFF Research Database (Denmark)

Bisgaard, H.C.; Holmskov, U.; Santoni-Rugiu, E.

2002-01-01

The regenerative capacity of mammalian adult liver reflects the ability of a number of cell populations within the hepatic lineage to take action. Limited information is available regarding factors and mechanisms that determine the specific lineage level at which liver cells contribute to liver......), were specifically associated with the emergence of ductular (oval) cell populations in injured liver. Subsequent cloning and characterization of the rat DMBT1 homologue revealed a highly inducible expression in ductular reactions composed of transit-amplifying ductular (oval) cells, but not in ductular...... reactions after ligation of the common bile duct. In human liver diseases, DMBT1 was expressed in ductular reactions after infection with hepatitis B and acetaminophen intoxication, but not in primary biliary cirrhosis, primary sclerosing cholangitis, and obstruction of the large bile duct. The expression...

Liver diseases and aging : friends or foes?

NARCIS (Netherlands)

Sheedfar, Fareeba; Di Biase, Stefano; Koonen, Debby; Vinciguerra, Manlio

2013-01-01

The liver is the only internal human organ capable of natural regeneration of lost tissue, as little as 25% of a liver can regenerate into a whole liver. The process of aging predisposes to hepatic functional and structural impairment and metabolic risk. Therefore, understanding how aging could

Assessment of chimeric mice with humanized livers in new drug development: generation of pharmacokinetics, metabolism and toxicity data for selecting the final candidate compound.

Science.gov (United States)

Kamimura, Hidetaka; Ito, Satoshi

2016-01-01

1. Chimeric mice with humanized livers are expected to be a novel tool for new drug development. This review discusses four applications where these animals can be used efficiently to collect supportive data for selecting the best compound in the final stage of drug discovery. 2. The first application is selection of the final compound based on estimated pharmacokinetic parameters in humans. Since chimeric mouse livers are highly repopulated with human hepatocytes, hepatic clearance values in vivo could be used preferentially to estimate pharmacokinetic profiles for humans. 3. The second is prediction of human-specific or disproportionate metabolites. Chimeric mice reproduce human-specific metabolites of drugs under development to conform to ICH guidance M3(R2), except for compounds that were extensively eliminated by co-existing mouse hepatocytes. 4. The third is identifying metabolites with distinct pharmacokinetic profiles in humans. Slow metabolite elimination specifically in humans increases its exposure level, but if its elimination is faster in laboratory animals, the animal exposure level might not satisfy ICH guidance M3(R2). 5. Finally, two examples of reproducing acute liver toxicity in chimeric mice are introduced. Integrated pharmacokinetics, metabolism and toxicity information are expected to assist pharmaceutical scientists in selecting the best candidate compound in new drug development.

Iodine 123-17-iodoheptadecanoic acid for metabolic liver studies in humans

International Nuclear Information System (INIS)

Hoeck, A.S.; Spohr, G.; Schmitz, M.; Notohamiprodjo, G.; Porschen, R.; Vyska, K.; Freundlieb, C.; Shreeve, W.W.; Feinendegen, L.E.

1986-01-01

(17- 123 I)-Iodoheptadecanoic acid ([ 123 I]HA) was used for dynamic planar scintigraphy of the liver in normal individuals (control I), in patients without liver disease but with elevated serum cholesterol and/or triglycerides (control II), and in patient groups with alcohol-induced fatty liver (PG I), fatty liver not due to alcohol (PG II), alcohol-induced liver cirrhosis (PG III), or liver cirrhosis of the posthepatitic type (PG IV). Tracer uptake and elimination time were assayed in different liver regions; mean elimination time was expressed for total liver. In control I, tracer uptake was homogeneous, and mean elimination time was 20.7 +/- 5.3 min without significant local variations. In control II, tracer uptake was reduced but homogeneous and mean elimination time was 59.4 +/- 35.8 min with some local variations. In PG I, uptake was reduced and inhomogeneous and elimination time was the same as in control I, irrespective of cholesterol and triglyceride values. In PG II, uptake was the same as in PG I but mean elimination time was 48 +/- 8.1 min with some local variations. In PG III, uptake was extremely reduced and spotty and elimination time correlated with the severity of disease from 19 to 881 min in different liver regions

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

Science.gov (United States)

Naldi, Marina; Baldassarre, Maurizio; Nati, Marina; Laggetta, Maristella; Giannone, Ferdinando Antonino; Domenicali, Marco; Bernardi, Mauro; Caraceni, Paolo; Bertucci, Carlo

2015-08-10

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

Energy Technology Data Exchange (ETDEWEB)

Watanabe, Yoko, E-mail: y-watanabe@nichiyaku.ac.jp [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Kojima, Hiroyuki; Takeuchi, Shinji [Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819 (Japan); Uramaru, Naoto [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Sanoh, Seigo [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Sugihara, Kazumi [Faculty of Pharmaceutical Science, Hiroshima International University, Koshingai 5-1-1, Kure, Hiroshima 737-0112 (Japan); Kitamura, Shigeyuki [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Ohta, Shigeru [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan)

2015-01-15

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes. - Highlights: • Metabolic modification of the endocrine-disrupting activity of BP-3 was examined. • 2,4,5-TriOH BP and 3-OH BP-3 were identified as new BP-3 metabolites. • 2,4-DiOH BP and 2,3,4-triOH BP exhibited high or similar estrogenic activities. • Estrogenic activity of BP-3 was enhanced by incubation with rat liver

Human liver segments: role of cryptic liver lobes and vascular physiology in the development of liver veins and left-right asymmetry

NARCIS (Netherlands)

Hikspoors, Jill P. J. M.; Peeters, Mathijs M. J. P.; Kruepunga, Nutmethee; Mekonen, Hayelom K.; Mommen, Greet M. C.; Köhler, S. Eleonore; Lamers, Wouter H.

2017-01-01

Couinaud based his well-known subdivision of the liver into (surgical) segments on the branching order of portal veins and the location of hepatic veins. However, both segment boundaries and number remain controversial due to an incomplete understanding of the role of liver lobes and vascular

Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI

Directory of Open Access Journals (Sweden)

Victoria Kegel

2015-01-01

Full Text Available Drug induced liver injury (DILI is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2±0.9×106 cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay and cell activity (XTT assay. The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production.

Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI

Science.gov (United States)

Kegel, Victoria; Pfeiffer, Elisa; Burkhardt, Britta; Liu, Jia L.; Zeilinger, Katrin; Nüssler, Andreas K.; Seehofer, Daniel; Damm, Georg

2015-01-01

Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 106 cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production. PMID:26491234

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

Directory of Open Access Journals (Sweden)

Walchli John

2009-04-01

Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

Science.gov (United States)

Yin, Shouliang; Li, Zilong; Wang, Xuefeng; Wang, Huizhuan; Jia, Xiaole; Ai, Guomin; Bai, Zishang; Shi, Mingxin; Yuan, Fang; Liu, Tiejun; Wang, Weishan; Yang, Keqian

2016-12-01

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

Long-Lasting Effects of BCG Vaccination on Both Heterologous Th1/Th17 Responses and Innate Trained Immunity

DEFF Research Database (Denmark)

Kleinnijenhuis, Johanneke; Quintin, Jessica; Preijers, Frank

2013-01-01

'. In the present study we assessed whether BCG was able to induce long-lasting effects on both trained immunity and heterologous T helper 1 (Th1) and Th17 immune responses 1 year after vaccination. The production of TNFα and IL-1β to mycobacteria or unrelated pathogens was higher after 2 weeks and 3 months...... in proinflammatory cytokine production after stimulation with the TLR4 ligand lipopolysaccharide. The heterologous production of Th1 (IFN-γ) and Th17 (IL-17 and IL-22) immune responses to nonmycobacterial stimulation remained strongly elevated even 1 year after BCG vaccination. In conclusion, BCG induces sustained...... changes in the immune system associated with a nonspecific response to infections both at the level of innate trained immunity and at the level of heterologous Th1/Th17 responses. © 2013 S. Karger AG, Basel....

Adaptation in the innate immune system and heterologous innate immunity.

Science.gov (United States)

Martin, Stefan F

2014-11-01

The innate immune system recognizes deviation from homeostasis caused by infectious or non-infectious assaults. The threshold for its activation seems to be established by a calibration process that includes sensing of microbial molecular patterns from commensal bacteria and of endogenous signals. It is becoming increasingly clear that adaptive features, a hallmark of the adaptive immune system, can also be identified in the innate immune system. Such adaptations can result in the manifestation of a primed state of immune and tissue cells with a decreased activation threshold. This keeps the system poised to react quickly. Moreover, the fact that the innate immune system recognizes a wide variety of danger signals via pattern recognition receptors that often activate the same signaling pathways allows for heterologous innate immune stimulation. This implies that, for example, the innate immune response to an infection can be modified by co-infections or other innate stimuli. This "design feature" of the innate immune system has many implications for our understanding of individual susceptibility to diseases or responsiveness to therapies and vaccinations. In this article, adaptive features of the innate immune system as well as heterologous innate immunity and their implications are discussed.

