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Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell–cell and cell–matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.

Liver fibrosis is characterised by excessive deposition of extracellular matrix that interrupts normal liver functionality. It is a pathological stage in several untreated chronic liver diseases such as the iron overload syndrome hereditary haemochromatosis, viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and diabetes. Interestingly, regardless of the aetiology, iron-loading is frequently observed in chronic liver diseases. Excess iron can feed the Fenton reaction to generate unquenchable amounts of free radicals that cause grave cellular and tissue damage and thereby contribute to fibrosis. Moreover, excess iron can induce fibrosis-promoting signals in the parenchymal and non-parenchymal cells, which accelerate disease progression and exacerbate liver pathology. Fibrosis regression is achievable following treatment, but if untreated or unsuccessful, it can progress to the irreversible cirrhotic stage leading to organ failure and hepatocellular carcinoma, where resection or transplantation remain the only curative options. Therefore, understanding the role of iron in liver fibrosis is extremely essential as it can help in formulating iron-related diagnostic, prognostic and treatment strategies. These can be implemented in isolation or in combination with the current approaches to prepone detection, and halt or decelerate fibrosis progression before it reaches the irreparable stage. Thus, this review narrates the role of iron in liver fibrosis. It examines the underlying mechanisms by which excess iron can facilitate fibrotic responses. It describes the role of iron in various clinical pathologies and lastly, highlights the significance and potential of iron-related proteins in the diagnosis and therapeutics of liver fibrosis.

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR-Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.

Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis. We developed a data-analysis pipeline that identifies with high confidence somatic variants that accumulated in vivo in the original ASC. These genome-wide mutation catalogs are valuable resources for the characterization of the underlying mutational mechanisms. In addition, this protocol can be used to determine the effects of culture conditions or mutagen exposure on mutation accumulation in ASCs in vitro. Here, we describe a protocol for human liver ASCs that can be completed over a period of 3-4 months with hands-on time of â 1/45 d.

The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process.To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I- and N-) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis.We found that all I + treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions. (C) 2017 Elsevier Inc. All rights reserved.

Sucrose overfeeding increases intrahepatocellular (IHCL) and intramyocellular (IMCL) lipid concentrations in healthy subjects. We hypothesized that these effects would be modulated by diet protein/fat content. Twelve healthy men and women were studied on two occasions in a randomized, cross-over trial. On each occasion, they received a 3-day 12% protein weight maintenance diet (WM) followed by a 6-day hypercaloric high sucrose diet (150% energy requirements). On one occasion the hypercaloric diet contained 5% protein and 25% fat (low protein-high fat, LP-HF), on the other occasion it contained 20% protein and 10% fat (high protein-low fat, HP-LF). IHCL and IMCL concentrations (magnetic resonance spectroscopy) and energy expenditure (indirect calorimetry) were measured after WM, and again after HP-LF/LP-HF. IHCL increased from 25.0 &plusmn, 3.6 after WM to 147.1 &plusmn, 26.9 mmol/kg wet weight (ww) after LP-HF and from 30.3 &plusmn, 7.7 to 57.8 &plusmn, 14.8 after HP-LF (two-way ANOVA with interaction: p &lt, 0.001 overfeeding x protein/fat content). IMCL increased from 7.1 &plusmn, 0.6 to 8.8 &plusmn, 0.7 mmol/kg ww after LP-HF and from 6.2 &plusmn, 0.6 to 6.9 &plusmn, 0.6 after HP-LF, (p &lt, 0.002). These results indicate that liver and muscle fat deposition is enhanced when sucrose overfeeding is associated with a low protein, high fat diet compared to a high protein, low fat diet.

