Liver fibrosis is defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requires in vitro and in vivo experimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

Human erythrovirus is a minute, single-stranded DNA virus causing many diseases, including erythema infectiosum, arthropathy, and fetal death. After primary infection, the viral genomes persist in solid tissues. Besides the prototype, virus type 1, two major variants (virus types 2 and 3) have been identified recently, the clinical significance and epidemiology of which are mostly unknown. We examined 523 samples of skin, synovium, tonsil, or liver (birth year range, 1913-2000), and 1,640 sera, by qualitative and quantitative molecular assays for the DNA of human erythroviruses. Virus types 1 and 2 were found in 132 (25%) and 58 (11%) tissues, respectively. DNA of virus type 1 was found in all age groups, whereas that of type 2 was strictly confined to those subjects born before 1973 (P < 0.001). Correspondingly, the sera from the past two decades contained DNA of type 1 but not type 2 or 3. Our data suggest strongly that the newly identified human erythrovirus type 2 as well as the prototype 1 circulated in Northern and Central Europe in equal frequency, more than half a century ago, whereafter type 2 disappeared from circulation. Type 3 never attained wide occurrence in this area during the past >70 years. The erythrovirus DNA persistence in human tissues is lifelong and represents a source of information about our past, the Bioportfolio, which, at the individual level, provides a registry of one's infectious encounters, and at the population level, a database for epidemiological and phylogenetic analyses.epidemiology ͉ gene therapy ͉ parvovirus ͉ phylogeny ͉ single-stranded DNA

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Infi ltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infi ltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4 R24C mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic infl ammatory response in the tumor microenvironment and signifi cantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibodymediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. SIGNIFICANCE:Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance. Cancer Discov; 4(6);

The toxic properties of various nitrosamines in animals and humans are well established. The parenteral or oral administration of the smallest quantities of diethylnitrosamine (DEN) or dimethylnitrosamine (DMN) results in severe liver damage. Most prominent are intense neutrophilic infiltration, extensive centrilobular haemorrhagic necrosis, bile duct proliferation, fibrosis, and bridging necrosis that ends in hepatocarcinogenesis. Due to the robustness of the induced hepatic alterations, the application of DEN in rodents has become an attractive experimental model for studies aimed at understanding the pathogenetic alterations underlying the formation of liver cancer, which represents one of the most common malignancies in humans worldwide. However, several studies have shown that the hepatocarcinogenic effects of nitrosamines might vary with the genetic background of the animals, their sex, their age, and other factors that might impact the outcome of experimentation. We present general guidelines for working with DEN, and a detailed protocol that allows the establishment of highly reproducible liver cancer in mice. The outcome of liver injury after the application of DEN in mice, as estimated by the formation of cirrhosis and cancer, appears to be a suitable animal model for the analysis of some aspects and processes that promote the pathogenesis of hepatocellular carcinoma in humans.

Autologous fibrin-based tissue-engineered heart valves have demonstrated excellent potential as patient-derived valve replacements. The present pilot study aims to evaluate the structure and mechanical durability of fibrin-based heart valves after implantation in a large-animal model (sheep). Tissue-engineered heart valves were molded using a fibrin scaffold and autologous arterial-derived cells before 28 days of mechanical conditioning. Conditioned valves were subsequently implanted in the pulmonary trunk of the same animals from which the cells were harvested. After 3 months in vivo, explanted valve conduits (n = 4) had remained intact and exhibited native tissue consistency, although leaflets demonstrated insufficiency because of tissue contraction. Routine histology showed remarkable tissue development and cell distribution, along with functional blood vessel ingrowth. A confluent monolayer of endothelial cells was present on the valve surface, as evidenced by scanning electron microscopy and positive von Willebrand factor staining. Immunohistochemistry and extracellular matrix (ECM) assay demonstrated complete resorption of the fibrin scaffold and replacement with ECM proteins. Transmission electron microscopy revealed mature collagen formation and viable, active resident tissue cells. The preliminary findings of implanted fibrin-based tissue-engineered heart valves are encouraging, with excellent tissue remodeling and structural durability after 3 months in vivo. The results from this pilot study highlight the potential for construction of completely "autologous" customized tissue-engineered heart valves based on a patient-derived fibrin scaffold.

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