Auswahl der wissenschaftlichen Literatur zum Thema „Liver cell models“

Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens—namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites—has hampered the development of disease models. These models are crucial for uncovering the molecular mechanisms underlying the biology of infection and governing host–pathogen interaction, as well as for fostering drug development. Bioengineered cell models better recapitulate the human liver microenvironment and extend hepatocyte viability and phenotype in vitro, when compared with conventional two-dimensional cell models. In this article, we review the bioengineering tools employed in the development of hepatic cell models for studying infection, with an emphasis on 3D cell culture strategies, and discuss how those tools contributed to the level of recapitulation attained in the different model layouts. Examples of host–pathogen interactions uncovered by engineered liver models and their usefulness in drug development are also presented. Finally, we address the current bottlenecks, trends, and prospect toward cell models’ reliability, robustness, and reproducibility.

Humanized liver mouse models are crucial tools in liver research, specifically in the fields of liver cell biology, viral hepatitis and drug metabolism. The livers of these humanized mouse models are repopulated by 3-dimensional islands of fully functional primary human hepatocytes (PHH), which are notoriously difficult to maintain in vitro. As low efficiency and high cost hamper widespread use, optimization is of great importance. In the present study, we analyzed experimental factors associated with Hepatitis E virus (HEV) infection and PHH engraftment in 2 xenograft systems on a Nod-SCID-IL2Ry-/- background: the alb-urokinase plasminogen activator mouse model (uPA-NOG, n=399); and the alb-HSV thymidine kinase model (TK-NOG, n = 198). In a first analysis, HEV fecal shedding in liver humanized uPA-NOG and TK-NOG mice with comparable human albumin levels was found to be similar irrespective of the mouse genetic background. In a second analysis, sex, mouse age at transplantation and hepatocyte donor were the most determinant factors for xenograft success in both models. The sexual imbalance for xenograft success was related to higher baseline ALT levels and lower thresholds for ganciclovir induced liver morbidity and mortality in males. These data call for sexual standardization of human hepatocyte xenograft models, but also provide a platform for further studies on mechanisms behind sexual dimorphism in liver diseases.

Abstract Purpose of Review To describe experimental liver injury models used in regenerative medicine, cell therapy strategies to repopulate damaged livers and the efficacy of liver bioengineering. Recent Findings Several animal models have been developed to study different liver conditions. Multiple strategies and modified protocols of cell delivery have been also reported. Furthermore, using bioengineered liver scaffolds has shown promising results that could help in generating a highly functional cell delivery system and/or a whole transplantable liver. Summary To optimize the most effective strategies for liver cell therapy, further studies are required to compare among the performed strategies in the literature and/or innovate a novel modifying technique to overcome the potential limitations. Coating of cells with polymers, decellularized scaffolds, or microbeads could be the most appropriate solution to improve cellular efficacy. Besides, overcoming the problems of liver bioengineering may offer a radical treatment for end-stage liver diseases.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure and accounts for the majority of regulatory actions on drugs. Furthermore, DILI is a relevant cause for project terminations in pharmaceutical development. The idiosyncratic form of DILI is especially a threat in late clinical development phases and postmarketing, respectively. Even the occurrence of only a few idiosyncratic DILI cases in late clinical development or postmarketing may suffice to terminate or withdraw an otherwise promising therapy. Despite advances in preclinical assessment of dose-dependent toxicity, idiosyncratic DILI is still a big challenge for in vitro research: it not only requires individualized models but also a huge number of tests. We have developed and investigated MetaHeps®, a technology involving hepatocyte-like cells generated from peripheral monocytes without genetic modifications. These cells exhibit several hepatocyte-like characteristics and show donor-specific activities of drug-metabolizing enzymes. With MetaHeps we have performed in vitro investigations in patients with DILI suspicion. By investigating MetaHeps derived from DILI patients we could show increased in vitro susceptibility to the drugs involved in the individual patients. MetaHeps testing could also rule out DILI and help to identify other causes of acute liver injury. Moreover, MetaHeps identified the causative agent in polymedicated patients. In conclusion, in vitro research of idiosyncratic DILI requires individual cell models which produce results comparable to the clinical situation. We suggest the MetaHeps technology as a novel tool to cope with these challenges of DILI.

Abstract The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver diseases. Hepatocyte-driven liver regeneration that involves the proliferation of preexisting hepatocytes is a primary regeneration mode. On the other hand, liver progenitor cell (LPC)-driven liver regeneration that involves dedifferentiation of biliary epithelial cells or hepatocytes into LPCs, LPC proliferation, and subsequent differentiation of LPCs into hepatocytes is a secondary mode. This secondary mode plays a significant role in liver regeneration when the primary mode does not effectively work, as observed in severe liver injury settings. Thus, promoting LPC-driven liver regeneration may be clinically beneficial to patients with severe liver diseases. In this review, we describe the current understanding of LPC-driven liver regeneration by exploring current knowledge on the activation, origin, and roles of LPCs during regeneration. We also describe animal models used to study LPC-driven liver regeneration, given their potential to further deepen our understanding of the regeneration process. This understanding will eventually contribute to developing strategies to promote LPC-driven liver regeneration in patients with severe liver diseases.

