Fibrosis is a common feature in most pathogenetic processes in the liver, and usually results from a chronic insult that depletes the regenerative capacity of hepatocytes and activates multiple inflammatory pathways, recruiting resident and circulating immune cells, endothelial cells, non-parenchymal hepatic stellate cells, and fibroblasts, which become activated and lead to excessive extracellular matrix accumulation. The ongoing development of liver fibrosis results in a clinically silent and progressive loss of hepatocyte function, demanding the constant need for liver transplantation in clinical practice, and motivating the search for other treatments as the chances of obtaining compatible viable livers become scarcer. Although initially cell therapy has emerged as a plausible alternative to organ transplantation, many factors still challenge the establishment of this technique as a main or even additional therapeutic tool. Herein, the authors discuss the most recent advances and point out the corners and some controversies over several protocols and models that have shown promising results as potential candidates for cell therapy for liver fibrosis, presenting the respective mechanisms proposed for liver regeneration in each case.

Seed maintenance of parental lines of red clover ( Trifolium pratense L.) was examined in an I 1 sib, population derived from a single clone to determine amounts of selfing and sibbing. Seeds per head produced by pseudoself‐compatibility (PSC) on uniformly heterozygous S‐allele clones under cage varied from 0 to 8.8. Twelve cages with paired leafmarked ( LL or Ll ) and non‐leafmarked ( ll ) clones produced progenies which, as shown by leafmarking, resulted more from selfing than sibbing. Cages with a high percentage of selfing produced fewer seeds than those with a high percentage of sibbing. Amount of PSC in field cages was comparable to that produced on excised stems at 40 C in the laboratory, indicating that the growing of red clover in warmer climates for PSC seed production may not be necessary. The prevalence of selfing rather than sibbing may result in high inbreeding depression manifested by loss of vigor and PSC. However, the possibility remains that selfs can be eliminated by selection and the line propagated by the remaining sibs. Further investigation of seed maintenance schemes seems justified.

Copper (Cu) is an essential trace element that is vital to the health of all living organisms. As a transition metal, it is involved in a myriad of biological processes. Balance studies estimated that the adult human requirement for copper is in the range of 1.3 to 2 mg per day. Cu deficiency alters immune function, neuropeptide synthesis and antioxidant defense, while the excess in Cu results in oxidative stress and progressive structural damage of mitochondrial and clinically in hepatic and/or neurological symptoms. This becomes particularly visible in Wilson’s disease (WD) representing a rare autosomal recessive inherited disorder with a disease prevalence of about 1 in 30,000 people. The affected gene, i.e., ATP7B , belongs to the class of ATP-dependent, P-type Cu-transporting ATPases. To understand the pathomechanism in WD, several experimental models for studying WD were established. Independent studies performed in these models showed that the inactivation of the Atp7b gene results in a gradual increase in Cu in many organs during life span. However, the exact distribution of Cu and the potential impact of elevated Cu concentrations on other metals within the tissue are only sparely analyzed. Recently, novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based protocols for metal bio-imaging in liver and brain were established. In the present review, we will discuss the methodological background of this innovative technique and summarize our experiences using LA-ICP-MS imaging in biological monitoring, exact measurement, and spatial assignment of metals within tissue obtained from Atp7b null mice and clinical specimens taken from patients suffering from genetically confirmed WD. Using WD as an example, the data discussed demonstrates that LA-ICP-MS has multi-element capability, allowing precise measurement and visualization of metals in the tissue with high spatial resolution, sensitivity, quantification ability, and exceptional reproducibility.