Quiescent hepatic stellate cells (HSCs), in response to liver injury, undergo characteristic morphological transformation into proliferative, contractile and ECM-producing myofibroblasts. In this study, we investigated the implication of canonical Wnt signaling pathway in HSCs and liver fibrogenesis. Canonical Wnt signaling pathway activation and inhibition using β-catenin/CBP inhibitor ICG001 was examined in-vitro in TGFβ-activated 3T3, LX2, primary human HSCs, and in-vivo in CCl4-induced acute liver injury mouse model. Fibroblasts-conditioned medium studies were performed to assess the Wnt-regulated paracrine factors involved in crosstalk between HSCs-macrophages and HSCs-endothelial cells. Canonical Wnt signaling pathway components were significantly up-regulated in-vitro and in-vivo. In-vitro, ICG-001 significantly inhibited fibrotic parameters, 3D-collagen contractility and wound healing. Conditioned medium induced fibroblasts-mediated macrophage and endothelial cells activation was significantly inhibited by ICG-001. In-vivo, ICG-001 significantly attenuated collagen accumulation and HSC activation. Interestingly, ICG-001 drastically inhibited macrophage infiltration, intrahepatic inflammation and angiogenesis. We further analyzed the paracrine factors involved in Wnt-mediated effects and found CXCL12 was significantly suppressed both in-vitro and in-vivo following Wnt inhibition. Wnt-regulated CXCL12 secretion from activated HSCs potentiated macrophage infiltration and activation, and angiogenesis. Pharmacological inhibition of canonical Wnt signaling pathway via suppression of stromal CXCL12 suggests a potential therapeutic approach targeting activated HSCs in liver fibrosis.

Tissue engineering with scaffolds to form transplantable organs is of wide interest. Decellularized tissues have been tested for this purpose, although supplies of healthy donor tissues, vascular recellularization for perfusion and tissue homeostasis in engineered organs pose challenges. We hypothesized decellularized human placenta will be suitable for tissue engineering. The universal availability and unique structures of placenta for accommodating tissue, including presence of embedded vessels, were major attractions. We found decellularized placental vessels were re-endothelialized by adjacent native cells and bridged vessel defects in rats. Also, implantation of liver fragments containing all cell types successfully hepatized placenta with maintenance of albumin and urea synthesis, as well as hepatobiliary transport of 99mTc-mebrofenin, over up to three days in vitro. After hepatized placenta containing autologous liver was transplanted into sheep, tissue units were well-perfused and self-assembled. Histological examination indicated transplanted tissue retained hepatic cord structures with characteristic hepatic organelles, such as gap junctions, and hepatic sinusoids lined by endothelial, Kupffer, and other cell types. Hepatocytes in this neo-organ expressed albumin and contained glycogen. Moreover, transplantation of hepatized placenta containing autologous tissue rescued sheep in extended partial hepatectomy-induced acute liver failure. This rescue concerned amelioration of injury and induction of regeneration in native liver. The grafted hepatized placenta was intact with healthy tissue that neither proliferated nor was otherwise altered. Conclusions: The unique anatomic structure and matrix of human placenta were effective for hepatic tissue engineering. This will advance applications ranging from biological studies, drug development and toxicology to therapies in people. This article is protected by copyright. All rights reserved.

Increasing evidence supports that modifications in the mitochondrial content, oxidative phosphorylation (OXPHOS) activity, and cell metabolism influence the fate of stem cells. However, the regulators involved in the crosstalk between mitochondria and stem cell fate remains poorly characterized. Here, we identified a transcriptional regulatory axis, composed of transcription factor 7-like 2 (TCF7L2) (a downstream effector of the Wnt/β-catenin pathway, repressed during differentiation) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) (the master regulator of mitochondrial biogenesis, induced during differentiation), coupling the loss of pluripotency and early commitment to differentiation, to the initiation of mitochondrial biogenesis and metabolic shift toward OXPHOS. PGC-1α induction during differentiation is required for both mitochondrial biogenesis and commitment to the hepatocytic lineage, and TCF7L2 repression is sufficient to increase PGC-1α expression, mitochondrial biogenesis and OXPHOS activity. We further demonstrate that OXPHOS activity is required for the differentiation toward the hepatocytic lineage, thus providing evidence that bi-directional interactions control stem cell differentiation and mitochondrial abundance and activity.