A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination.

Science.gov (United States)

León-Ortiz, Ana María; Panier, Stephanie; Sarek, Grzegorz; Vannier, Jean-Baptiste; Patel, Harshil; Campbell, Peter J; Boulton, Simon J

2018-01-18

Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The classification of secondary colorectal liver cancer in human biopsy samples using angular dispersive x-ray diffraction and multivariate analysis

International Nuclear Information System (INIS)

Theodorakou, Chrysoula; Farquharson, Michael J

2009-01-01

The motivation behind this study is to assess whether angular dispersive x-ray diffraction (ADXRD) data, processed using multivariate analysis techniques, can be used for classifying secondary colorectal liver cancer tissue and normal surrounding liver tissue in human liver biopsy samples. The ADXRD profiles from a total of 60 samples of normal liver tissue and colorectal liver metastases were measured using a synchrotron radiation source. The data were analysed for 56 samples using nonlinear peak-fitting software. Four peaks were fitted to all of the ADXRD profiles, and the amplitude, area, amplitude and area ratios for three of the four peaks were calculated and used for the statistical and multivariate analysis. The statistical analysis showed that there are significant differences between all the peak-fitting parameters and ratios between the normal and the diseased tissue groups. The technique of soft independent modelling of class analogy (SIMCA) was used to classify normal liver tissue and colorectal liver metastases resulting in 67% of the normal tissue samples and 60% of the secondary colorectal liver tissue samples being classified correctly. This study has shown that the ADXRD data of normal and secondary colorectal liver cancer are statistically different and x-ray diffraction data analysed using multivariate analysis have the potential to be used as a method of tissue classification.

Partial protection of SIV-infected rhesus monkeys against superinfection with a heterologous SIV isolate

Energy Technology Data Exchange (ETDEWEB)

Korber, Bette [Los Alamos National Laboratory

2009-01-01

Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virus for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T-cell count, CD4(+) memory T-cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4(+) T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.

Effect of short-term carbohydrate overfeeding and long-term weight loss on liver fat in overweight humans.

Science.gov (United States)

Sevastianova, Ksenia; Santos, Alexandre; Kotronen, Anna; Hakkarainen, Antti; Makkonen, Janne; Silander, Kaisa; Peltonen, Markku; Romeo, Stefano; Lundbom, Jesper; Lundbom, Nina; Olkkonen, Vesa M; Gylling, Helena; Fielding, Barbara A; Rissanen, Aila; Yki-Järvinen, Hannele

2012-10-01

Cross-sectional studies have identified a high intake of simple sugars as an important dietary factor predicting nonalcoholic fatty liver disease (NAFLD). We examined whether overfeeding overweight subjects with simple sugars increases liver fat and de novo lipogenesis (DNL) and whether this is reversible by weight loss. Sixteen subjects [BMI (kg/m²): 30.6 ± 1.2] were placed on a hypercaloric diet (>1000 kcal simple carbohydrates/d) for 3 wk and, thereafter, on a hypocaloric diet for 6 mo. The subjects were genotyped for rs739409 in the PNPLA3 gene. Before and after overfeeding and after hypocaloric diet, metabolic variables and liver fat (measured by proton magnetic resonance spectroscopy) were measured. The ratio of palmitate (16:0) to linoleate (18:2n-6) in serum and VLDL triglycerides was used as an index of DNL. Carbohydrate overfeeding increased weight (±SEM) by 2% (1.8 ± 0.3 kg; P fat by 27% from 9.2 ± 1.9% to 11.7 ± 1.9% (P = 0.005). DNL increased in proportion to the increase in liver fat and serum triglycerides in subjects with PNPLA3-148IIbut not PNPLA3-148MM. During the hypocaloric diet, the subjects lost 4% of their weight (3.2 ± 0.6 kg; P fat content (from 11.7 ± 1.9% to 8.8 ± 1.8%; P Carbohydrate overfeeding for 3 wk induced a >10-fold greater relative change in liver fat (27%) than in body weight (2%). The increase in liver fat was proportional to that in DNL. Weight loss restores liver fat to normal. These data indicate that the human fatty liver avidly accumulates fat during carbohydrate overfeeding and support a role for DNL in the pathogenesis of NAFLD. This trial was registered at www.hus.fi as 235780.

Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice

Directory of Open Access Journals (Sweden)

Shanzheng Lu

2016-10-01

Full Text Available Abstract Background The endometrial regenerative cell (ERC is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4–induced acute liver injury (ALI. Methods An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction. Results ERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT and aspartate aminotransferase (AST activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1β, IL-6, and TNF-α but higher level of IL-10 in both serum and liver. Conclusions Human ERCs protect the liver from acute injury

Organ slices as an in vitro test system for drug metabolism in human liver, lung and kidney

NARCIS (Netherlands)

Olinga, Peter; de Jager, M.H; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

1999-01-01

Metabolism of xenobiotics occurs mainly in the liver, but in addition, the lungs and kidneys may contribute considerably. The choice of the animal species during drug development as a predictive model for the human condition is often inadequate due to large interspecies differences. Therefore, a

From fresh heterologous oocyte donation to autologous oocyte banking.

Science.gov (United States)

Stoop, D

2012-01-01

Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock.

Detecting gallbladders in chicken livers using spectral analysis

DEFF Research Database (Denmark)

Jørgensen, Anders; Mølvig Jensen, Eigil; Moeslund, Thomas B.

2015-01-01

This paper presents a method for detecting gallbladders attached to chicken livers using spectral imaging. Gallbladders can contaminate good livers, making them unfit for human consumption. A data set consisting of chicken livers with and without gallbladders, has been captured using 33 wavelengths...

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

International Nuclear Information System (INIS)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2007-01-01

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are ∼ 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (∼ 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

NARCIS (Netherlands)

Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

2015-01-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential

Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

Directory of Open Access Journals (Sweden)

Ju-Hyun Kim

2017-05-01

Full Text Available Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP enzyme activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4’-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4’-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1, CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1, and CYP3A4-catalyzed midazolam 1’-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1 in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1’-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

Stereoselective sulfate conjugation of racemic 4-hydroxypropranolol by human and rat liver cytosol

Energy Technology Data Exchange (ETDEWEB)

Walle, T.; Walle, U.K. (Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston (USA))

1991-03-01

The objective of this study was to determine the stereochemistry of sulfoconjugation of a chiral phenolic amine drug, 4-hydroxypropranolol (HOP), by the human liver. The reaction was catalyzed by the 100,000 g cytosol as the phenolsulfotransferase (PST) enzyme source with PAP35S as the co-substrate. The enantiomers of the intact sulfate conjugate formed, (+)-HOP35S and (-)-HOP35S, were separated by HPLC and measured by liquid scintillation spectrometry. Complex velocity vs. substrate concentration curves were obtained with two peaks of activity, one at 3 microM (high affinity) and one at 500 microM (low affinity). The high-affinity reaction demonstrated a high degree of stereoselectivity. Whereas the affinity of the enantiomers for this reaction was identical, with a very low apparent KM value of 0.59 microM, the apparent Vmax value for (+)-HOPS formation was 4.6-fold higher than for (-)-HOPS. In sharp contrast, the low-affinity reaction, with an apparent KM of 65 microM, was not stereoselective. Inhibition of the high-affinity reaction by elevated temperature, but not by dichloronitrophenol, indicated that this activity was due to a monoamine form of PST. Inhibition of the low-affinity reaction by dichloronitrophenol, but not by elevated temperature, indicated that this activity was due to a phenol form of PST. As a comparison, experiments with the rat liver cytosol demonstrated only one activity, with apparent KM values of 50 microM for both enantiomers and opposite stereoselectivity in maximum velocity compared to humans, {plus minus}-HOPS ratio 0.72. The results of this study demonstrate stereoselectivity in human hepatic sulfation of a chiral phenolic amine, with clear differences between PST isoenzymes.

Evaluation of regeneration of liver function in pig model of auxiliary partial liver transplantation

International Nuclear Information System (INIS)

Li Jiaxin; Chen Xiaopeng; Rui Ging; Shong Qun; Chen Fangman; Lu Meijing; Chen Yongquan

2010-01-01

Objective: To establish a pig model of auxiliary partial liver transplantation and observe the liver function regeneration of host liver and graft. Methods: The portal vein providing for the host liver were gradually contracted; the donor hepatic veins were eng-to-side anastomosed to inferior vena cava in host caudal; graft was transplanted into the space under the host liver, part of receivers relieved portal vein angiography and color Doppler flow imaging was performed 3 days after surgery. Liver function of double livers in relievers was checked up, 3 days and 1 week after surgery respectively. Results: After surgery 10 relievers survived over 1 week, blood enzymology from hepatic vein of grafts 1 week after surgery were not ameliorative significantly compared with those 3 days after surgery (P > 0.05). Blood enzymology indexes from hepatic veins of grafts 1 week after surgery were were improved significantly compared with 3 days after surgery (P < 0.05). The graft did not reveal atrophic and gained favorable function. Conclusion: Favorable regeneration in the auxiliary partial liver transplantation model has achieved. Ideal foundation has been established for simulating and investigating human auxiliary liver transplantation. (authors)

Absolute Quantification of Human Liver Phosphorus-Containing Metabolites In Vivo Using an Inhomogeneous Spoiling Magnetic Field Gradient

Science.gov (United States)

Bashir, Adil; Gropler, Robert; Ackerman, Joseph

2015-01-01

Purpose Absolute concentrations of high-energy phosphorus (31P) metabolites in liver provide more important insight into physiologic status of liver disease compared to resonance integral ratios. A simple method for measuring absolute concentrations of 31P metabolites in human liver is described. The approach uses surface spoiling inhomogeneous magnetic field gradient to select signal from liver tissue. The technique avoids issues caused by respiratory motion, chemical shift dispersion associated with linear magnetic field gradients, and increased tissue heat deposition due to radiofrequency absorption, especially at high field strength. Methods A method to localize signal from liver was demonstrated using superficial and highly non-uniform magnetic field gradients, which eliminate signal(s) from surface tissue(s) located between the liver and RF coil. A double standard method was implemented to determine absolute 31P metabolite concentrations in vivo. 8 healthy individuals were examined in a 3 T MR scanner. Results Concentrations of metabolites measured in eight healthy individuals are: γ-adenosine triphosphate (ATP) = 2.44 ± 0.21 (mean ± sd) mmol/l of wet tissue volume, α-ATP = 3.2 ± 0.63 mmol/l, β-ATP = 2.98 ± 0.45 mmol/l, inorganic phosphates (Pi) = 1.87 ± 0.25 mmol/l, phosphodiesters (PDE) = 10.62 ± 2.20 mmol/l and phosphomonoesters (PME) = 2.12 ± 0.51 mmol/l. All are in good agreement with literature values. Conclusions The technique offers robust and fast means to localize signal from liver tissue, allows absolute metabolite concentration determination, and avoids problems associated with constant field gradient (linear field variation) localization methods. PMID:26633549

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Science.gov (United States)

Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

Liver slices in in vitro pharmacotoxicology with special reference to the use of human liver tissue

NARCIS (Netherlands)

Olinga, Peter; Meijer, D.K F; Slooff, M.JH; Groothuis, Geny

In the early years of research in in vitro pharmacotoxicology liver slices have been used. After a decline in the application of slices in favour of the use of isolated hepatocytes and the isolated perfused liver preparation, the development of the Krumdieck slicer in the 1980s led to a 'comeback'

Comparison of Tc-99m labeled liver and liver pate as markers for solid-phase gastric emptying

International Nuclear Information System (INIS)

Christian, P.E.; Moore, J.G.; Datz, F.L.

1984-01-01

A radionuclide marker for studies of solid-phase gastric emptying should have a high labeling efficiency and remain relatively stable during gastric emptying. The availability of materials and the ease of preparation are also considerations in selecting radionuclide markers. The stability of intracellularly labeled chicken liver, surface-labeled chicken liver, and labeled pureed meat (liver pate) incubated with hydrochloric acid solution or gastric juice have been compared. Intracellularly labeled chicken liver and labeled liver pate were also compared in gastric emptying studies in humans. In vitro results demonstrated labeling efficiencies greater than 92% for both intracellularly labeled liver and labeled liver pate. The pate labeled with Tc-99m sulfur colloid was more stable than Tc-99m surface-labeled liver in vitro and its prepartion was easier than with the intracellular labeling technique. Gastric emptying studies on normal subjects demonstrated equal performance of the intracellularly labeled liver and the labeled liver pate. Labeled liver pate is thus an alternative to intracellularly labeled chicken liver in measuring solid-phase gastric emptying

Nomenclature of the finer branches of the biliary tree : Canals, ductules, and ductular reactions in human livers

NARCIS (Netherlands)

Roskams, TA; Theise, ND; Balabaud, C; Bhagat, G; Bhathal, PS; Bioulac-Sage, P; Brunt, EM; Crawford, JM; Crosby, HA; Desmet, [No Value; Finegold, MJ; Geller, SA; Gouw, ASH; Hytiroglou, P; Knisely, AS; Kojiro, M; Letkowitch, JH; Nakanuma, Y; Olynyk, JK; Park, YN; Portmann, B; Saxena, R; Scheuer, PJ; Strain, AJ; Thung, SN; Wanless, IR; West, AB

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what

Proliferative human cell sources applied as biocomponent in bioartificial livers: a review

NARCIS (Netherlands)

Nibourg, Geert A. A.; Chamuleau, Robert A. F. M.; van Gulik, Thomas M.; Hoekstra, Ruurdtje

2012-01-01

Introduction: Bioartificial livers (BALs) are urgently needed to bridge severe liver failure patients to liver transplantation or liver regeneration. When based on primary hepatocytes, their efficacy has been shown in animal experiments and their safety was confirmed in clinical trials. However, a

Metabolism of ginger component [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human.

Science.gov (United States)

Chen, Huadong; Soroka, Dominique; Zhu, Yingdong; Sang, Shengmin

2013-05-01

There are limited data on the metabolism of [6]-shogaol (6S), a major bioactive component of ginger. This study demonstrates metabolism of 6S in liver microsomes from mouse, rat, dog, monkey, and human. The in vitro metabolism of 6S was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with 6S, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E,4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites, M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than 6S. We conclude that 6S is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning preclinical trials toward 6S chemoprevention. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function

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Phillips R

2006-04-01

Full Text Available Abstract Background Dendritic cells (DCs are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host's systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusion Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

DEFF Research Database (Denmark)

Jørgensen, Mikael Skaanning; Skovlund, Dominique Aubert; Johannesen, Pia Francke

2014-01-01

ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools...

In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis.

Science.gov (United States)

Zhang, Guo-Zun; Sun, Hui-Cong; Zheng, Li-Bo; Guo, Jin-Bo; Zhang, Xiao-Lan

2017-12-14

To investigate the hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and to evaluate their therapeutic effect on liver fibrosis/cirrhosis. A CCl 4 -induced liver fibrotic/cirrhotic rat model was used to assess the effect of hUC-MSCs. Histopathology was assessed by hematoxylin and eosin (H&E), Masson trichrome and Sirius red staining. The liver biochemical profile was measured using a Beckman Coulter analyzer. Expression analysis was performed using immunofluorescent staining, immunohistochemistry, Western blot, and real-time PCR. We demonstrated that the infused hUC-MSCs could differentiate into hepatocytes in vivo . Functionally, the transplantation of hUC-MSCs to CCl 4 -treated rats improved liver transaminases and synthetic function, reduced liver histopathology and reversed hepatobiliary fibrosis. The reversal of hepatobiliary fibrosis was likely due to the reduced activation state of hepatic stellate cells, decreased collagen deposition, and enhanced extracellular matrix remodeling via the up-regulation of MMP-13 and down-regulation of TIMP-1. Transplanted hUC-MSCs could differentiate into functional hepatocytes that improved both the biochemical and histopathologic changes in a CCl 4 -induced rat liver fibrosis model. hUC-MSCs may offer therapeutic opportunities for treating hepatobiliary diseases, including cirrhosis.