Background: Recent clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with ectopic manifestations including neuroinflammation and neurodegeneration, but the mediators and critical pathways involved are not well understood. Lipocalin 2 (Lcn2) is one of the important mediators exclusively produced in the liver and circulation during NASH pathology. Methods: Using murine model of NASH, we studied the role of Lcn2 as a potent mediator of neuroinflammation and neurodegeneration in NASH pathology via the liver-brain axis.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers worldwide. Therefore, current global research focuses on molecular tools for early diagnosis of HCC, which can lead to effective treatment at an early stage. Perilipin 5 (PLIN5) has been studied as one of the main proteins of the perilipin family, whose role is to maintain lipid homeostasis by inhibiting lipolysis. In this study, we show for the first time that PLIN5 is strongly expressed in tumors of human patients with HCC as well as in mouse livers, in which HCC was genetically or experimentally induced by treatment with the genotoxic agent diethylnitrosamine. Moreover, the secreted acute phase glycoprotein Lipocalin 2 (LCN2) established as a biomarker of acute kidney injury, is also proven to indicate liver injury with upregulated expression in numerous cases of hepatic damage, including steatohepatitis. LCN2 has been studied in various cancers, and it has been assigned roles in multiple cellular processes such as the suppression of the invasion of HCC cells and their metastatic abilities. The presence of this protein in blood and urine, in combination with the presence of α -Fetoprotein (AFP), is hypothesized to serve as a biomarker of early stages of HCC. In the current study, we show in humans and mice that LCN2 is secreted into the serum from liver cancer tissue. We also show that AFP-positive hepatocytes represent the main source for the massive expression of LCN2 in tumoral tissue. Thus, the strong presence of PLIN5 and LCN2 in HCC and understanding their roles could establish them as markers for diagnosis or as treatment targets against HCC.

Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4-/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4-/- mice were unable to generate activated Protein C, a protein with anti-inflammatory activities on monocytes/macrophages. In vitro, PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of activated Protein C. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases.

Unfolded protein response (UPR) is an adaptive response, allowing the endoplasmic reticulum (ER) responds to an accumulation of unfolded proteins in its lumen, also known as ER stress. The ER reacts to ER stress through ER transmembrane protein sensors, thus activating intracellular signal transduction pathways. The UPR is interconnected with inflammation through reactive oxygen species production, activation of nuclear factor-kB (NF-kB) and JUN N-terminal kinase (JNK) via inositol-requiring enzyme 1 (IRE1) and induction of acute-phase response. LCN2 is one of the acute phase proteins that are induced under inflammatory conditions and up-regulated during ER stress. We therefore examined the ER stress responses in LCN2-/- condition.

Supplementary Figure 3 from Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Supplementary Figure from Colorectal Cancer Is Associated with the Presence of Cancer Driver Mutations in Normal Colon

<div>Abstract<p>Gene amplification is one of the most frequent manifestations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of drug resistance. To better understand the factors involved in acquired resistance to cytotoxic drugs via gene amplification, we have analyzed the structure and dynamics of dihydrofolate reductase (<i>DHFR</i>) gene amplification in HT29 cells treated with methotrexate (MTX). Analysis of the <i>DHFR</i> gene amplification process shows that the amplicon exhibits a complex structure that is consistently reproduced in independent treatments. The cytogenetic manifestation of the amplification in advanced stages of the treatment may be in the form of double minutes or as a homogeneously stained region. To get insights into the mechanisms of resistance, we have also investigated the sensitization to MTX of MTX-resistant cells after drug withdrawal and reexposure to MTX. Passive loss of the DHFR amplicon by withdrawal of the drug results in MTX-sensitive cells exhibiting a substantial reduction of their capacity or even an incapacity to generate resistance when submitted to a second cycle of MTX treatment. On a second round of drug administration, the resistant cells generate a different amplicon structure, suggesting that the formation of the amplicon as in the first cycle of treatment is not feasible. These results indicate that <i>DHFR</i> gene amplification is a “wear and tear” process in HT29 cells and that MTX-resistant cells may become responsive to a second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009;8(2):424–32]</p></div>

