Four-And-A-Half LIM-Domain Protein 2 (FHL2) Deficiency Aggravates Cholestatic Liver Injury

Cholestasis occurs in different clinical circumstances and leads to severe hepatic disorders. The four-and-a-half LIM-domain protein 2 (FHL2) is a scaffolding protein that modulates multiple signal transduction pathways in a tissue- and cell context-specific manner. In this study, we aimed to gain insight into the function of FHL2 in cholestatic liver injury. FHL2 expression was significantly increased in the bile duct ligation (BDL) model in mice. In Fhl2-deficient (Fhl2-ko) mice, BDL caused a more severe portal and parenchymal inflammation, extended portal fibrosis, higher serum transaminase levels, and higher pro-inflammatory and pro-fibrogenic gene expression compared to wild type (wt) mice. FHL2 depletion in HepG2 cells with siRNA resulted in a higher expression of the bile acid transporter Na+-taurocholate cotransporting polypeptide (NTCP) gene. Furthermore, FHL2-depleted HepG2 cells showed higher expression of markers for oxidative stress, lower B-cell lymphoma 2 (Bcl2) expression, and higher Bcl2-associated X protein (BAX) expression after stimulation with deoxycholic acid (DCA). In hepatic stellate cells (HSCs), FHL2 depletion caused an increased expression of TGF-beta and several pro-fibrogenic matrix metalloproteinases. In summary, our study shows that deficiency in FHL2 aggravates cholestatic liver injury and suggests FHL2-mediated effects on bile acid metabolisms and HSCs as potential mechanisms for pronounced hepatocellular injury and fibrosis

Diagnostic free text analysis in biobanks with CRIP.CodEx: Automated matching of classifications

Biobanks represent key resources for biomedical research. To be accessible, e.g. over web-based query tools or trans-institutional metabiobanks, the stored human biospecimens have to be annotated with clinical data, transformed into harmonized and structured form, e.g. ICD codes, while currently only available from free text records. The Biobank under Administration of Human Tissue and Cell Research Foundation HTCR at the University of Munich Medical Centre is routinely collecting remnant tissues and blood samples from treatments of patients. For diagnostic classification of the corresponding cases, a biobank specific classification was developed, but not yet matched to ICD codes. So far done manually, we now used the automated knowledge extraction software CRIP.CodEx, not needing a training set or access to external resources, to recodify the textual description of the specialized HTCR biobank classification with ICD. We show that the information contained in the nomenclature of the individual biobank specific catalogue of diagnoses is sufficient for a mapping towards ICD-10 as well as ICD-O-3 catalogues, and deliver an automated matching of two different classification systems using CRIP.CodEx

Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa.

The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies

260/ 280 and 260/ 230 ratios after a time course of warm (A, C) or cold (B, D) ischaemia.

<p>Values represent means ± SEM. All values were <i>N</i> = 6 except for 3 values that were <i>N</i> = 5; 260/ 280 ratio for warm ischaemia at 30 min (snap frozen), 260/ 230 ratios for warm ischaemia at 0 min (RNAlater) and 30 min (snap frozen). ^aSignificantly different from corresponding 0 min condition, <i>p</i> < 0.05. ^bSignificantly different from corresponding 90 min condition, <i>p</i> < 0.05.</p

Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.

<p>Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.</p

Description of the TaqMan Gene Expression Assays used.

<p>Description of the TaqMan Gene Expression Assays used.</p

Normalised gene expressions of <i>MYC</i> (A, B), <i>HIF1α</i> (C, D), <i>CDX2</i> (E, F), <i>HMOX1</i> (G, H) or <i>IL1β</i> (I, J) after a time course of warm or cold ischaemia.

<p>Values represent means ± SEM. All values were <i>N</i> = 6 unless otherwise stated. The following values were <i>N</i> = 5; <i>HMOX1</i> expression at 0 and 90 min warm ischaemia (RNAlater) and 0 min cold ischaemia (RNAlater), <i>IL1β</i> expression at 90 min warm ischaemia (RNAlater) and 30 min cold ischaemia (snap frozen). The following values were <i>N</i> = 4; <i>IL1β</i> expression at 0 min warm or cold ischaemia (RNAlater). Four technical replicates were done per donor for each data point. *Significantly different from corresponding snap-frozen condition, <i>p</i> < 0.05.</p

A representative electropherogram is shown for the highest (A) and lowest (B) average RNA Integrity Number (RIN) obtained in this study. RIN of ileum mucosa specimens collected by snap-freezing in liquid nitrogen or banked using RNA stabilization solution after a time course of warm (C) or cold ischaemia (D).

<p>Values represent means ± SEM with <i>N</i> = 6. *Significantly different from corresponding snap-frozen condition, <i>p</i> < 0.05.</p

Xanthohumol, a Prenylated Chalcone Derived from Hops, Inhibits Growth and Metastasis of Melanoma Cells

Simple Summary Melanoma is an aggressively growing form of skin cancer. It has a high metastatic potential, and the liver is one of the most common sites for visceral metastasis. Patients with hepatic metastases have a very poor prognosis, and effective forms of treatment are urgently needed. Xanthohumol is a natural compound of the hop plant with a wide range of beneficial health effects. In the present study, we show in cell culture experiments with human melanoma cells that xanthohumol inhibits several pro-tumorigenic mechanisms that are critical for melanoma metastasis. Furthermore, we analyzed the effect of xanthohumol application in a mouse model of melanoma metastasis and found that xanthohumol significantly inhibited the growth of melanoma metastases in the liver. Together with previous studies indicating that xanthohumol application is well tolerated, our present findings point to this natural compound as a promising novel treatment of melanoma patients with liver metastases. Melanoma is one of the most aggressive and lethal cancers worldwide. Despite recent progress in melanoma therapy, the prognosis for metastasized melanoma continues to be poor. Xanthohumol (XN), a prenylated chalcone derived from hop cones, is known to possess a broad spectrum of chemopreventive and anticancer activities. However, few studies have analyzed functional XN effects on melanoma cells and there have been no previous in vivo studies of its effects on metastasis. The aim of this study was to investigate the impact of XN on the tumorigenic and liver metastatic activity of melanoma cells. XN exhibited dose-dependent cytotoxic effects on human melanoma cell lines (Mel Ju; Mel Im) in vitro. Functional analysis in the subtoxic dose-range revealed that XN dose-dependently inhibited proliferation, colony formation, and migratory activity of melanoma cells. Subtoxic XN doses also induced markers of endoplasmic reticulum stress but inhibited the phosphorylation of the protumorigenic c-Jun N-terminal kinases (JNK). Furthermore, XN effects on hepatic metastasis were analyzed in a syngeneic murine model (splenic injection of murine B16 melanoma cells in C57/BL6 mice). Here, XN significantly reduced the formation of hepatic metastasis. Metastases formed in the liver of XN-treated mice revealed significantly larger areas of central necrosis and lower Ki67 expression scores compared to that of control mice. In conclusion, XN inhibits tumorigenicity of melanoma cells in vitro and significantly reduced hepatic metastasis of melanoma cells in mice. These data, in conjunction with an excellent safety profile that has been confirmed in previous studies, indicate XN as a promising novel agent for the treatment of hepatic (melanoma) metastasis