Liver bioengineering: Current status and future perspectives

Institute of Scientific and Technical Information of China (English)

Christopher Booth; Tom Soker; Pedro Baptista; Christina L Ross; Shay Soker; Umar Farooq; Robert J Stratta

2012-01-01

The present review aims to illustrate the strategies that are being implemented to regenerate or bioengineer livers for clinical purposes.There are two general pathways to liver bioengineering and regeneration.The first consists of creating a supporting scaffold,either synthetically or by decellularization of human or animal organs,and seeding cells on the scaffold,where they will mature either in bioreactors or in vivo.This strategy seems to offer the quickest route to clinical translation,as demonstrated by the development of liver organoids from rodent livers which were repopulated with organ specific cells of animal and/or human origin.Liver bioengineering has potential for transplantation and for toxicity testing during preclinical drug development.The second possibility is to induce liver regeneration of dead or resected tissue by manipulating cell pathways.In fact,it is well known that the liver has peculiar regenerative potential which allows hepatocyte hyperplasia after amputation of liver volume.Infusion of autologous bone marrow cells,which aids in liver regeneration,into patients was shown to be safe and to improve their clinical condition,but the specific cells responsible for liver regeneration have not yet been determined and the underlying mechanisms remain largely unknown.A complete understanding of the cell pathways and dynamics and of the functioning of liver stem cell niche is necessary for the clinical translation of regenerative medicine strategies.As well,it will be crucial to elucidate the mechanisms through which cells interact with the extracellular matrix,and how this latter supports and drives cell fate.

Critical concentrations of cadmium in human liver and kidney measured by prompt-gamma neutron activation

International Nuclear Information System (INIS)

Cohn, S.H.; Vartsky, D.; Yasumura, S.; Zanzi, I.; Ellis, K.J.

1979-01-01

Few data exist on Cd metabolism in human beings. In particular, data are needed on the role of parameters such as age, sex, weight, diet, smoking habits, and state of health. Prompt-gamma neutron activation analysis (PGNAA) provides the only currently available means for measuring in vivo levels of liver and kidney Cd. The method employs an 85 Ci, 235 Pu,Be neutron source and a gamma ray detection system consisting of two Ge(Li) detector. The dose delivered to the liver and left kidney is 666 mrem (detection limit is 1.4 μg/g Cd in the liver and 2.0 mg Cd for one kidney). Absolute levels of Cd in the kidney and concentrations of Cd in the liver were measured in vivo in twenty healthy adult males using 238 Pu,Be neutron sources. Organ Cd levels of smokers were significantly elevated above those of nonsmokers. Biological half-time for Cd in the body was estimated to be 15.7 yr. Cigarette smoking was estimated to result in the absorption of 1.9 μg of Cd per pack. No relationship was bound between body stores of Cd (liver and kidney) and Cd or β-microglobulin levels in urine and blood. Currently the above neutron activation facility is being mounted on a 34-ft mobile trailer unit. This unit will be used to monitor levels of Cd in industrial workers. It is anticipated that critically important data, particularly on industrially exposed workers, will provide a better basis for determining critical concentrations and for the setting or revision of standards for industrial and environmental Cd pollution

Multiparameter analysis of fall-out plutonium burdens in human liver

International Nuclear Information System (INIS)

Griffith, W.C.; Guilmette, R.A.

1991-01-01

The effect of multiple factors on Pu liver burdens is estimated for a group of 310 people who were selected to have relative uniform exposure to fall-out plutonium ( 239 Pu plus 240 Pu), based on age in 1952, the start of atmospheric testing of thermonuclear weapons, and based on residence history in eastern Colorado, where they died between 1975 and 1979. The data were analysed using multiple linear regression of the logarithm of the total liver plutonium burden on other available covariates. The results of the regression indicated that the liver burden was increased by 34% in very heavy smokers (100 pack year history) compared to non-smokers, decreased by 27% in females compared to males, decreased by 24% in people with a neoplasm in the liver compared to those without a neoplasm, and decreased by 64% in people with cirrhosis compared to those without cirrhosis. However, all of those parameters accounted for only 26% of the variability in liver burdens observed among these people, indicating that there remains a large unexplained variation. (author)

Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

Science.gov (United States)

Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

2015-11-01

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes

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Feng Li

2018-05-01

Full Text Available Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM using metabolomic approaches. In human liver microsomes (HLM, we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

Heterologous expression of cellobiohydrolases in filamentous fungi

DEFF Research Database (Denmark)

Zoglowek, Marta; Lübeck, Peter S.; Ahring, Birgitte K.

2015-01-01

Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well...... into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering...... promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation...

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

Science.gov (United States)

Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

2017-11-15

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human.

Science.gov (United States)

Sato, Masaya; Ikeda, Hitoshi; Uranbileg, Baasanjav; Kurano, Makoto; Saigusa, Daisuke; Aoki, Junken; Maki, Harufumi; Kudo, Hiroki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

2016-08-26

The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

The K Domain Mediates Homologous and Heterologous Interactions Between FLC and SVP Proteins of Brassica juncea

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Ma Guanpeng

2015-07-01

Full Text Available The transcription factors FLOWERING LOCUS C (FLC and SHORT VEGETATIVE PHASE (SVP can interact to form homologous and heterologous protein complexes that regulate flowering time in Brassica juncea Coss. (Mustard.Previous studies showed that protein interactions were mediated by the K domain, which contains the subdomains K1, K2 and K3. However, it remains unknown how the subdomains mediate the interactions between FLC and SVP. In the present study, we constructed several mutants of subdomains K1–K3 and investigated the mechanisms involved in the heterologous interaction of BjFLC/BjSVP and in the homologous interaction of BjFLC/BjFLC or BjSVP/BjSVP. Yeast two-hybrid and β-Galactosidase activity assays showed that the 19 amino acids of the K1 subdomain in BjSVP and the 17 amino acids of the K1 subdomain in BjFLC were functional subdomains that interact with each other to mediate hetero-dimerization. The heterologous interaction was enhanced by the K2 subdomain of BjSVP protein, but weakened by its interhelical domain L2. The heterologous interaction was also enhanced by the K2 subdomain of BjFLC protein, but weakened by its K3 subdomain. The homologous interaction of BjSVP was mediated by the full K-domain. However, the homologous interaction of BjFLC was regulated only by its K1 and weakened by its K2 and K3 subdomains. The results provided new insights into the interactions between FLC and SVP, which will be valuable for further studies on the molecular regulation mechanisms of the regulation of flowering time in B. juncea and other Brassicaceae.

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

Science.gov (United States)

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human.

Science.gov (United States)

Krauskopf, Julian; de Kok, Theo M; Schomaker, Shelli J; Gosink, Mark; Burt, Deborah A; Chandler, Patricia; Warner, Roscoe L; Johnson, Kent J; Caiment, Florian; Kleinjans, Jos C; Aubrecht, Jiri

2017-01-01

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic "liquid biopsies" enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as "liquid biopsies".

Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human.

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Julian Krauskopf

Full Text Available MicroRNAs (miRNAs released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic "liquid biopsies" enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as "liquid biopsies".

Noninvasive assessment of the rheological behavior of human organs using multifrequency MR elastography: a study of brain and liver viscoelasticity

Energy Technology Data Exchange (ETDEWEB)

Klatt, Dieter [Department of Radiology, Charite-Universitaetsmedizin Berlin, Campus Charite Mitte, Chariteplatz 1, 10117 Berlin (Germany); Hamhaber, Uwe [Institute of Medical Informatics, Charite-Universitaetsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin (Germany); Asbach, Patrick [Department of Radiology, Charite-Universitaetsmedizin Berlin, Campus Charite Mitte, Chariteplatz 1, 10117 Berlin (Germany); Braun, Juergen [Institute of Medical Informatics, Charite-Universitaetsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin (Germany); Sack, Ingolf [Department of Radiology, Charite-Universitaetsmedizin Berlin, Campus Charite Mitte, Chariteplatz 1, 10117 Berlin (Germany)

2007-12-21

MR elastography (MRE) enables the noninvasive determination of the viscoelastic behavior of human internal organs based on their response to oscillatory shear stress. An experiment was developed that combines multifrequency shear wave actuation with broad-band motion sensitization to extend the dynamic range of a single MRE examination. With this strategy, multiple wave images corresponding to different driving frequencies are simultaneously received and can be analyzed by evaluating the dispersion of the complex modulus over frequency. The technique was applied on the brain and liver of five healthy volunteers. Its repeatability was tested by four follow-up studies in each volunteer. Five standard rheological models (Maxwell, Voigt, Zener, Jeffreys and fractional Zener model) were assessed for their ability to reproduce the observed dispersion curves. The three-parameter Zener model was found to yield the most consistent results with two shear moduli {mu}{sub 1} = 0.84 {+-} 0.22 (1.36 {+-} 0.31) kPa, {mu}{sub 2} = 2.03 {+-} 0.19 (1.86 {+-} 0.34) kPa and one shear viscosity of {eta} = 6.7 {+-} 1.3 (5.5 {+-} 1.6) Pa s (interindividual mean {+-} SD) in brain (liver) experiments. Significant differences between the rheological parameters of brain and liver were found for {mu}{sub 1} and {eta} (P < 0.05), indicating that human brain is softer and possesses a higher viscosity than liver.

Noninvasive assessment of the rheological behavior of human organs using multifrequency MR elastography: a study of brain and liver viscoelasticity

International Nuclear Information System (INIS)

Klatt, Dieter; Hamhaber, Uwe; Asbach, Patrick; Braun, Juergen; Sack, Ingolf

2007-01-01

MR elastography (MRE) enables the noninvasive determination of the viscoelastic behavior of human internal organs based on their response to oscillatory shear stress. An experiment was developed that combines multifrequency shear wave actuation with broad-band motion sensitization to extend the dynamic range of a single MRE examination. With this strategy, multiple wave images corresponding to different driving frequencies are simultaneously received and can be analyzed by evaluating the dispersion of the complex modulus over frequency. The technique was applied on the brain and liver of five healthy volunteers. Its repeatability was tested by four follow-up studies in each volunteer. Five standard rheological models (Maxwell, Voigt, Zener, Jeffreys and fractional Zener model) were assessed for their ability to reproduce the observed dispersion curves. The three-parameter Zener model was found to yield the most consistent results with two shear moduli μ 1 = 0.84 ± 0.22 (1.36 ± 0.31) kPa, μ 2 = 2.03 ± 0.19 (1.86 ± 0.34) kPa and one shear viscosity of η = 6.7 ± 1.3 (5.5 ± 1.6) Pa s (interindividual mean ± SD) in brain (liver) experiments. Significant differences between the rheological parameters of brain and liver were found for μ 1 and η (P < 0.05), indicating that human brain is softer and possesses a higher viscosity than liver

Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

Science.gov (United States)

Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

2016-04-01

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

Remote assessment and guidance of liver harvesting for transplantation.

OpenAIRE

Eadie, L. H.

2005-01-01

The harvesting of livers for transplantation involves assessment of the liver's suitability, including an examination of the colour and general appearance of the liver. If the organ is to be split for transplantation into two recipients, the vasculature of the liver must be studied and recorded. Remote assessment of livers and telesurgical guidance could save time and money. This thesis highlights the importance of colour in liver diagnosis, using animal and human models to examine the colour...

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice

Science.gov (United States)

Vaughan, Ashley M.; Mikolajczak, Sebastian A.; Wilson, Elizabeth M.; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H.I.

2012-01-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase–deficient mouse (Fah–/–, Rag2–/–, Il2rg–/–, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS–to–blood-stage transition of a human malaria parasite. PMID:22996664

Two insulin-like growth factor I messenger RNAs are expressed in human liver

International Nuclear Information System (INIS)

Rotwein, P.

1986-01-01

Through use of a synthetic radiolabelled oligonucleotide probe, human insulin-like growth factor I (IGF-I) cDNA clones were isolated from a liver library. Two types of cDNAs were defined by restriction enzyme analysis and DNA sequencing. Both encode IGF-I precursors of either 195 or 153 amino acids. The two predicted protein precursors are identical from their amino terminus to a lysine residue 16 codons beyond the IGF-I sequence, and then they diverge. Both cDNAs predict additional unique carboxyl-terminal extension peptides. Since there is only one IGF-I gene in the human genome, the finding of two different cDNAs suggests that alternative RNA processing plays a role in IGF-I gene expression. The functions of the different extension peptides remain to be elucidates

TWEAK induces liver progenitor cell proliferation

Science.gov (United States)

Jakubowski, Aniela; Ambrose, Christine; Parr, Michael; Lincecum, John M.; Wang, Monica Z.; Zheng, Timothy S.; Browning, Beth; Michaelson, Jennifer S.; Baestcher, Manfred; Wang, Bruce; Bissell, D. Montgomery; Burkly, Linda C.

2005-01-01

Progenitor (“oval”) cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors. PMID:16110324

Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids

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Steven D. Forsythe

2018-04-01

Full Text Available IntroductionEnvironmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future.MethodsWe employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate, and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM–10 mM, mercury (200 nM–200 µM, thallium (10 nM–10 µM, or glyphosate (25 µM–25 mM for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity.ResultsAs expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures.Conclusion3D organoids have significant utility to be

Human Mesenchymal Stem Cell Transfusion Is Safe and Improves Liver Function in Acute-on-Chronic Liver Failure Patients

Science.gov (United States)

Shi, Ming; Zhang, Zheng; Xu, Ruonan; Lin, Hu; Fu, Junliang; Zou, Zhengsheng; Zhang, Aimin; Shi, Jianfei; Chen, Liming; Lv, Sa; He, Weiping; Geng, Hua; Jin, Lei; Liu, Zhenwen

2012-01-01

Acute-on-chronic liver failure (ACLF) is a severe, life-threatening complication, and new and efficient therapeutic strategies for liver failure are urgently needed. Mesenchymal stem cell (MSC) transfusions have been shown to reverse fulminant hepatic failure in mice and to improve liver function in patients with end-stage liver diseases. We assessed the safety and initial efficacy of umbilical cord-derived MSC (UC-MSC) transfusions for ACLF patients associated with hepatitis B virus (HBV) infection. A total of 43 ACLF patients were enrolled for this open-labeled and controlled study; 24 patients were treated with UC-MSCs, and 19 patients were treated with saline as controls. UC-MSC therapy was given three times at 4-week intervals. The liver function, adverse events, and survival rates were evaluated during the 48-week or 72-week follow-up period. No significant side effects were observed during the trial. The UC-MSC transfusions significantly increased the survival rates in ACLF patients; reduced the model for end-stage liver disease scores; increased serum albumin, cholinesterase, and prothrombin activity; and increased platelet counts. Serum total bilirubin and alanine aminotransferase levels were significantly decreased after the UC-MSC transfusions. UC-MSC transfusions are safe in the clinic and may serve as a novel therapeutic approach for HBV-associated ACLF patients. PMID:23197664

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

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Cyrill Géraud

Full Text Available Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ, i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

Liver Development, Regeneration, and Carcinogenesis

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Janet W. C. Kung

2010-01-01

Full Text Available The identification of putative liver stem cells has brought closer the previously separate fields of liver development, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bioartificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers.

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

Science.gov (United States)

Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

2014-10-01

Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Pathway to Personalizing Therapy for Metastases Using Liver-on-a-Chip Platforms.

Science.gov (United States)

Khazali, A S; Clark, A M; Wells, A

2017-06-01

Metastasis accounts for most cancer-related deaths. The majority of solid cancers, including those of the breast, colorectum, prostate and skin, metastasize at significant levels to the liver due to its hemodynamic as well as tumor permissive microenvironmental properties. As this occurs prior to detection and treatment of the primary tumor, we need to target liver metastases to improve patients' outcomes. Animal models, while proven to be useful in mechanistic studies, do not represent the heterogeneity of human population especially in drug metabolism lack proper human cell-cell interactions, and this gap between animals and humans results in costly and inefficient drug discovery. This underscores the need to accurately model the human liver for disease studies and drug development. Further, the occurrence of liver metastases is influenced by the primary tumor type, sex and race; thus, modeling these specific settings will facilitate the development of personalized/targeted medicine for each specific group. We have adapted such all-human 3D ex vivo hepatic microphysiological system (MPS) (a.k.a. liver-on-a-chip) to investigate human micrometastases. This review focuses on the sources of liver resident cells, especially the iPS cell-derived hepatocytes, and examines some of the advantages and disadvantages of these sources. In addition, this review also examines other potential challenges and limitations in modeling human liver.

A new liver function test using the asialoglycoprotein-receptor system on the liver cell membrane, 3

International Nuclear Information System (INIS)

Hazama, Hiroshi; Kawa, Soukichi; Kubota, Yoshitsugu

1986-01-01

We evaluated the vilidity of a new liver function test using liver scintigraphy based on the asialoglycoprotein (ASGP) receptor system on the liver cell membrane in rats with galactosamine-induced acute liver disorder and those with carbon tetra-chloride-induced chronic liver disorder. Neoglycoprotein (GHSA) produced by combining human serum albumin with 32 galactose units was labeled with 99m Tc and administered (50 μg/100 g body weight) to rats with acute or chronic liver disorder. Clearance curves were produced based on liver scintigrams and analysed using the two-compartment model to obtain parameters. In acute liver disorder, the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities correlated well to the increase in serum GOT and the decrease in the esterified to total cholesterol ratio (E/T ratio); in chronic liver disorder, they correlated significantly to the increase in the content of liver hydroxyproline (Hyp) which increased in proportion to the severity of liver fibrosis studied histologically, and to the decrease in the contents of cytochrome P-450 and cytochrome b 5 in liver microsomes. Significant correlation was observed between the prolongation of 99m Tc-GHSA clearance and the decrease in ASGP receptor activities in both acute and chronic liver disorders. These findings indicate that the measurement of 99m Tc-GHSA clearance can be a new liver function test sensitively reflecting the severity of liver damage. (author)

Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

International Nuclear Information System (INIS)

Berasain, Carmen; Latasa, María Ujue; Urtasun, Raquel; Goñi, Saioa; Elizalde, María; Garcia-Irigoyen, Oihane; Azcona, María; Prieto, Jesús; Ávila, Matías A.

2011-01-01

Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment

Assessment of possible carcinogenicity of oxyfluorfen to humans using mode of action analysis of rodent liver effects.

Science.gov (United States)

Stagg, Nicola J; LeBaron, Matthew J; Eisenbrandt, David L; Gollapudi, B Bhaskar; Klaunig, James E

2012-08-01

Oxyfluorfen is a herbicide that is not genotoxic and produces liver toxicity in rodents, following repeated administration at high dose levels. Lifetime rodent feeding studies reported in 1977 with low-purity oxyfluorfen (85%) showed no increase in any tumor type in rats (800 ppm, high dose) and only a marginally increased incidence of hepatocellular tumors in male CD-1 mice at the highest dose (200 ppm). To evaluate the potential carcinogenicity of the currently registered oxyfluorfen (> 98% purity), we conducted a series of short-term liver mode of action (MOA) toxicology studies in male CD-1 mice administered dietary doses of 0, 40, 200, 800, and 1600 ppm for durations of 3, 7, 10, or 28 days. MOA endpoints examined included liver weight, histopathology, cell proliferation, nuclear receptor-mediated gene expression, and other peroxisome proliferator-specific endpoints and their reversibility. Minimal liver effects were observed in mice administered doses at or below 200 ppm for up to 28 days. Increased liver weight, single-cell necrosis, cell proliferation, and peroxisomal acyl-CoA oxidase (ACO) were observed at 800 ppm after 28 days, but there was no increase in peroxisomes. Expression of Cyp2b10 and Cyp4a10 transcripts, markers of constitutive androstane receptor and peroxisome proliferator activated receptor α nuclear receptor activation, respectively, were increased at 800 and 1600 ppm after 3 or 10 days. Collectively, these data along with the negative genotoxicity demonstrate that oxyfluorfen (> 98% purity) has the potential to induce mouse liver tumors through a nongenotoxic, mitogenic MOA with a clear threshold and is not predicted to be carcinogenic in humans at relevant exposure levels.

Development of the liver during the fetal period

International Nuclear Information System (INIS)

Albay, S.; Malas, Mehmet A.; Cetin, E.; Cankara, N.; Karahan, N.

2005-01-01

To investigate the development of the liver in human fetuses aged between 9-40 weeks. We studied 121 human fetuses (62 males, 59 females) with no external anomalies aged between 9-40 postmenstrual weeks during 2003-2004 in Suleyman Demirel University, Isparta, Turkey. The fetuses were divided into four groups as 1st, 2nd and 3rd trimesters and full term fetuses. We measured fetal weight, length, width, thickness, and volume of the liver. We established localization of the liver and its relation with the neighboring structures, its ligaments, and size of itself and its lobes, shapes of the liver and the localization of the porta hepatis on the visceral surface of the liver. We found significant correlations between the size, weight, volume of the liver, sizes of its lobe and gestational age (p 0.05). However, the proportion of the distance between the porta hepatis and the upper margin to the distance between the porta hepatis and the lower margin decreased significantly with gestational age (p<0.05). Type 3 liver (square) was the most commonly observed type of fetal liver (53%). Our opinion is that the parameters obtained can be useful to diagnose pathologies of liver development and anomalies concerning several branches of medicine such as anatomy, pathologic anatomy (fetopathology), forensic medicine, medical imaging, obstetrics and pediatrics. (author)

Alteration in Haematological and Liver Function Indices during Human Infection with Fasciola spp. Post Treatment with Triclabendazole

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M.I. Edalatzadeh

2006-07-01

Full Text Available Introduction & Objective: Fascioliasis is a zoonotic parasitic disease, caused by the liver fluke, Fasciola spp.. Human is occasional host when ingesting the metacercaria by eating contaminated aquatic vegetable. In the two past decades, human fasciolasis was emerging as a problem of public health in the Guilan province; in Anzali city. Triclabendazole is a novel anti-helmenthic that during recent years has been used for fascioliasis treatment in this region. The aim of the present work is to study alteration in haematological and liver function indices during human infection with Fasciola spp. pre and post treatment with triclabendazoleMaterials & Methods: The present work is a longitudinal clinical trail. In this regard, fifty confirmed fasciolasis patients, were chosen for parasitological, hematological and biochemical examinations pre-therapy as well as 1 and 6 months post-therapy. Formalin-ether and modified Telemann methods were used for stool examination. For Fasciola antibody detection ELISA technique was employed. Hematological and biochemical tests were performed by standard methods. Results: Results indicated that, triclabendazole efficacy was 74% after usage as one dose of 20mg/kg and reached to 88% after repeating in the next month. Before triclabebdazole therapy the Hb and HCT of the patients were slightly found lower than normal ranges, meanwhile the ESR and eosinophil percentages were higher. However following receiving the drug, in the cured individuals, the indices returned to the normal ranges but in the non-cured individuals were not shifted to the normal. On the other hand liver function indices of the patients mostly were at normal ranges before and following drug therapy.Conclusion: In conclusion haematological indices could be valuable indicator for successful therapy of patients treated with triclabendazole.

Fetal liver blood flow distribution: role in human developmental strategy to prioritize fat deposition versus brain development.

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Keith M Godfrey

Full Text Available Among primates, human neonates have the largest brains but also the highest proportion of body fat. If placental nutrient supply is limited, the fetus faces a dilemma: should resources be allocated to brain growth, or to fat deposition for use as a potential postnatal energy reserve? We hypothesised that resolving this dilemma operates at the level of umbilical blood distribution entering the fetal liver. In 381 uncomplicated pregnancies in third trimester, we measured blood flow perfusing the fetal liver, or bypassing it via the ductus venosus to supply the brain and heart using ultrasound techniques. Across the range of fetal growth and independent of the mother's adiposity and parity, greater liver blood flow was associated with greater offspring fat mass measured by dual-energy X-ray absorptiometry, both in the infant at birth (r = 0.43, P<0.001 and at age 4 years (r = 0.16, P = 0.02. In contrast, smaller placentas less able to meet fetal demand for essential nutrients were associated with a brain-sparing flow pattern (r = 0.17, p = 0.02. This flow pattern was also associated with a higher degree of shunting through ductus venosus (P = 0.04. We propose that humans evolved a developmental strategy to prioritize nutrient allocation for prenatal fat deposition when the supply of conditionally essential nutrients requiring hepatic inter-conversion is limited, switching resource allocation to favour the brain if the supply of essential nutrients is limited. Facilitated placental transfer mechanisms for glucose and other nutrients evolved in environments less affluent than those now prevalent in developed populations, and we propose that in circumstances of maternal adiposity and nutrient excess these mechanisms now also lead to prenatal fat deposition. Prenatal developmental influences play important roles in the human propensity to deposit fat.

Purification and characterization of akr1b10 from human liver: role in carbonyl reduction of xenobiotics.

Science.gov (United States)

Martin, Hans-Jörg; Breyer-Pfaff, Ursula; Wsol, Vladimir; Venz, Simone; Block, Simone; Maser, Edmund

2006-03-01

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM-1 min-1 for dolasetron and 1.3, 0.43, and 0.47 mM-1 min-1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km=112 mM-1 min-1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

Hepatic mitochondrial function analysis using needle liver biopsy samples.

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Michael J J Chu

Full Text Available BACKGROUNDS AND AIM: Current assessment of pre-operative liver function relies upon biochemical blood tests and histology but these only indirectly measure liver function. Mitochondrial function (MF analysis allows direct measurement of cellular metabolic function and may provide an additional index of hepatic health. Conventional MF analysis requires substantial tissue samples (>100 mg obtained at open surgery. Here we report a method to assess MF using <3 mg of tissue obtained by a Tru-cut® biopsy needle making it suitable for percutaneous application. METHODS: An 18G Bard® Max-core® biopsy instrument was used to collect samples. The optimal Tru-cut® sample weight, stability in ice-cold University of Wisconsin solution, reproducibility and protocol utility was initially evaluated in Wistar rat livers then confirmed in human samples. MF was measured in saponin-permeabilized samples using high-resolution respirometry. RESULTS: The average mass of a single rat and human liver Tru-cut® biopsy was 5.60±0.30 and 5.16±0.15 mg, respectively (mean; standard error of mean. Two milligram of sample was found the lowest feasible mass for the MF assay. Tissue MF declined after 1 hour of cold storage. Six replicate measurements within rats and humans (n = 6 each showed low coefficient of variation (<10% in measurements of State-III respiration, electron transport chain (ETC capacity and respiratory control ratio (RCR. Ischemic rat and human liver samples consistently showed lower State-III respiration, ETC capacity and RCR, compared to normal perfused liver samples. CONCLUSION: Consistent measurement of liver MF and detection of derangement in a disease state was successfully demonstrated using less than half the tissue from a single Tru-cut® biopsy. Using this technique outpatient assessment of liver MF is now feasible, providing a new assay for the evaluation of hepatic function.