Immune checkpoint inhibitors have shown clinical benefit in several cancer entities including metastatic microsatellite instable colorectal carcinomas. However, for the majority of metastatic colorectal carcinomas the potential and limitations of immune checkpoint inhibition is not fully understood. In this study, the effects of sole and dual CTLA-4 and PD-L1 blockade were investigated in a microsatellite stable highly aggressive orthotopic mouse model of colon cancer. Dual CTLA-4 and PD-L1 inhibition resulted in tumor growth stagnation and completely blocked liver metastasis. Sole CTLA-4 and PD-L1 inhibition only moderately reduced metastatic spread of the colon cancer cells, though CTLA-4 blockade being superior to PD-L1 inhibition. Dual immune checkpoint blockade and sole CTLA-4 inhibition significantly increased intratumoral CD8+ and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells. This was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12. Moreover, tumors treated with combined immune checkpoint blockade showed the strongest increase in intratumoral iNOS+ macrophages, reduction of PD-L1+ and Tie2+ macrophages and the lowest expression of M2/Th2-related IL-4, TARC and COX-2. The assessment of further microenvironmental changes by DCE-MRI and immunohistology revealed no alterations in functional tumor vascularization upon combined immune checkpoint blockade, but a significant increase in intratumoral fibroblasts and collagen I deposition. Thus, the synergistic inhibitory effects of dual immune checkpoint inhibition can be explained by anti-tumorigenic T cell responses mediated by CTLA-4 inhibition and M1 macrophage polarization predominantly induced by PD-L1 blockade. This was accompanied by pronounced fibroblast activation highlighting the interconnection between immunogenicity and desmoplasia.

<div>Abstract<p><b>Background:</b> Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers.</p><p><b>Methods:</b> We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls.</p><p><b>Results:</b> Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of <i>EN1</i>, <i>SCTR</i>, and <i>INHBB</i> and corresponding loss of H3K27me3. <i>EN1</i> and <i>SCTR</i> were frequently methylated (65% and 53%, respectively), whereas <i>INHBB</i> was less frequently methylated.</p><p><b>Conclusions:</b> Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of <i>EN1</i> and <i>SCTR</i> was able to differentiate cancer from normal (<i>P</i> < 0.0001) and improved the diagnostic specificity of <i>GSTP1</i> methylation for prostate cancer detection by 26%.</p><p><b>Impact:</b> For the first time we show that DNA methylation of <i>EN1</i> and <i>SCTR</i> promoters provide potential novel biomarkers for prostate cancer detection and in combination with <i>GSTP1</i> methylation can add increased specificity and sensitivity to improve diagnostic potential. <i>Cancer Epidemiol Biomarkers Prev; 20(1); 148–59. ©2011 AACR</i>.</p></div>

Mass spectrometry imaging (MSI) using laser ablation (LA) inductively coupled plasma (ICP) is an innovative and exciting methodology to perform highly sensitive elemental analyses. LA-ICP-MSI of metals, trace elements or isotopes in tissues has been applied to a range of biological samples. Several LA-ICP-MSI studies have shown that metals have a highly compartmentalized distribution in some organs, which might be altered in consequence of genetic diseases, intoxication, or malnutrition. Although metal imaging by LA-ICP-MSI is an established methodology, potential pitfalls in the determination of metal concentrations might result from erroneous calibration, standardization, and normalization. In addition, for simple display of final imaging results, most LA-ICP-MSI users prefer to process their measurements by commercial processing software. Such programs typically visualize the regional metal differences in colorful and vivid imaging maps, but might not represent the actual signal densities correctly. There is a great abundance of such MSI data processing programs available differing in quality, usability, integrated features, workflow, reliability, system requirements, speed of data processing, and price. Some software packages contain a multitude of features which are superfluous for most users. In contrast, often only few data formats are used, in case of commercial programs even only the instrument provider's own raw data format. Therefore, first time and average users are often confused and helpless in choosing the correct software for processing their data. Here we have briefly summarized software packages, data routines, macros, programming tools, scripts, algorithms, or self-written patches and updates for existing programs presently in use for mining LA-ICP-MSI data.

Supplementary Figure 1 from Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers