Electrical Impedance-Based Characterization of Hepatic Tissue with Early-Stage Fibrosis

Liver fibrosis is a key pathological precondition for hepatocellular carcinoma in which the severity is confidently correlated with liver cancer. Liver fibrosis, characterized by gradual cell loss and excessive extracellular matrix deposition, can be reverted if detected at the early stage. The gold standard for staging and diagnosis of liver fibrosis is undoubtedly biopsy. However, this technique needs careful sample preparation and expert analysis. In the present work, an ex vivo, minimally destructive, label-free characterization of liver biopsies is presented. Through a custom-made experimental setup, liver biopsies of bile-duct-ligated and sham-operated mice were measured at 8, 15, and 21 days after the procedure. Changes in impedance were observed with the progression of fibrosis, and through data fitting, tissue biopsies were approximated to an equivalent RC circuit model. The model was validated by means of 3D hepatic cell culture measurement, in which the capacitive part of impedance was proportionally associated with cell number and the resistive one was proportionally associated with the extracellular matrix. While the sham-operated samples presented a decrease in resistance with time, the bile-duct-ligated ones exhibited an increase in this parameter with the evolution of fibrosis. Moreover, since the largest difference in resistance between healthy and fibrotic tissue, of around 2 kW, was found at 8 days, this method presents great potential for the study of fibrotic tissue at early stages. Our data point out the great potential of exploiting the proposed needle setup in clinical applications

Characteristics of neutrophilic granulocytes of peripheral blood in patients with mechanical jaundice caused by cholangiocarcinoma

Obstructive jaundice caused by cholangiocarcinoma takes a special place among malignant disorders and surgical pathology in Russia. Involvement of nonspecific immunity and the role of neutrophils in carcinogenesis are ambiguously evaluated. The aim of this study was to study functional activity of peripheral blood neutrophilic granulocytes and their phenotype in obstructive jaundice caused by cholangiocarcinoma. The study included 56 patients with obstructive jaundice associated with cholangiocarcinoma at the T2- 3N0- 1M0 stage (clinical stages II-III), and 90 apparently healthy volunteers of similar age group. Neutrophilic granulocytes were isolated from peripheral blood by means of double-density Ficoll-Urografin gradient.Venous blood was collected in patients into vacutainers with heparin upon admission to the hospital, before the surgery was performed. Spontaneous cytokine production (IL-8, IFNá) was determined by enzyme-linked immunosorbent assay using Vector-Best diagnostic kits. To assess phagocytic activity of neutrophilic granulocytes, the phagocytic index (according to Hamburger), number of phagocytes (according to Wright) and the index of completed phagocytosis (according to Rudik, 2006) were calculated. Immunophenotyping of neutrophilic granulocytes was carried out using an FC500 flow cytometer (Beckman Coulter, USA) with monoclonal antibodies to CD11b+, CD16+, CD95+. The results were statistically analyzed using the Statistica v.12.0 software (StatSoft Inc., USA). To assess intergroup differences, the nonparametric Kruskal-Wallis tests (for three or more comparison groups) and Mann-Whitney tests (for pairwise comparison) were used. Comparison of groups for a qualitative binary trait was carried out using the two-sided Fisher’s exact test. Data are presented as Median (25 quartile-75 quartile).The study of the functional activity of peripheral blood neutrophiles in obstructive jaundice patients caused by cholangiocarcinoma revealed an increase in their relative and absolute number, increased phagocytic index and decreased phagocytic number of neutrophilic granulocytes, increased expression of CD11b+, CD16+, CD95+ immunological markers. The changes in neutrophil secretory activity were characterized by a decrease in cytokine production (IL-2, IFNá). An increase in functional activity of neutrophilic granulocytes, along with a decrease in their cytokine production suggests that, in obstructive jaundice observed in cholangiocarcinoma at clinical stage T2-3N0-1M0, an equilibrium stage is revealed between the cells of immune system and malignant tumor