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen

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Tomáš Zárybnický

2018-06-01

Full Text Available Monoterpenes R-pulegone (PUL and R-menthofuran (MF, abundant in the Lamiaceae family, are frequently used in herb and food products. Although their hepatotoxicity was shown in rodent species, information about their effects in human liver has been limited. The aim of our study was to test the effects of PUL, MF and acetaminophen (APAP, as a reference compound on cell viability and microRNA (miRNA expression in human precision-cut liver slices. Slices from five patients were used to follow up on the inter-individual variability. PUL was toxic in all liver samples (the half-maximal effective concentration was 4.0 µg/mg of tissue, while MF and surprisingly APAP only in two and three liver samples, respectively. PUL also changed miRNA expression more significantly than MF and APAP. The most pronounced effect was a marked decrease of miR-155-5p expression caused by PUL even in non-toxic concentrations in all five liver samples. Our results showed that PUL is much more toxic than MF and APAP in human liver and that miR-155-5p could be a good marker of PUL early hepatotoxicity. Marked inter-individual variabilities in all our results demonstrate the high probability of significant differences in the hepatotoxicity of tested compounds among people.

Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

DEFF Research Database (Denmark)

Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

2014-01-01

Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as stre...

Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice.

Science.gov (United States)

Hsu, Tsai-Ching; Chiu, Chun-Ching; Chang, Shun-Chih; Chan, Hsu-Chin; Shi, Ya-Fang; Chen, Tzy-Yen; Tzang, Bor-Show

2016-01-01

Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1β and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection.

Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

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Porntip Pinlaor

Full Text Available The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and

Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

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Hui Xu

2015-01-01

Full Text Available The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA and Csac_1078 (celB from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA and TM0070 (xynB, resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

Mitochondrial alterations in children with chronic liver disease

African Journals Online (AJOL)

Rabah M. Shawky

chondrial function and structure in livers from humans with chronic liver disease ... ease, 2 with lipid storage disease, one with type I autoimmune hepatitis, one ..... a classification scheme for mitochondrial hepatopathies into primary and ...

Heterologous expression, purification and characterization of human β-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

Science.gov (United States)

Miyazaki, Takatsugu; Kato, Tatsuya; Park, Enoch Y

2018-02-03

β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Association of human cytomegalovirus viremia with human leukocyte antigens in liver transplantation recipients

Institute of Scientific and Technical Information of China (English)

Jianhua Hu; Jun Fan; Xueqin Meng; Hong Zhao; Xuan Zhang; Hainv Gao; Meifang Yang; Yadan Ma; Minhuan Li; Weihang Ma

2011-01-01

Human cytomegalovirus (HCMV) reactivation is a common complication after liver transplantation (LT).Here, we investigated whether human leukocyte antigen (HLA)-matching was related to HCMV infection and subsequent graft failure after LT for hepatitis B virus cirrhosis. This retrospective study reviewed 91 LT recipients.All the patients were grouped according to HLA-A, HLA-B, and HLA-DR locus matching. Clinical data were collected, including complete HLA-typing, HCMV viremia, graft failure, and the time of HCMV viremia.HLA typing was performed using a sequence-specific primer-polymerase chain reaction kit. HCMV was detected by pp65 antigenemia using a commercial kit.The incidence of HCMV infection post-LT was 81.32%.Graft failure was observed in 16 of 91 (17.6%) patients during the 4-year study. The incidence of HCMV viremia was 100% (5/5), 91.4% (32/35), and 72.5% (37/51) in HLA-A two locus, one locus, and zero locus compatibility,respectively. Nevertheless, the degree of the HLA-A,HLA-B, or HLA-DR match did not influence the time of HCMV viremia, graft failure, or the time of graft failure after a diagnosis of HCMV viremia (all P＞ 0.05). An interesting discovery was that the risk of HCMV viremia tended to be higher in patients with better HLA-A compatibility. Graft failure, time of HCMV viremia, and graft failure after a diagnosis of HCMV viremia appear to be independent of HLA allele compatibility.

Proteomic analysis of tyrosine phosphorylation during human liver transplantation

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Boutros Tarek

2007-01-01

Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

NARCIS (Netherlands)

Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

2008-01-01

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1¿-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

The risk of liver tumors in dogs and man from radioactive aerosols

International Nuclear Information System (INIS)

Muggenburg, B.A.; Boecker, B.B.; Hahn, F.F.; Griffith, W.G.; McClellan, R.O.

1986-01-01

Life-span studies in progress using beagle dogs that inhaled relatively soluble or relatively insoluble forms of radionuclides will provide information from which we may project the risk to humans for liver cancer from inhaled radioactive material. Twenty-two liver tumors have been observed in dogs exposed to beta-emitting radionuclides, mainly 144 Ce, and one liver tumor in a dog exposed to 238 Pu. Two liver cancers were also observed in control dogs. The risk of liver cancer in dogs that inhaled beta-emitting radionuclides was calculated to be 90 liver cancers per million rads. The risk of liver cancers in dogs in our studies and in studies at the University of Utah, when compared to the incidence of liver tumors in humans exposed to Thorotrast, suggest that the risk of liver cancer from an inhaled beta-emitting radionuclide in people is about 30 liver cancers per million person-rads. 19 refs., 3 tabs

Dynamics of the vascular profile of the finer branches of the biliary tree in normal and diseased human livers

NARCIS (Netherlands)

Gouw, Annette S. H.; van den Heuvel, Marius C.; Boot, Marcel; Slooff, Maarten J. H.; Poppema, Sibrand; de Jong, Koert P.

Background/Aims: Results of our previous studies supported the concept that in the human liver, the smallest ramification of the biliary tree, the bile ductules, might contain hepatic progenitor cells. An insufficient proliferative response and loss of bile ductules preceded bile duct loss whereas

Granulin Secreted by the Food-Borne Liver Fluke Opisthorchis viverrini Promotes Angiogenesis in Human Endothelial Cells

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Brandon Haugen

2018-02-01

Full Text Available The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke.

Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model

DEFF Research Database (Denmark)

Kermanizadeh, Ali; Løhr, Mille; Roursgaard, Martin

2014-01-01

BackgroundThe liver has a crucial role in metabolic homeostasis as well as being the principal detoxification centre of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated...... with accumulative production of nanomaterials, there is an urgent need to consider the possibility of detrimental health consequences of engineered NM exposure. It has been shown that exposure via inhalation, intratracheal instillation or ingestion can result in NM translocation to the liver. Traditional in vitro...... or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e. reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.).MethodsIn order to rectify these issues and for the very first time we have utilised a 3D human liver...

Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

International Nuclear Information System (INIS)

Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

2003-01-01

Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

Science.gov (United States)

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-01-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

DEFF Research Database (Denmark)

Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...... discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed...... a multi-omic physiological characterization including metabolic foot printing of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H-/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers...

Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

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Ebru YABAŞ

2017-08-01

Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

Heterologous expression of a rice metallothionein isoform (OsMTI-1b in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance

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Zahra Ansarypour

Full Text Available Abstract Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

Targeting a heterologous protein to multiple plant organelles via rationally designed 5? mRNA tags

NARCIS (Netherlands)

Voges, M.J.; Silver, P.A.; Way, J.C.; Mattozzi, M.D.

2013-01-01

Background Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments. Results We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in

Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

CSIR Research Space (South Africa)

Crampton, Michael C

2010-07-01

Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

A search for losses of chromium and other trace elements during lyophilization of human liver tissue

International Nuclear Information System (INIS)

Goeij, J.J.M. de; Volkers, K.J.; Tjioe, P.S.

1979-01-01

Human liver tissues were investigated for possible trace-element losses during lyophilization by comparison of concentrations of lyophilized and untreated (wet) samples. When destructive neutron activation analysis (n.a.a.) was used, no significant losses were observed for As, Br, Cd, Co, Cr, Cu, Fe, Hg, Mo, Sb, Se, and Zn. The advantages of n.a.a. over radio-tracer techniques for studies of trace-element volatility are discussed. (Auth.)