IFN-gamma is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-gamma reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-gamma-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-g by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common gamma-chain (gamma c) genes (Rag2(-/-) gamma c(-/-)), indicating their innate nature and their gc cytokine independence. Rag2(-/-) gamma c(-/-) mice are as resistant to Mycobacterium avium as Rag2(-/-) mice, whereas Rag2(-/-) mice lacking IFN-g are more susceptible than either Rag2(-/-) or Rag2(-/-) gamma c(-/-). These lineage-negative CD45(+)/Thy1.2(+) cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2(-/-) gamma c(-/-) animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-gamma-producing cells. Accordingly, Thy1.2specific mAb administration to infected Rag2(-/-) gamma c(-/-) animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2(+) non-NK innate-like cells present in the liver expresses IFN-g and can confer protection against M. avium infection in immunocompromised mice., Supported by a structured program on bioengineered therapies for infectious diseases and tissue regeneration (Grant NORTE-01-0145-FEDER-000012) supported by Norte Portugal Regional Operational Programme (NORTE 2020, under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund); by Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation, Portugal 2020; by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência (FCT), Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (Grant POCI-01-0145-FEDER-007274); by the Trudeau Institute, Inc.; and by FCT Grant SFRH/BD/89871/2012 (to M.R.), info:eu-repo/semantics/publishedVersion

UNLABELLED: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting. Immunocytochemistry and qPCR analyses on freshly isolated ALDH(+) cells reveal an enrichment in cells expressing liver stem cell markers such as EpCAM, CK19, CD133, and Sox9. In culture, the ALDH(+) population can give rise to functional hepatocyte-like cells as illustrated by albumin and urea secretion and cytochrome P450 activity. ALDH1A1 expression can be detected in canals of Hering and bile duct epithelial cells and is increased on liver injury. Finally, we showed that the isolation and differentiation toward hepatocyte-like cells of LPCs with high ALDH activity is also successfully applicable to human liver samples. CONCLUSION: High ALDH activity is a feature of LPCs that can be taken advantage of to isolate these cells from untreated mouse as well as human liver tissues. This novel protocol is practically relevant, because it provides an easy and nontoxic method to isolate liver stem cells from normal tissue for potential therapeutic purposes.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.

Genome-wide rhythms in RNA polymerase II loading and dynamic chromatin remodeling underlie periodic gene expression during diurnal cycles in the mouse liver., Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver., Author Summary In mammalian organs such as the liver, many metabolic and physiological processes occur preferentially at specific times during the 24-hour daily cycle. The timing of these rhythmic functions depends on a complex interplay between the endogenous circadian clock and environmental timing cues relayed through the master circadian clock in the suprachiasmatic nucleus, or via feeding rhythms. These rhythms can be implemented on several regulatory levels, and here we aimed at a better understanding of the transcriptional and epigenetic changes that regulate diurnal rhythms. We performed genome-wide analysis of the locations of RNA polymerase II (Pol II) and the epigenetic histone modifications H3K4me3 and H3K36me3 at specific times of day, relating these data to mRNA expression levels. Our analyses show that Pol II transcriptional rhythms are biphasic in mouse liver, having predominant peak activities in the morning and evening. Moreover, dynamic changes in histone marks lag transcription rhythms genome-wide, indicating that the epigenetic landscape can be remodeled during the 24-hour cycle. Finally, a quantitative analysis of temporal Pol II and mRNA accumulation profiles indicates that posttranscriptional regulation significantly contributes to the amplitude and phase of mRNA accumulation profiles. While many studies have analyzed how transcription and chromatin states are modified during irreversible cell differentiation processes, our work highlights how these states can evolve reversibly in a system exhibiting periodicity in time.