Over the last decade a huge amount of experimental data on biological systems has been generated by modern high-throughput methods. Aided by bioinformatics, the '-omics' (genomics, transcriptomics, proteomics, metabolomics and interactomics) have listed, quantif ed and analyzed molecular components and interactions on all levels of cellular regulation. However, a comprehensive framework, that does not only list, but links all those components, is still largely missing. The biology-based but highly interdisciplinary field of systems biology aims at such a holistic understanding of complex biological systems covering the length scales from molecules to whole organisms. Spanning the length scales, it has to integrate the data from very different fields and to bring together scientists from those fields. For linking experiments and theory, hypothesis-driven research is an indispensable concept, formulating a cycle of experiment, modeling, model predictions for new experiments and, fi nally, their experimental validation as the start of the new iteration. On the hierarchy of length scales certain unique entities can be identi fied. At the nanometer scale such functional entities are molecules and at the micrometer level these are the cells. Cells can be studied in vitro as independent individuals isolated from an organism, but their interplay and communication in vivo is crucial for tissue function. Control over such regulation mechanisms is therefore a main goal of medical research. The requirements for understanding cellular interplay also illustrate the interdisciplinarity of systems biology, because chemical, physical and biological knowledge is needed simultaneously. Following the notion of cells as the basic units of life, the focus of this thesis are mathematical multi-scale models of multi-cellular systems employing the concept of individual (or agent) based modeling (IBM). This concept accounts for the entity cell and their individuality in function and space. Motivated by experimental observations, cells are represented as elastic and adhesive spheres. Their interaction is given by a model for elastic homogeneous spheres, which has been established for analysis of the elastic response of cells, plus an adhesion term. Cell movement is modeled by an equation of motion for each cell which is based on the balance of interaction, friction and active forces on the respective cell. As a fi rst step the model was carefully examined with regard to the model assumptions, namely, spherical shape, homogeneous isotropic elastic body and apriori undirected movement. The model examination included simulations of cell sorting and compression of multicellular spheroids. Cell sorting could not be achieved with only short range adhesion. However, it sorting completed with long range interactions for small cell numbers, but failed for larger aggregates. Compression dynamics of multi-cellular spheroids was apparently reproduced qualitatively by the model. But in a more detailed survey neither the time scales nor the rounding after compression could be reproduced. Based on these results, the applications consistent with the assumed simpli cations are discussed. One already established application is colony growth in two-dimensional cell cultures. In order to model cell growth and division, a two-phase model of the cell cycle was established. In a growth phase the cell doubles its volume by stochastic increments, and in a mitotic phase it divides into two daughter cells of equal volume. Additionally, control of the cell cycle by contact inhibition is included in the model. After examination of its applicability, the presented model is used for simulations of in vitro growth of mesenchymal stem cells (MSC) and subsequent cartilage formation in multi-cellular spheroids. A main factor for both processes is the oxygen concentration. Experimental results have shown, that i) MSC grow much better in vitro at low than at high oxygen concentrations and ii) the MSC progeny harvested from low oxygen culture produce higher amounts of the cartilage components aggrecan and collagen II in multicellular spheroids than the ones from high oxygen culture. In order to model these processes, IBM was extended by a stochastic model for cellular differentiation. In this model cellular differentiation is captured phenomenologically by two additional individual properties, the degree of differentiation and the lineage or cell type, which are subject to fl uctuations, that are state and environment dependent. After fitting the model parameters to the experimental results on MSC growth in monoclonal expansion cultures at low and high oxygen concentrations, the resulting simulated cell populations were used for initialization of the simulations of cartilage formation in multi-cellular spheroids. The model nicely reproduced the experimental results on growth dynamics and the observed number of functional cells in the spheroids and suggests the following explanation for the difference between the two expansion cultures: due to the stronger pre-differentiation found after expansion in high oxygen, the plasticity of these cells is smaller and less cell adopt the chondrogenic phenotype and start to produce cartilage. Moreover, the model predicts an optimal oxygen concentration for cartilage formation independent of expansion culture and a de-differentiating effect of low oxygen culture within 24h. Because all simulations comply with the concept of hypothesis-driven research and follow closely the experimental protocols, they can easily be tested and are currently used for optimization of a bioreactor for cartilage production. Cell populations are composed of individual cells and regulation of population properties is performed by individual cell, but knowledge about individual cell fates is largely missing due to the problem of single cell tracking. The IBM modeling approach used for modeling MSC growth and differentiation generically includes information of each individual cell and is therefore perfectly suited for tackling this question. Based on the validated parameter set, the model was used to generate predictions on plasticity of single cells and related population dynamics. Single cell plasticity was quantifi ed by calculating transition times into stem cell and differentiated cell states at high and low oxygen concentrations. At low oxygen the results predict a frequent exchange between all subpopulations, while at high oxygen a quasi-deterministic differentiation is found. After quantifying the plasticity of single cells at low and high oxygen concentration, the plasticity of a cell population is addressed in a simulation closely following a regeneration experiment of populations of hematopoietic progenitor cells. In the simulation the regeneration of the distribution of differentiation states in the population is monitored after selection of subpopulations of stem cells and differentiated cells. Simulated regeneration occurs on the time scales estimated from the single cell transition times except the unexpectedly fast regeneration from differentiated cells in the high oxygen environment, which favors differentiation. The latter case emphasizes the importance of single outlier cells in such system, which in this case repopulate less differentiated states with their progeny. In general, cell proliferation and regeneration behavior are in uenced by biomechanical and geometrical properties of the environment e.g. matrix stiffness or cell density. Because in the model cells are represented as physical objects, a variation of friction is linked to cell motility. The cultures of less motile cells become denser at the same size and the effects of contact inhibition of growth more pronounced. This variation of friction coe fficients allows the comparison of cultures with varying degrees of contact inhibition regarding their differentiation structure and the results suggest, that stalled proliferation is su fficient to explain the well-known differentiation effects in confl uent colonies. In addition, the composition of the simulated stem cell pool was analyzed regarding differentiation. In contrast to the established pedigree models, where stem cell can only be produced by asymmetric division, this model predicts that most of the cells in stem cell states descend from progenitor cells of intermediate differentiation states. A more detailed analysis of single cell derived clones revealed properties that could not be described by the model so far. First, a differentiation gradient was observed in larger colonies, that was the opposite of the one predicted by the model. Second, the proliferative activity turned out to depend not only on oxygen, but also to be a property of individual clones persisting over many generations. Because the relation slow growth/pre-differentiation also holds for single cell derived clones, the general model of differentiation is extended by another heritable individual property. Motivated by the decline of proliferation and differentiation in culture and the high metabolic and epigenetic activity during cell division, each division event is assumed to de-stabilize stem cell states. Consequently, in the model the cells age in terms of cell divisions determines the fl uctuations in stem cell states and the environment the mean fl uctuation strength. Including this novel concept, that links aging to growth and differentiation dynamics, into the model reproduces the experimental results regarding differentiation gradient and persistent clonal heterogeneity. The spatial differentiation pattern can largely be explained by the spatio-temporal growth pattern of the mono-clonal cell assembly: cells close to the border of the cell assembly have undergone more cell divisions than those in the interior and therefore their stem cell states are less stable. Heterogeneity of single-cell derived clones depends on the age of the first cell in the clone. When the stem cell fluctuations equal the mean fl uctuations strength, the proliferative activity passes a maximum at a certain age due to the destabilization of stem cell states. Thereafter the proliferative activity decreases, because more time is spent in non-proliferative differentiated states. Considering the number of divisions the cells have already undergone in vivo and after the initial expansion in vitro, it can be assumed that all cells have already passed this maximum. Interestingly, the model also predicts an optimal age for directed differentiation, when cells stably differentiate, but have not lost the required plasticity. According to the model, this clonal heterogeneity may be caused purely in vitro, but hypothetical simulation of in vivo aging yielded results consistent with experiments on MSC from rats of varying age. Finally, the detailed molecular regulation mechanisms in a multi-scale tissue model of liver zonation was studied, in which the key molecular components were explicitly modeled. Hence, this model resolved the intracellular regulation in higher resolution than the above considered differentiation models which had summarized the intracellular control and differentiation mechanisms by a few phenomenological, dynamical variables. The metabolic zonation of the liver is essential for many of the complex liver functions. One of the vitally important enzymes, glutamine synthetase, (GS) is only synthesized in a strictly defi ned pattern. Experimental evidence has shown that a particular pathway, the canonical wnt pathway, controls expression of the gene for GS. A model for transport, receptor dynamics and intracellular regulation mechanism has been set up for modeling the spatio-temporal formation of this pattern. It includes membrane-bound transport of the morphogen and an enzyme kinetics approach to fibeta-catenin-regulation in the interior of the cell. As an IBM this model reproduces the results of co-culture experiments in which two-dimensional arrangements of liver cells and an epithelial liver cell line give rise to different patterns of GS synthesis. The two main predictions of the model are: First, GS-synthesis requires a certain local cell number of wnt releasing cells. And second, a simple inversion of geometry explains the difference between the specifi c GS pattern found in the liver and in the co-culture experiments. Summarizing the results presented in this thesis, it can be concluded that properties such as the occurrence of memory effects and single cells pursuing fates far off the population average could be essential for biological function. Considering the role of single cells in many tissues, the use of individual based methods, that are able to take such effects into account, can be expected to be a very valuable tool for the problems of systems biology.