PROGRESSION OF LIVER FIBROSIS IN MONOINFECTED PATIENTS BY HEPATITIS C VIRUS AND COINFECTED BY HCV AND HUMAN IMMUNODEFICIENCY VIRUS

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Cristiane Valle TOVO

2013-03-01

Full Text Available Context The progression of liver fibrosis in patients coinfected by hepatitis C virus and human immunodeficiency virus (HCV/HIV has been increasingly studied in the past decade. Studies made before the highly active antiretroviral therapy suggest that HIV can change the natural history of the HCV infection, leading to a faster progression of the liver fibrosis. Objective To evaluate and compare the fibrosis progression in two groups of patients (HCV/HIV coinfected and HCV monoinfected Methods Seventy patients HCV monoinfected and 26 patients HCV/HIV coinfected who had not undertaken HCV treatment and were submitted to serial percutaneous liver biopsies were retrospectively evaluated. There was no difference in the fibrosis progression between the two groups. Conclusion The fibrosis grade evolution was not worse in the coinfected patients. The immunosuppression absence and the shortest time period between the biopsies in the coinfected group are possible explanations.

A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges.

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Nagarajan Vinod

Full Text Available Salmonella enteritidis and Salmonella typhimurium are important food-borne bacterial pathogens, which are responsible for diarrhea and gastroenteritis in humans and animals. In this study, S. typhimurium bacterial ghost (STG was generated based on minimum inhibitory concentration (MIC of sodium hydroxide (NaOH. Experimental studies performed using in vitro and in vivo experimental model systems to characterize effects of STG as a vaccine candidate. When compared with murine macrophages (RAW 264.7 exposed to PBS buffer (98.1%, the macrophages exposed to formalin-killed inactivated cells (FKC, live wild-type bacterial cells and NaOH-induced STG at 1 × 108 CFU/mL showed 85.6%, 66.5% and 84.6% cell viability, respectively. It suggests that STG significantly reduces the cytotoxic effect of wild-type bacterial cells. Furthermore, STG is an excellent inducer for mRNAs of pro-inflammatory cytokine (TNF-α, IL-1β and factor (iNOS, anti-inflammatory cytokine (IL-10 and dual activities (IL-6 in the stimulated macrophage cells. In vivo, STG vaccine induced humoral and cellular immune responses and protection against homologous and heterologous challenges in rats. Furthermore, the immunogenicity and protective efficacy of STG vaccine were compared with those of FKC and non-vaccinated PBS control groups. The vaccinated rats from STG group exhibited higher levels of serum IgG antibody responses, serum bactericidal antibodies, and CD4+ and CD8+ T-cell populations than those of the FKC and PBS control groups. Most importantly, after challenge with homologous and heterologous strains, the bacterial loads in the STG group were markedly lower than the FKC and PBS control groups. In conclusion, these findings suggest that the STG vaccine induces protective immunity against homologous and heterologous challenges.

Genomic instability induced by 60Co γ ray radiation in normal human liver cells

International Nuclear Information System (INIS)

Gen Xiaohua; Guo Xianhua; Zuo Yahui; Wang Xiaoli; Wang Zhongwen

2007-01-01

Objective: To explore the genomic instability induced by 60 Co γ rays. Methods: The cloning efficiency and micronucleus efficiency of normal human liver cell irradiated by 60 Co γ rays were detected, and the method of single cell gel electrophoresis (SCGE) was carried out to measure DNA chains damage. The fast-growing cells were divided into different dose-groups and then irradiated by 60 Co γ rays. After 40 populations doubling, the progenies were secondly irradiated with 2 Gy 60 Co γ rays. Results: The cloning efficiency decreased with the increase of doses after the initial irradiation. After the survival cells were given second irradiation, both results of SCGE and micronucleus frequency showed that the second damage was correlated with the original irradiation doses. Conclusions: 60 Co γ rays can not only induce the immediate biological effects in liver cells, but also lead to the genomic instability in the descendants that leads to an enhanced frequency of genetic changes occurring among the progeny of the original irradiated cell. The expanding effect of second event helps to study the genomic instability. (authors)

Induction of Morphological Changes in Human Embryo Liver Cells by the Pyrrolizidine Alkaloid Lasiocarpine

Science.gov (United States)

Armstrong, Sylvia J.; Zuckerman, A. J.; Bird, R. G.

1972-01-01

The pyrrolizidine alkaloids have been implicated in the aetiology of liver disease in man and in animals. Studies of the effects of lasiocarpine indicate that they have several and perhaps independent effects on human liver cells in culture. These may be summarized as follows: 1. Nuclear and nucleolar changes which are probably related to the alkylation of DNA and ensuing inhibition of nucleic acid and protein synthesis. 2. The induction of possible chromosomal damage and mutation. 3. A generalized reduction of the metabolic activities of the cells due to membrane and mitochondrial damage, and to alkylation and inactivation of cell enzymes and proteins. 4. A long-term inhibition of mitosis leading to the formation of giant cells (“megalocytes”). The morphological effects induced by a number of the pyrrolizidine alkaloids were very similar but the pattern of metabolic changes varied somewhat. It is believed that the hepatotoxic effects are not due to the pyrrolizidine alkaloids themselves but to metabolic derivatives formed by the cell. ImagesFigs. 3-5Figs. 1-2 PMID:5032090

Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

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Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

2013-01-01

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE. PMID:23555760

Disease modeling using human induced pluripotent stem cells: lessons from the liver.

Science.gov (United States)

Gieseck, Richard L; Colquhoun, Jennifer; Hannan, Nicholas R F

2015-01-01

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Cross-amplification of heterologous microsatellite markers in Rhamdia quelen and Leporinus elongatus

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Nelson Mauricio Lopera-Barrero

2016-02-01

Full Text Available Native fish species in Brazil are an asset in fish farming, but their natural stocks have been significantly reduced in recent years. To mitigate this negative impact, studies on fish conservation are being conducted and genetic tools for the discrimination of population parameters are increasingly achieving great importance. Current analysis evaluates a set of microsatellite heterologous primers in the jundiá (Rhamdia quelen and in the piapara (Leporinus elongatus. Samples from the caudal fin of 15 broodstock from each species were analyzed. DNA extraction was performed with NaCl protocol and the integrity of the extracted DNA was checked with agarose gel 1%. Twenty primers developed for Piaractus mesopotamicus, Colossoma macropomum, Prochilodus lineatus, Brycon opalinus and Oreochromis niloticus were evaluated. Cross amplification of four primers of the B. opalinus and P. lineatus species (BoM12, Pli43 and Pli60 in R. quelen and BoM2, Pli43 and Pli60 in L. elongatus was assessed. Primers of P. mesopotamicus, C. macropomum and O. niloticus showed no cross amplification in the two species analyzed. Results revealed the possibility of using the four amplified heterologous primers in genetic studies for R. quelen and L. elongatus.

Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

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Fabian Machens

2017-10-01

Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

Science.gov (United States)

Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

2014-11-01

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. © The

Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae

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Akira Yoshimi

2018-04-01

Full Text Available A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb. The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.

Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae.

Science.gov (United States)

Yoshimi, Akira; Yamaguchi, Sigenari; Fujioka, Tomonori; Kawai, Kiyoshi; Gomi, Katsuya; Machida, Masayuki; Abe, Keietsu

2018-01-01

A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata . It consists of 10 amino acid residues, including five N -methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae . The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR , were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae , gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata . Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae , although there may be unknown factors limiting productivity in this species.

Pyrroloquinoline-quinone suppresses liver fibrogenesis in mice.

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Dongwei Jia

Full Text Available Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injuries, and its progression toward cirrhosis is the major cause of liver-related morbidity and mortality worldwide. However, anti-fibrotic treatment remains an unconquered area for drug development. Accumulating evidence indicate that oxidative stress plays a critical role in liver fibrogenesis. In this study, we found that PQQ, a natural anti-oxidant present in a wide variety of human foods, exerted potent anti-fibrotic and ROS-scavenging activity in Balb/C mouse models of liver fibrosis. The antioxidant activity of PQQ was involved in the modulation of multiple steps during liver fibrogenesis, including chronic liver injury, hepatic inflammation, as well as activation of hepatic stellate cells and production of extracellular matrix. PQQ also suppressed the up-regulation of RACK1 in activated HSCs in vivo and in vitro. Our data suggest that PQQ suppresses oxidative stress and liver fibrogenesis in mice, and provide rationale for the clinical application of PQQ in the prevention and treatment of liver fibrosis.

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

NARCIS (Netherlands)

Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

2008-01-01

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

Energy Technology Data Exchange (ETDEWEB)

Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya, E-mail: inagaki@metab.kuhp.kyoto-u.ac.jp

2015-05-15

Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action.

DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

International Nuclear Information System (INIS)

Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya

2015-01-01

Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action