GATA6 modulates the ductular reaction to bile duct ligation

Background GATA6, a transcription factor expressed in cholangiocytes, has been implicated in the response to liver injury. In biliary atresia, a disease characterized by extrahepatic bile duct obstruction, liver expression of GATA6 increases with pathological bile duct expansion and decreases after successful Kasai portoenterostomy. The aim of this study was to garner genetic evidence that GATA6 is involved in ductular formation/expansion. Methods The murine Gata6 gene was conditionally deleted using Alb-cre, a transgene expressed in hepatoblasts (the precursors of hepatocytes and cholangiocytes) and mature hepatocytes. Bile duct ligation (BDL) was used to model biliary obstruction. Results Alb-Cre;Gata6(flox/flox) mice were viable and fertile. Cre-mediated recombination of Gata6 in hepatocytes had little impact on cellular structure or function. GATA6 immunoreactivity was retained in a majority of biliary epithelial cells in adult Alb-Cre;Gata6(flox/flox) mice, implying that surviving cholangiocytes were derived from hepatoblasts that had escaped biallelic Cre-mediated recombination. Although GATA6 immunoreactivity was preserved in cholangiocytes, Alb-cre;Gata6(flox/flox) mice had a demonstrable biliary phenotype. A neutrophil-rich infiltrate surrounded newly formed bile ducts in neonatal Alb-Cre;Gata6(flox/flox) mice. Foci of fibrosis/necrosis, presumed to reflect patchy defects in bile duct formation, were observed in the livers of 37% of adult Alb-cre;Gata6(flox/flox) mice and 0% of controls (p <0.05). Most notably, Alb-cre;Gata6(flox/flox) mice had an altered response to BDL manifest as reduced survival, impaired bile ductule proliferation, increased parenchymal necrosis, reduced fibrosis, and enhanced macrophage accumulation in the portal space. Conclusions GATA6 orchestrates intrahepatic biliary remodeling and mitigates liver injury following extrahepatic bile duct obstruction. Graphic abstractPeer reviewe

Fetal androgen exposure is a determinant of adult male metabolic health

Androgen signalling is a critical driver of male development. Fetal steroid signalling can be dysregulated by a range of environmental insults and clinical conditions. We hypothesised that poor adult male health was partially attributable to aberrant androgen exposure during development. Testosterone was directly administered to developing male ovine fetuses to model excess prenatal androgenic overexposure associated with conditions such as polycystic ovary syndrome (PCOS). Such in utero androgen excess recreated the dyslipidaemia and hormonal profile observed in sons of PCOS patients. 1,084 of 15,134 and 408 of 2,766 quantifiable genes and proteins respectively, were altered in the liver during adolescence, attributable to fetal androgen excess. Furthermore, prenatal androgen excess predisposed to adolescent development of an intrahepatic cholestasis-like condition with attendant hypercholesterolaemia and an emergent pro-fibrotic, pro-oxidative stress gene and protein expression profile evident in both liver and circulation. We conclude that prenatal androgen excess is a previously unrecognised determinant of lifelong male metabolic health

Animal models for liver disease – A practical approach for translational research

Animal models are crucial for improving our understanding of human pathogenesis, enabling researchers to identify therapeutic targets and test novel drugs. In the current review, we provide a comprehensive summary of the most widely used experimental models of chronic liver disease, starting from early stages of fatty liver disease (non-alcoholic and alcoholic) to steatohepatitis, advanced cirrhosis and end-stage primary liver cancer. We focus on aspects such as reproducibility and practicality, discussing the advantages and weaknesses of available models for researchers who are planning to perform animal studies in the near future. Additionally, we summarise current and prospective models based on human tissue bioengineering

Untersuchung des antifibrotischen Potenzials von Foxf1-siRNA im Modell der akuten und chronischen CCl4-Leberschädigung

In der vorliegenden Dissertation wird ein gentherapeutischer Ansatz unter der Nutzung des Prinzips der RNA-Interferenz verwendet, um die Progression der Leberzirrhose im akuten und chronischen CCl4-Schädigungsmodell in vivo zu untersuchen. Foxf1, als aktivierender Transkriptionsfaktor der Hepatischen Sternzelle, war Target des antifibrotischen Ansatzes. Die entsprechende therapeutische siRNA wurde mittels Lipoplex-basiertem Vektor in die Zielzelle eingebracht. Trotz eines Foxf1-Silencings im akuten Modell, zeigten sich weder im akuten noch im chronischen Versuchsmodell antifibrotische Effekte