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Each year liver cancer kills more than half a million people, making it the third leading cause of cancer-related death worldwide. Annual incidence continues to rise steadily, both domestically and globally, increasing the burden of this disease. Advancements in the ability to obtain detailed molecular profiles of tumors have led to the successful development of targeted therapies for a number of different cancers. Unfortunately, however, the molecular pathogenesis of liver cancer is poorly understood relative to many other types of malignancies. Thus, the identification of factors contributing to the development and progression of liver tumors is a major goal of current research. In pursuit of this goal, I have utilized the Sleeping Beauty (SB) transposon system as a tool for forward genetic mutagenesis screening in mice. The SB system recapitulates the kinetics of spontaneous tumor development in humans by providing a stepwise accumulation of mutations. Micro-evolutionary processes within a developing tumor lead to the selective expansion of cells harboring mutations that confer some kind of selective advantage. By identifying the most prevalent mutation events within a specific tumor type across a large number of independent samples, a list of genes implicated as being involved in tumorigenesis can be generated. Using this approach, the Dlk1-Dio3 imprinted domain was identified as a site of frequent mutation in SB-induced hepatocellular carcinomas (HCCs). I discovered that the mechanistic basis for recurrent selection of transposon insertion within this domain in liver tumors involved activated expression of Retrotransposon-like 1 (Rtl1). I also found that RTL1 activation is a common event in human HCC, suggesting that it could potentially be beneficial as a therapeutic target in a subset of patients. Etiological factors related to liver cancer development are varied, but are linked by the fact that each provides a chronic liver injury stimulus that promotes the development of hepatic fibrosis. In fact, ˜ 90% of human HCC occurs in this context, and yet the majority of mouse liver cancer models fail to account for this important environmental component of the disease. I have conducted a screen for genetic drivers of liver cancer in the presence or absence of hepatic fibrosis. Comparison of mutation profiles between fibrotic and non-fibrotic tumors revealed largely non-overlapping sets of candidate genes, indicative of a differential selective pressure for mutations depending on the fibrotic context of the liver. Driver mutations identified preferentially in the presence of liver fibrosis have a high likelihood of relevance to human disease, given the similarities in environmental context and kinetics of mutation acquisition. Consistent with this idea, multiple genes with well-established roles in human HCC were found to be preferentially mutated in SB-induced tumors developed in a fibrotic liver. Before a candidate cancer gene identified in an animal model system can have an impact on human disease, its proposed role in tumorigenesis must be validated. Existing techniques for validation of putative liver cancer genes suffer from significant limitations including high cost, low throughput, and a level of complexity that prohibits widespread utilization. I have contributed to the generation of a novel tool for in vivo validation of candidate genes that is not subject to these limitations. By combining elements of recombinant adenoviral vectors and the piggyBac transposition system, we have generated a highly flexible gene delivery system with significant advantages over existing techniques. The Ad-PB system has broad accessibility and applicability, making it a valuable tool for advancing efforts to improve cancer therapies.