Obesity with insulin resistance and alcohol are the most frequent causes of steatohepatitis. This work investigates the contribution of bioactive TNF and Th1 type cytokines in a mouse model of steatohepatitis induced by FAT alone or FAT+EtOH and endotoxin. The extent of liver injury and cytokine activation induced by endotoxin in chronic FAT-fed mice, FAT+EtOH-fed mice, or mice fed standard chow were analyzed. Endotoxin administration to either FAT-fed or FAT+EtOH-fed mice increased serum ALT and AST compared to standard chow mice. Immunoreactive TNF was strongly activated by LPS in FAT-fed and FAT+EtOH-fed mice which presented the highest levels, but low levels were found in standard chow mice. In contrast, bioactive TNF was only present in serum of FAT-fed and in particular the highest levels were found in FAT+EtOH-fed mice. Moreover, soluble TNFR2 but not TNFR1 was found in lower amounts in serum of FAT+EtOH-fed mice compared to FAT-fed mice. Steatohepatitis was associated with increased IL-6, IFN-gamma, and iNOS mRNA and proteins. Data show that a moderately FAT diet and low-dose EtOH concur to generate steatohepatitis and TNF liver expression after LPS. In this model, changes in the regulation of TNF are associated with increased expression of IL-6, IFN-gamma, and iNOS.

We have amplified a (CA)n:(GT)n microsatellite from the TNF promoters of a panel of mouse strains using the polymerase chain reaction. The length of the microsatellites was polymorphic, with eight alleles observed among 15 inbred strains bearing seven distinct H-2 haplotypes, and four outbred strains. In B10 congenic strains, the TNF allele detected by microsatellite polymorphism segregated with the MHC, and in recombinant haplotypes (NOD, NZW), it segregated with H-2D. The TNF allele found in the NZW strain (H-2z) was distinct from those of all other haplotypes, consistent with the hypothesis that this strain may carry a genetic defect in TNF production.

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver. Bos C, Delmas Y, Desmouliere A, Solanilla A, Hauger O, Grosset C, Dubus I, Ivanovic Z, Rosenbaum J, Charbord P, Combe C, Bulte JW, Moonen CT, Ripoche J, Grenier N. Purpose To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs). Materials and Methods This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically. Results SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48h with 50μg of iron per milliliter was 50sec (−1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability ( P >27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100μg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs. Conclusions MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver. [Abstract reproduced by permission of Radiology 2004;233:781–9]

The effects of agents that could manipulate the lysosomal calcium such as phorbol myristate acetate, ionophore A23187, and phentolamine on the lysosomal glycogen degradation were studied by electron microscopy, morphometric analysis, and biochemical assays in newborn rat hepatocytes. Phorbol myristate acetate, which promotes the input of calcium to lysosomes, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid alpha 1,4 glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles and also decreased the activity of acid mannose 6-phosphatase. Ionophore A23187, which releases lysosomal calcium, produced opposite results in these enzyme activities. Phentolamine, an alpha-adrenergic blocking agent which interferes with the generation of phosphoinositides and may activate the lysosomal calcium uptake pump, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles. The results of this study constitute evidence that changes in lysosomal calcium may influence certain aspects of autophagy, including the degradation of glycogen inside the autophagic vacuoles. They also support our previous postulate [Kalamidas and Kotoulas (2000a,b) Histol Histopathol 15:29–35, 1011–1018] that stimulation of autophagic mechanisms in newborn rat hepatocytes may be associated with acid mannose 6-phosphatase activity-deficient lysosomes. Microsc. Res. Tech. 57:507–511, 2002. © 2002 Wiley-Liss, Inc.

Purpose: Hepatocyte transplantation (HcTX) has been investigated for many years as an alternative therapy to orthotopic liver transplantation to treat hepatic congenital enzymatic deficiency disease. The animal model most used is the Gunn rat, which presents a hyperbilirubinemia caused by the lack of uridine-diphosphate-glucuronyl-transferase. Some investigators have clearly described a hepatotrophic effect mediated by islets of Langerhans (IL) when transplanted with hepatocytes (Hc). In this study, the functional effect of cotransplanted IL on hepatocytes (co-HcTX) in Gunn rats in an isograft model is assessed. Methods: Two groups are compared: group 1 (n = 6), HcTX to group 2 (n = 6), co-HcTX. Cells isolated by enzymatic digestion are transplanted directly into the splenic parenchyma. Blood samples are taken regularly until day 100 to measure the unconjugated bilirubin (UB). Histological examination of the spleen is performed at the end of the experiment. Results: Both groups show a significant decrease of the UB: group 1, 47%; group 2, 65%. The decrease is statistically more pronounced with co-HcTX. The histological analysis shows a trophic effect of the IL on the grafted hepatocytes in the co-HcTX group. Conclusions: The HcTX and the co-HcTX correct partially the hyperbilirubinemia of the Gunn rat. A functional assessment has been performed to evaluate the effect of cotransplanted IL on HcTX.

