This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Procalcitonin (PCT) is a 13 kD protein of which plasma concentrations are strongly increased in inflammatory states. PCT concentrations are claimed to have a more powerful discriminatory value for bacterial infection than the acute phase proteins serum amyloid A (SAA) or C-reactive protein (CRP). The source of production and its mechanism of induction are unknown. We investigated the inducibility of PCT both in vivo and in vitro and compared the behavior of PCT with those of SAA and CRP. A prospective descriptive patient sample study and a controlled liver tissue culture study. A university hospital. Cancer patients who were treated with human tumor necrosis factor-alpha (rhTNF-alpha; 5 patients) or interleukin-6 (rhIL-6; 7 patients). Serial serum samples were collected for analysis of concentrations of PCT, SAA, and CRP. In the TNF-alpha group, frequent sampling was performed on the first day to allow analysis of initial responses. In a human liver slice model, the release of PCT, SAA, and CRP was measured on induction with rhTNF-alpha and rhIL-6 for 24 hrs. We found that PCT displayed acute phase reactant behavior in vivo after administration of both rhTNF-alpha and rhIL-6. After rhTNF-alpha-administration, PCT reached half-maximal concentrations within 8 hrs, 12 hrs earlier than either SAA or CRP did. PCT, SAA, and CRP were produced in detectable quantities by liver tissue in vitro. PCT production by liver slices was enhanced after stimulation with rhTNF-alpha or rhIL-6; SAA and CRP concentrations were elevated after stimulation with rhTNF-alpha. We found that PCT and acute phase proteins such as CRP are induced by similar pathways. The liver appears to be a major source of PCT production. Thus, PCT may be considered an acute phase protein. The different kinetics of PCT, rather than a fundamentally different afferent pathway, may explain its putative diagnostic potential to discriminate bacterial infection from other causes of inflammation.

Endotoxin-induced cholestasis in rodents is caused by hepatic downregulation of transporters, including the basolateral Na⁺-dependent taurocholate transporter (ntcp) and the canalicular bile salt export pump (bsep) and multidrug resistance-associated protein 2 (mrp2). Details about the regulation of the human transporter proteins during this process are lacking. We used precision-cut human and rat liver slices to study the regulation of transporter expression during LPS-induced cholestasis. We investigated the effect of LPS on nitrate/nitrite and cytokine production in relation to the expression of inducible nitric oxide synthase, NTCP, BSEP, and MRP2 both at the level of mRNA with RT-PCR and protein using immunofluorescence microscopy. In liver slices from both species, LPS-induced expression of inducible nitric oxide synthase was detected within 1–3 h and remained increased over 24 h. In rat liver slices, this was accompanied by a significant decrease of rat ntcp and mrp2 mRNA levels, whereas bsep levels were not affected. These results are in line with previous in vivo studies and validate our liver slice technique. In LPS-treated human liver slices, NTCP mRNA was downregulated and showed an inverse correlation with the amounts of TNF-α and Il-1β produced. In contrast, MRP2 and BSEP mRNA levels were not affected under these conditions. However, after 24-h LPS challenge, both proteins were virtually absent in human liver slices, whereas marker proteins remained detectable. In conclusion, we show that posttranscriptional mechanisms play a more prominent role in LPS-induced regulation of human MRP2 and BSEP compared with the rat transporter proteins.

Objective: The aim of the present study is to investigate the association of gut microbiota, depending on treatment method, with the development of colorectal anastomotic leakage (AL). Background: AL is a major cause for morbidity and mortality after colorectal surgery, but the mechanism behind this complication still is not fully understood. Methods: Bacterial DNA was isolated from 123 “donuts” of patients where a stapled colorectal anastomosis was made and was analyzed using 16S MiSeq sequencing. In 63 patients, this anastomosis was covered with a C-seal, a bioresorbable sheath stapled to the anastomosis. Results: In non-C-seal patients, AL development was associated with low microbial diversity (P = 0.002) and correspondingly with a high abundance of the dominant Bacteroidaceae and Lachnospiraceae families (P = 0.008 and 0.010, respectively). In C-seal samples, where AL rates were slightly higher (25% vs 17%), an association with the gut microbiota composition was almost undetectable. Only a few opportunistic pathogenic groups of low abundance were associated with AL in C-seal patients, in particular Prevotella oralis (P = 0.007). Conclusions: AL in patients without a C-seal can be linked to the intestinal microbiota, in particular with a low microbial diversity and a higher abundance of especially mucin-degrading members of the Bacteroidaceae and Lachnospiraceae families. In C-seal patients, however, it seems that any potential protective benefits or harmful consequences of the gut microbiota composition in regard to wound healing are negated, as progression to AL is independent of the initially dominant bacterial composition.