L’un des enjeux de l’industrie pharmaceutique aujourd’hui est de développer des modèles de foie in vitro fidèles pour améliorer la prédictivité des études précliniques, notamment l’étude de la toxicité et de l’efficacité des médicaments candidats. Ces dernières années, l’ingénierie tissulaire, approche multidisciplinaire pour développer des tissus, a mené au développement de nouvelles méthodes de culture cellulaire. Parmi elles, les cultures de cellules en 3D ou en perfusion ont permis d’obtenir des activités hépatiques similaires à celles observées in vivo. L’objectif de cette thèse est de combiner ces deux méthodes de culture cellulaire pour créer un modèle de foie in vitro encore plus fidèle. Pour cela, nous cherchons à développer un cryogel d’alginate intégré en micropuce avec des propriétés mécaniques adaptables à celles du foie en fonction de l’état physiologique à reproduire (foie sain ou pathologique). Dans la première partie, nous développons et caractérisons le cryogel d’alginate au niveau microscopique et macroscopique, à l’extérieur (échantillons cylindriques) puis à l’intérieur de la biopuce. Trois paramètres sont étudiés ici : la température de cryopolymérisation, la concentration d’alginate ainsi que la quantité d’agents réticulants. Les propriétés mécaniques, la porosité, l’absorption, l’interconnectivité des pores et la résistance au flux sont analysés.La deuxième partie vise à cultiver des cellules hépatiques au sein de ce nouveau dispositif. Pour cette étude de faisabilité la lignée cellulaire HepG2/C3A est utilisée. Les résultats montrent des cellules viables et fonctionnelles (production d’albumine, transformation d’APAP). De plus, nous observons une structure tissulaire 3D, qui se maintient après retrait du cryogel d’alginate. La dernière partie a pour but de complexifier le modèle hépatique, notamment par des co-cultures. Pour se rapprocher de la structure du sinusoïde, des cellules hépatiques sont cultivées avec des cellules endothéliales (HUVEC) selon deux approches. De plus, la possibilité de suivre des cellules tumorales circulantes (MDA-MB-231) dans le système est étudiée
Today, one of the challenges for the pharmaceutical industry is to develop accurate in vitro liver models to improve the predictability of preclinical studies, in particular the study of the toxicity and efficacy of drug candidates. In recent years, tissue engineering, a multidisciplinary approach to develop tissues, has led to the development of new cell culture methods. Among them, cell cultures in 3D or in perfusion allowed to obtain hepatic activities similar to those observed in vivo. The objective of this thesis is to combine these two cell culture methods to create an even more accurate in vitro liver model. To do so, we are seeking to develop an alginate cryogel integrated into a microchip with mechanical properties adaptable to those of the liver depending on the physiological state to be reproduced (healthy or pathological liver).In the first part, we develop and characterize the alginate cryogel at the microscopic and macroscopic level, outside (cylindrical samples) and then inside the biochip. Three parameters are studied here: the cryopolymerization temperature, the alginate concentration and the quantity of cross-linking agents. Mechanical properties, porosity, absorption, pore interconnectivity and flow resistance are analyzed. The second part aims to culture liver cells within this new device. For this feasibility study the HepG2/C3A cell line is used. The results show viable and functional cells (albumin production, APAP transformation). In addition, we observe a 3D tissue structure, which is maintained after removal of the alginate cryogel. The last part aims to complexify the hepatic model, in particular by co-cultures. To get closer to the sinusoid structure, liver cells are cultured with endothelial cells (HUVEC) according to two approaches. In addition, the possibility to follow circulating tumor cells (MDA-MB-231) in the system is studied