OBJECTIVES: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis. DESIGN AND METHODS: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/ sample were analyzed by transmission electron microscopy. RESULTS: Apoptotic cells were observed (mmol/L EtOH): 40: 6 +/-0.5%, 60:13 +/- 2% (p < 0.05), 80: 26 +/- 1% (p < 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 +/- 3% (p < 0.0001 vs. control and p < 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 (p < 0.05). CONCLUSIONS: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure.

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.

Acute hepatic insufficiency (AHF) is one of the major challenges of intensive care medicine. Liver transplantation is the current solution to unsuccessful medical management. Owing to the lack of organ donors, other methods such as hepatocyte transplantation (HcTX) and bioartificial livers need to be explored. The aim of our experimental study is to evaluate the effect of hepatocyte transplantation on the survival in AHF animals intoxicated with D-Galactosamine (D-Gal).The first step consists of the determination of the dose of D-Gal needed to induce at least 80% mortality between 48 and 72 hours. Two groups of a single strain of male Wistar rats are then compared, one being intoxicated with D-Gal (control group), the other receiving and HcTX in the splenic parenchyma 48 hours after intoxication.The required dose to achieve AHF is 3 gr/kg body weight (Gr. 0). Survival rates are as follow: Gr. 1: D0: 93%; from D1 to D28: 13%. Gr. 2: D0: 80%; D1 and D2: 33%; D3: 20%; D4: 13%, from D5 to D28: 6%. (D0 = Day of transplantation). Liver enzymes show a peak of deterioration at 24 hours, then return to normal values in both groups. Histological examination of those animals still alive and sacrificed on day 28 demonstrates a restitutio ad integrum of hepatic structure. In Group 2, it is possible to observe remaining living hepatocytes in the splenic parenchyma.When HcTX is performed 48 hours after D-Gal intoxication, i.e., when the animals begin to develop AHF symptoms, animal survival only significantly improves between days 0 and 3. Unlike other trials, we cannot demonstrate an improvement in long-term survival. Thus, according to this particular experimental model, HcTX is not an alternative for the treatment of AHF.

p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below.

The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.

1. Sex difference in the hepatic uptake of sulphobromophthalein (BSP) was investigated in male and female rats in three different experimental models. 2. In the intact animal the BSP plasma disappearance rate was significantly higher (P < 0.01) in females than in males when 0.15 or 1.5 μmol/kg body wt. was injected. Comparable values were found at the highest BSP dose (15 μmol/kg body wt.) used. 3. In the perfused liver, the first-pass hepatic extraction and the uptake velocity were significantly higher (P < 0.001) in female rats at low BSP doses (0.3–750 μmol/g of liver) whereas identical values were found at higher concentrations. 4. In hepatocytes isolated by collagenase perfusion, the BSP uptake occurs via two different uptake sites in both sexes. The Km of the high affinity sites was lower in females than in males (3.67 ± 0.58 vs 7.24±0.68 Mmol/l, P < 0.001) whereas Vmax. showed comparable values (2.70 ± 0.36 vs 2.47 ± 0.45 nmol of BSP/mg of protein, NS). In contrast, no difference was found in the kinetic parameters of the low affinity sites (Km 50.6±31.1 vs 61.0±17.5 μmol/l; Vmax. 21.9 ± 13.2 vs 25.0±3.6 nmol of BSP/mg of protein, mean ± sd, NS, females and males respectively). 5. Taken together these data show that low doses of BSP are taken up by the liver more efficiently in female than in male rats and are consistent with a sex-related difference in the affinity but not in the number of the BSP high affinity uptake sites.

The aim of this work was to study the effect of a sustained activation of AMP-activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA-riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c-Jun N-terminal kinase (JNK) and caspase-3. Experiments with iodotubercidin, dicoumarol and z-VAD-fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase-3.