Antifibrotische Effekte von DBTC-Foxf1 siRNA-Formulierungen im murinen Modell der cholestatischen Leberfibrose

Ein wesentlicher Schritt in der Entstehung der Leberfibrose ist die Aktivierung hepatischer Sternzellen (HSC), wobei hierbei unter anderem eine veränderte Expression von Transkriptionsfaktoren vorliegt. Für den Transkriptionsfaktor forkhead box f1 (Foxf1) ist bekannt, dass dessen verstärkte Expression aktivierend auf HSC wirkt. In dieser Arbeit wurde daher mit dem Konzept der Gen-Interferenz durch small interfering RNA (siRNA) im murinen Modell der cholestatischen Leberfibrose untersucht, ob ein Foxf1-Silencing in den HSC zu antifibrotischen Effekten führt

Analyse des therapeutischen Potentials von Resolvin D1 im murinen Modell der cholestatischen Leberfibrose

Leberfibrose ist eine Vorstufe chronischer Lebererkrankungen. Bei weltweit steigender Inzidenz ist die einzige kurative Therapie die Lebertransplantation. Die Entdeckung neuer Therapien ist somit essentiell. Resolvine sind Omega-3-Fettsäure-Metabolite, die in zahlreichen Studien anti-inflammatorische Eigenschaften bei verschiedenen Erkrankungen zeigten. In dieser Studie wurde im murinen Modell der Gallengangsligatur-(BDL)-induzierten cholestatischen Leberfibrose die Wirkung von Resolvin D1 hinsichtlich Leberschaden, Inflammation, Fibrose und Proliferation untersucht

TGF-β2 abundance in mice and men: A successful anti-TGF-β2 strategy in biliary-derived liver disease

TGF-β1 is a key player in the onset, the progress, and end stages of CLD promoting fibrogenesis and tumorigenesis. To date, the expression and function of TGF-β2 have not been investigated thoroughly in liver disease. In this thesis, we provide evidence that TgfB2 and not, as formerly known, only TgfB1 correlate with fibrogenesis and liver cancer development. In a comparative analysis, we analyzed TgfB2 and TgfB1 expression and secretion in murine and human HSCs, hepatocytes, and HCC/hepatoblastoma cell lines. In various mouse models reflecting regeneration, acute and chronic liver disease, and human HCC sample cohorts, we demonstrated that both isoforms are expressed in different types of liver cells and that expression is elevated during the progression of CLD in mouse models in most cases. Although TgfB2 is mostly secreted at lower levels than TgfB1, its expression patterns largely follow similar profiles. However, the secretion of TgfB2 exceeded that of TgfB1 in some HCC cell lines. Our data indicates a more prominent implication of TgfB2 in biliary-derived liver disease models. In this thesis, the anti-fibrotic and immunoregulatory effects of TgfB2 silencing in cholestatic MDR2-KO mice have been delineated for the first time. TgfB2 silencing by AONs specifically reduced collagen deposition and αSMA expression, but induced anti-fibrotic PparG expression. Accumulation of TGF-β2-specific AON was detected in macrophage-activated fibroblasts, LSEC, and activated HSCs in mice. This was in accordance with TgfB2 expression in these cell types. CD45-positive immune cell infiltration was reduced upon TGF-β2-specific AON treatment in the livers of MDR2-KO mice. Furthermore, TGF-β2 levels were found to be elevated and correlated with CD45-positive immune cell infiltration in PSC and PBC patients. In summary, the data presented, provides a strong rationale to examine anti-TgfB2-directed treatment in patients with cholestatic liver damage as PSC or PBC. Taken together, this thesis points towards TGF-β2 as a promising therapeutic target in CLD especially of biliary origin. It provides a direct rationale for TGF-β2-directed drug development an further suggests to initiate a clinical trial testing TGF-β2 inhibition in PSC and PBC patients