La mort cellulaire joue un rôle central dans le développement et la progression des maladies du foie. Quel qu’en soit l’agent étiologique, il en résulte une destruction des hépatocytes, conduisant à une inflammation et à une prolifération compensatoire. En outre, la disparition continue de cellules peut aboutir au développement d’une fibrose, d’une cirrhose, voire d’un carcinome hépatocellulaire, la 3ème cause de décès par cancer. L'expression ou la sécrétion de ligands de mort, tels que le TNF-α, le FAS L et le TRAIL, par les cellules inflammatoires constitue le principal facteur de la progression des maladies du foie. En aval des récepteurs de ces ligands de mort ou de motifs moléculaires associés aux pathogènes (PAMP), la protéine kinase 1 interagissant avec le récepteur (RIPK1) influence le destin de la cellule, que ce soit pour survivre ou pour mourir par apoptose dépendante de caspases ou par nécroptose dépendante de RIPK3 / MLKL. Elle pourrait donc constituer une cible thérapeutique potentielle pour réguler le destin des hépatocytes. RIPK1 peut jouer un rôle distinct sur la mort ou la survie, grâce, respectivement, à ses fonctions kinase ou d’échafaudage. Dans cette optique, nous avons déjà montré le rôle protecteur de RIPK1 dans des modèles animaux d'hépatite aiguë induite par la ConA et le LPS. Au cours de mon travail de thèse, l'objectif était d'évaluer le rôle de RIPK1 dans des modèles murins d’hépatites aiguës (hépatite virale fulminante, dommages au foie induits par le CCl4 ou l'acétaminophène [APAP]) et d'hépatites chroniques (régime à haute teneur en gras et en cholestérol [HFHCD]). Nos résultats ont démontré que RIPK1 protège les hépatocytes du TNF-α sécrété par les macrophages au cours de l'hépatite fulminante induite par le virus MHV3. Ces données révèlent les risques potentiels d'aggravation d'une infection par le VHB chez les personnes atteintes de polymorphisme ou de mutations amorphes homozygotes déjà décrites pour le gène RIPK1. En outre, nous avons établi que RIPK1 dans les cellules parenchymateuses du foie n’influence pas les lésions hépatiques induites par l’APAP chez la souris. L'inhibition supplémentaire de l'activité kinase de RIPK1 chez les souris Ripk1LPC-KO n'a pas limitée les dommages hépatiques, révélant que l'activité kinase de la RIPK1 dans les cellules hépatiques non-parenchymateuses ne contribue pas aux lésions hépatiques induites par l’APAP. Sinon, nous avons démontré que RIPK1 dans les cellules parenchymateuses du foie préserve partiellement le foie des lésions induites par le CC14, lésions ne dépendant pas du TNF-α. Enfin, nous avons montré que RIPK1 dans les cellules parenchymateuses hépatiques avait tendance à limiter le développement de la fibrose induite par HFHCD dans la NASH murine et qu'une intervention alimentaire pouvait améliorer la fibrose hépatique chez la souris atteinte de NASH. Quant au rôle de l'activité kinase de la RIPK1 dans la NASH, elle reste à être explorée pour évaluer son intérêt thérapeutique
Cell death plays central role in the development and progression of liver diseases. Irrespective of the etiological agents, it results in hepatocyte destruction, leading to inflammation and compensatory proliferation. In addition, the persistent cell demise can lead into fibrosis and ultimately hepatocellular carcinoma, the 3rd leading cause of cancer related death. Expression or release of death ligands, such as TNF-α, FAS L and TRAIL, by inflammatory cells remains the key players in the progression of liver diseases. Downstream of death ligand receptors or PAMPs, receptor interacting protein kinase 1 (RIPK1) influences the fate of cell, whether to survive or to die by caspase-dependent apoptosis or by RIPK3/MLKL-dependent necroptosis and could therefore be potential targets in regulating cell death. RIPK1 can have distinct pro-death or pro-survival role, regulated by its kinase or scaffolding functions, respectively. In line with this, we have already shown the protective role of RIPK1 in animal models of acute hepatitis induced by ConA, LPS. In my PhD work, the objective was to assess the role of RIPK1 in animal models of acute (fulminant viral hepatitis, CCl4 and acetaminophen [APAP] induced liver damage) and chronic hepatitis (High Fat High Cholesterol diet [HFHCD]-induced NASH). Our results demonstrated that RIPK1 protects hepatocytes from TNF-α secreted from macrophages during viral induced fulminant hepatitis. These data emphasize the potential worsening risks of an HBV infection in people with polymorphism or homozygous amorphic mutations already described for the RIPK1 gene. Besides, we established that RIPK1 in liver parenchymal cells does not influence APAP-induced liver injury in mice. Additional inhibition of RIPK1 kinase activity in Ripk1LPC-KO mice did not improve hepatic damage, revealing that RIPK1 kinase activity in liver non-parenchymal cells does not contribute to APAP-induced liver injury. Otherwise, we demonstrated that RIPK1 of liver parenchymal cells partly preserves the liver from CCl4-induced damage, lesions that do not depend on TNF-α . Finally, we showed that RIPK1 in liver parenchymal cells has a tendency to protect from HFHCD-induced fibrosis in murine NASH and that dietary intervention can improve liver fibrosis in mice with NASH. As for the role of RIPK1-kinase activity in NASH, it remains to be explored to evaluate its therapeutic interest

The last chapter focuses on daily prison life. It starts in interrogation rooms and moves to prison cells. Women prisoners undertook various activities to distract themselves from the idleness of their world. They spent their days learning, reading, and engaging in their own cultural activities. As they recreated their lives in prison, they chose traditionally female roles of sharing, providing for, and taking care of their cellmates. These new cell roles appeared to be stable. When they laughed at and ridiculed each other, they challenged this supportive model. Close attention is paid to the importance of religion. For Poles, religion is closely linked to nationalism, but religion and nationalism were not as important as expected. The role of religion became more prominent in the meaning of imprisonment for these women’s post-prison lives. This chapter takes place predominantly in the post-trial cells, in such prisons as Fordon and Inowrocław.

The liver with its parenchymal and non-parenchymal cells plays a key role in the organism with manifold functions of metabolism, synthesis, detoxification, excretion, and host response. This requires a portfolio of different tests to obtain an overview of hepatic function. In the critically ill hepatic dysfunction is common and potentially leading to extrahepatic organ dysfunctions culminating in multi-organ failure. Conventional laboratory measures are used to evaluate hepatocellular damage, cholestasis, or synthesis. They provide valuable (differential) diagnostic data and can yield prognostic information in chronic liver diseases, especially when used in scoring systems such as the ‘model for end-stage liver disease’. However, they have short-comings in the critically ill in assessing rapid changes in hepatic function and liver blood flow. In contrast, dynamic quantitative liver function tests measure current liver function with respect to the ability to eliminate and/or metabolize a specific substance. In addition, they are dependent on sinusoidal blood flow. Liver function tests have prognostic significance in the critically ill and may be used to guide therapy.

Within their cosmic ecology, the Athenians took it for granted that their polis was a “communion” (koinonia) of households, so in their experience there could be no equivalents of our modern distinctions between state and society or political and social realms. Households (oikoi) functioned as the cells of the social body, such that the vitality of the parts was inseparable from the vitality of the whole. Thus, the human “government” of the polis began not with assembly meetings but with the management of its constituent oikoi, which were the primary means of life and livelihood for all Athenians. The Athenians also took it for granted that the gods had deliberately designed males and females to play different, but complementary roles in the reproduction of social being. Women were expected to serve as “partners” to their husbands in the business of household management, performing a wide range of functions that were essential to the lives of their oikoi and therefore to the life of their polis. While they may not look like “citizens” to us, they were considered full members of the polis (politides) at the time. Terms like “patriarchy” and “misogyny,” so common in the modern literature, are accordingly unhelpful when describing gender relations in classical Athens.

Triheptanoin, the triglyceride of heptanoate (C7 fatty acid), is a novel treatment that is being used to treat patients with rare genetic metabolic disorders. When taken orally, triheptanoin is hydrolyzed in the gastrointestinal tract to heptanoate, which is thought to diffuse into the blood and body. Heptanoate and its liver ketone metabolites are then metabolized within cells to propionyl-CoA, which after carboxylation produces succinyl-CoA, resulting in anaplerosis—the refilling of a deficient tricarboxylic acid cycle. Here, data are summarized and discussed in relation to triheptanoin’s anticonvulsant effects in rodent seizure models. Biochemical data reveal that metabolic alterations found in brains of rodent seizure models can be restored by triheptanoin. Moreover, there are increasing preclinical and clinical studies indicating that triheptanoin is beneficial in other neurological and neuromuscular disorders, which are summarized here. Thus, triheptanoin seems to be a promising treatment for a variety of clinical conditions.

This book contains the first English translation of the lost dream diary of Santiago Ramón y Cajal (1852–1934), the Nobel Prize-winning “father of modern neuroscience.” In the late nineteenth century, while scientific psychologists searched the inner world of human beings for suitable objects of study, Cajal discovered that the nervous system, including the brain, is composed of distinctly independent cells, later termed neurons. “The mysterious butterflies of the soul,” he romantically called them, “whose beating of wings may one day reveal to us the secrets of the mind.” Cajal was contemporary with Sigmund Freud (1856–1939), whose “secrets of the mind” radically influenced a century of thought. Although the two men never met, their lives and works were intimately related, and each is identified with the foundation of a modern intellectual discipline—neurobiology and psychoanalysis—still in conversation and conflict today. In personal letters, Cajal insulted Freud and dismissed his theories as lies. In order to disprove his rival, Cajal returned to an old research project and started recording his own dreams. For the last five years of his life, he abandoned his own neurobiological research and concentrated on psychological manuscripts, including a new “dream book.” Although his intention was to publish, the project was never released. The unfinished work was thought to be lost, until recently, when a Spanish book appeared claiming to feature the dreams of Cajal, along with the untold story of their complex journey into print.

Female patients undergoing treatment for cancer often experience significant changes in their sexuality due to the disease and its treatment. Sexuality relates to the sexual habits and desires of each individual. It varies according to age-related sexual needs. Many women with cancer consider their sexuality an important aspect of their lives. Yet, they may refrain from sex or enjoy it less following treatment, whether it be surgical or by irradiation, and accompanied by adjunctive chemotherapy or hormonal therapy. Chapter 11 discusses these issues, with a vignette illustrating the impact of an unexpected diagnosis of cancer. Multiple studies have examined sexual dysfunction following treatment of gynaecological cancers, including breast cancer, and several proposed solutions are available. However, the information has not been implemented by many health providers, and patients often experience anxiety and embarrassment when planning to discuss sexuality. The patients may be concerned that their sexual habits might interfere with the treatment outcome, and cause a recurrence of cancer. Reproductive dysfunction is only one of the manifold problems in the female undergoing cancer therapy. It can lead to infertility but certain treatment methods could help retain fertility. Ethical concerns pertaining to the preservation, and use of germ cells, need to be addressed. Ideally, a team of healthcare providers should handle sexual rehabilitation of the cancer survivor based on the patient's history. Unfamiliarity with such matters makes many medical professionals hesitant in discussing their patients' sexuality. The PLISSIT model can help initiate the assessment of sexual dysfunction in these patients.

This paper presents a novel liver model platform that mimics the liver sinusoid, a functional unit of the liver where most liver activities occur. A key component of the current liver model is a layered co-culture of primary rat hepatocytes (PRH) and primary rat liver sinusoidal endothelial cells (LSEC) or a bovine aortic endothelial cells (BAEC) as an alternative. Poly-dimethylsiloxane (PDMS) microchannels were fabricated and attached to transwell membranes that contain submicroscale pores. Cells were cultured either on one side or on both sides of the transwell membrane, and in both cases cells formed confluent layers. A thin matrigel coating or micro porous membrane was applied between the two cell layers in order to mimic the Space of Disse. We used three different methods to check cell viability: recombinant adenovirus expressing green fluorescent protein, mito-tracker red to stain live mitochondria, and an expression plasmid expressing red fluorescent protein (RFP). It was shown that PRH retained normal morphology and remained viable for about 3 days with BAEC in the PDMS microchannel, about 57 days with BAEC on the transwell, and about 39 days with primary LSEC on the transwell. Preliminary observation suggests that there is formation of structures between hepatocytes that appear similar to bile canaliculi when PRH are co-cultured with endothelial cells. The layered co-culture system seems to be a promising method to generate accurate liver models.

Physiological tissue-on-a-chip technology is enabled by adapting microfluidics to create micro scale drug screening platforms that replicate the complex drug transport and reaction processes in the human liver. The ability to incorporate three-dimensional (3d) tissue models using layered fabrication approaches into devices that can be perfused with drugs offer an optimal analog of the in vivo scenario. The dynamic nature of such in vitro metabolism models demands reliable numerical tools to determine the optimum tissue fabrication process, flow, material, and geometric parameters for the most effective metabolic conversion of the perfused drug into the liver microenvironment. Thus, in this modeling-based study, the authors focus on modeling of in vitro 3d microfluidic microanalytical microorgan devices (3MD), where the human liver analog is replicated by 3d cell encapsulated alginate hydrogel based tissue-engineered constructs. These biopolymer constructs are hosted in the chamber of the 3MD device serving as walls of the microfluidic array of channels through which a fluorescent drug substrate is perfused into the microfluidic printed channel walls at a specified volumetric flow rate assuring Stokes flow conditions (Re<<1). Due to the porous nature of the hydrogel walls, a metabolized drug product is collected as an effluent stream at the outlet port. A rigorous modeling approached aimed to capture both the macro and micro scale transport phenomena is presented. Initially, the Stokes Flow Equations (free flow regime) are solved in combination with the Brinkman Equations (porous flow regime) for the laminar velocity profile and wall shear stresses in the whole shear mediated flow regime. These equations are then coupled with the Convection-Diffusion Equation to yield the drug concentration profile by incorporating a reaction term described by the Michael-Menten Kinetics model. This effectively yields a convection-diffusion–cell kinetics model (steady state and transient), where for the prescribed process and material parameters, the drug concentration profile throughout the flow channels can be predicted. A key consideration that is addressed in this paper is the effect of cell mechanotransduction, where shear stresses imposed on the encapsulated cells alter the functional ability of the liver cell enzymes to metabolize the drug. Different cases are presented, where cells are incorporated into the geometric model either as voids that experience wall shear stress (WSS) around their membrane boundaries or as solid materials, with linear elastic properties. As a last step, transient simulations are implemented showing that there exists a tradeoff with respect the drug metabolized effluent product between the shear stresses required and the residence time needed for drug diffusion.

Due to the growing shortage of donor livers, more patients are waiting for liver transplantation. Efforts to expand the donor pool include the use of living donor liver transplantation (LDLT) and split liver transplantation. LDLT involves a healthy person undergoing a partial hepatectomy to donate a part of his liver to a patient with severe liver failure. Afterwards, the regenerative capacity of the organ allows the livers of both donor and recipient to regrow to normal liver masses. The procedure is not without risk as serious complications may occur (such as cholestasis, ascites, gastrointestinal bleeding and renal impairment). An inadequate liver mass compared to the body mass may result in the small-for-size syndrome (SFSS). In both donor and recipient, LDLT may lead to portal hypertension associated with the elevated intrahepatic resistance of a smaller liver, and an increased portal venous (PV) inflow per gram of liver tissue compared to the total liver before resection. Excessive hyperperfusion and shear stress may damage the sinusoidal endothelial cells and lead to graft dysfunction.

The ability to incorporate three-dimensional (3D) hepatocyte-laden hydrogel constructs using layered fabrication approaches into devices that can be perfused with drugs enables the creation of dynamic microorgan devices (DMDs) that offer an optimal analog of the in vivo liver metabolism scenario. The dynamic nature of such in vitro metabolism models demands reliable numerical tools to determine the optimum process, material, and geometric parameters for the most effective metabolic conversion of the perfused drug into the liver microenvironment. However, there is a current lack of literature that integrates computational approaches to guide the optimum design of such devices. The groundwork of the present numerical study has been laid by our previous study [1], where the authors modeled in 2D an in vitro DMD of arbitrary dimensions and identified the modeling challenges towards meaningful results. These constructs are hosted in the chamber of the microfluidic device serving as walls of the microfluidic array of channels through which a fluorescent drug substrate is perfused into the microfluidic printed channel walls at a specified volumetric flow rate assuring Stokes flow conditions (Re<<1). Due to the porous nature of the hydrogel walls, a metabolized drug product is collected at the outlet port. A rigorous FEM based modeling approach is presented for a single channel parallel model geometry (1 free flow channel with 2 porous walls), where the hydrodynamics, mass transfer and pharmacokinetics equations are solved numerically in order to yield the drug metabolite concentration profile at the DMD outlet. The fluid induces shear stresses are assessed both in 3D, with only 27 cells modeled as single compartment voids, where all of the enzymatic reactions are assumed to take place. In this way, the mechanotransduction effect that alters the hepatocyte metabolic activity is assessed for a small scale model. This approach overcomes the numerical limitations imposed by the cell density (∼1012 cells/m3) of the large scale DMD device. In addition, a compartmentalization technique is proposed in order to assess the metabolism process at the subcellular level. The numerical results are validated with experiments to reveal the robustness of the proposed modeling approach and the necessity of scaling the numerical results by preserving dynamic and biochemical similarity between the small and large scale model.

Abstract Some of the most effective methods to separate circulating tumor cells (CTCs) from normal blood cells can be implemented using ultra-filtration, and/or electro-magnetic fields. As well known, each biological cell presents, on both sides of its membrane, different concentrations of ionic species that produce an electric charge concentration with respect to the lipid double layer (impermeable to ions). In this way, the bio-cell can be seen as an electric capacitor, which has the lipid double layer acting as an insulator inserted between two conductive plates, concentrated on the lipid double layer inner and outer surfaces. In this paper, firstly, the electrical capacitor equivalent system is used to treat different types of bio-cells normally flowing in blood vessels (red blood cells, lymphocytes and various types of CTCs-like), and to transform their biological characteristics into digital twin information useful for engineering applications. After, the preliminary 3D geometric analysis of the bio-cells shapes allowed to associate each bio-cell to a different capacitor model, and to predict the electric-equivalent dimensions characterizing its electric behavior. Finally, the equivalent capacitor model is used to study the influence of bio-cells characteristics variation on human blood cells, with particular attention devoted to liver and lung CTCs-like ones.

Filament wound composite cylinders are much expected as fuel gas containers for hydrogen fuel cell vehicles, hydrogen transportation containers, or pressurizing hydrogen accumulators due to their high performances in strength and lightness. Stress distribution in the cylinder can be controlled by the winding modes of the filaments and the liner thickness design. However, small fabrication defects may sometimes result irregular changes in stress distribution in the composite and liner layers and influences much upon the strength and lives of the composite cylinders. Stress distributions can be analyzed by a finite element method by modeling the mechanical anisotropy in composite layers and elasto-plasticity in the liner layer. The deviation of the position of the hoop layer ends influences much upon the basic performance of the vessel.

As part of a research program focusing on the role of human skin as a transport barrier, we formulate a coarse-grained model of the epidermis consisting of free extracellular water, live cells, and inert extracellular matrix subject to the coupling between molecular diffusive flux and electrokinetic flux (or electrodiffusion, as expressed by the Nernst-Planck equation). This polyphasic mathematical model accounts for active transport of physiologically-relevant solutes across the membrane of the live cells (keratinocytes), diffusion in the extracellular matrix and redistribution of water. The volume of the cell phase is regulated by the fluxes of water and Na+, K+ and Cl− ions across the cell membrane and is controlled according to the time-delay scheme introduced in the model of Hernandez & Cristina (1998). Computing the transient response of a 100 μm-thick viable epidermis layer exposed to a hyposmotic shock reveals that accounting for the electrokinetic flux in the extracellular domain has negligible effect on the results. This result suggests that a significant simplification of the model can be made in terms of decoupling the extracellular variation of the electrostatic field from the diffusion problem during the study of complex transepidermal transport.

The present work describes the implementation of an in-vitro model for the evaluation of the injury mechanisms of lung epithelial cells during simulated airway reopening events. Collagen-coated polyacrylamide substrates with different rigidities served as cell culture substrates, and a parallel plate perfusion chamber was used for simulating the reopening of fluid-filled airways. Human alveolar epithelial cell (A549) monolayers were grown to confluence on the different substrates and the cellular response was evaluated in terms of cytoskeletal distribution, proliferation rate, and cell stiffness. One and five reopening events were simulated, and cell response was characterized via live/dead fluorescence imaging. Cells cultured on stiffer substrates showed higher proliferation rate, as well as a more spread and tightly packed morphology. Airway reopening simulations showed that cells cultured on softer substrates are less susceptible to cell injury.