Gonzalo Carrasco

Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(-/-)) by CCl(4) intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl(4)-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.

Overexpression of 11β-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue and portal hypercortisolism in non-alcoholic fatty liver disease.

The enzyme 11β‐hydroxysteroid‐dehydrogenase type 1 (11β‐HSD1) catalyses the reactivation of intracellular cortisol. We explored the potential role of 11β‐HSD1 overexpression in visceral adipose tissue (VAT) in non‐alcoholic fatty liver disease (NAFLD) assessing sequential changes of enzyme expression, in hepatic and adipose tissue, and the occurrence of portal hypercortisolism in obese mice. 11β‐HSD1 expression was also assessed in tissues from obese patients undergoing bariatric surgery.

Helicobacter pylori-Induced Chronic Gastritis and Assessing Risks for Gastric Cancer

Chronic gastritis is an inflammation of the gastric mucosa and has multiple etiologies. Here we discuss the pathological alterations induced by Helicobacter pylori (HP) leading to chronic gastritis and the epigenetic bases underlying these changes. We review the histology of the normal gastric mucosa and overview the role of HP in the multistep cascade of GC. We attempt to define the role of the Operative Link for Gastritis Assessment (OLGA) staging system in assessing the risk of GC. The epigenetic bases of chronic gastritis, mainly DNA methylation, are presented through examples such as (i) the methylation of the promoter region of E-cadherin in HP-induced chronic gastritis and its reversion after HP eradication and (ii) the association of methylation of the promoter region of Reprimo, a p53-mediated cell cycle arrest gene, with aggressive HP strains in high risk areas for GC. In addition, we discuss the finding of RPRM as a circulating cell-free DNA, offering the opportunity for noninvasive risk assessment of GC. Finally, the integration of OLGA and tissue biomarkers, by systems pathology approach, suggests that severe atrophy has a greater risk for GC development if, in addition, overexpressed p73. This trial is registered with ClinicalTrials.gov NCT01774266.

Helicobacter pylori—Induced Loss of the Inhibitor-of-Apoptosis Protein Survivin Is Linked to Gastritis and Death of Human Gastric Cells

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

Overexpression of p73 as a Tissue Marker for High-Risk Gastritis

Purpose: Histologic assessment of high-risk gastritis for the development of gastric cancer is not well defined. The identification of tissue markers together with the integration of histologic features will be required for this assessment. Experimental Design: Matched tumor/nontumor adjacent mucosa (NTAM) of 91 early gastric cancer and 148 chronic gastritis cases were evaluated for histologic characteristics (atrophy, intestinal metaplasia, chronic inflammation, polymorphonuclear infiltration, and Helicobacter pylori) by the Sydney System. Atrophy risk assessment was also evaluated by the Operative Link on Gastritis Assessment (OLGA) staging system. Eight tissue markers (BRCA1, HSP90, STAT1, FHIT, EGFR, p73, p53, p16INK4a) and EBV were also evaluated by tissue microarray/immunohistochemistry/in situ hybridization platform. Data were analyzed by contingency tables (2 × 2) using Fishers exact two-tailed test (P < 0.001) and integrated by Significance Analysis of Microarrays (SAM) and clustering analysis. Results: Histologically, NTAM have severe intestinal metaplasia/chronic inflammation and severe atrophy assessed by Sydney and OLGA staging systems. H. pylori infection was similar in both groups, and EBV was found only in 5.5% of the tumor samples. Overexpression of p73 was higher in NTAM (50.5%) than in chronic gastritis (10.8%; P < 0.0001). Integration of histologic features and tissue markers showed that overexpression of p73, severe atrophy, and OLGA stage 4 were the most relevant features in NTAM. Clustering analysis correctly assigned NTAM and control cases (P < 0.0001). Conclusions: Overexpression of p73 should be considered for the assessment of high-risk chronic gastritis. SAM allows the integration of histology and tissue markers for this assessment. Clin Cancer Res; 16(12); 3253–9. ©2010 AACR.

The Genetic and Epigenetic Bases of Gastritis

Gastritis, the inflammation of the mucosal layer of the stomach, is a major clinical entity due to its association with gastric cancer and peptic ulcer disease. The primary cause of gastritis is the infection with the microaerofilic gram negative Helicobacter pylori that during the early phases elicits an acute inflammatory response which eventually evolves to a longstanding chronic gastritis (Ruggiero 2012). In the case of gastric cancer development, chronic gastritis is the first step of the so-called multistep cascade of gastric cancer. This sequence includes the non-atrophic chronic gastritis, multifocal atrophic gastritis, intestinal metapla‐ sia, dysplasia and invasive carcinoma as were described in detail by Correa as the “human model of gastric carcinogenesis” (Fig. 1) (Correa et al. 2007). This multistep model hypothe‐ sizes that the sequence of these lesions reflects a dynamic process from a naive inflammation caused by H. pylori infection to a fully malignant neoplasm of the stomach (Correa et al. 1976; Cuello et al. 1976; Haenszel et al. 1976; Correa et al. 1990). Independent epidemiologi‐ cal studies have confirmed that non-atrophic, atrophic, intestinal metaplasia and dysplasia are all linked through a sequential cause-effect relationship, thus supporting the concept of a human model for gastric carcinogenesis (Ohata et al. 2004). However, the risk of malignant transformation of these lesions is poorly defined. Long-term follow-up studies have shown a risk from 10% to 17% in the case of dysplasia (Saraga et al. 1987; Coma del Corral et al. 1990; Koch et al. 1990; Whiting et al. 2002; Rugge et al. 2003). For intestinal metaplasia, the risk assessment has conflicting results and therefore a limited clinical value (Ramesar et al. 1987; Silva et al. 1990; Rokkas et al. 1991; Conchillo et al. 2001; Vannella et al. 2012). The re‐ cently developed Operative Link for Gastritis Assessment (OLGA) staging system (Rugge et al. 2007), through the evaluation of the extension and site of the atrophic changes, is an at‐ tempt to evaluate the risk of chronic gastritis to progress to intestinal metaplasia and gastric cancer (Rugge et al. 2008; Capelle et al. 2010; Rugge et al. 2010). In this scenario, the identifi‐

Molecular Pathology of Gastritis

Gastritis is an inflammation of the mucosa of the stomach, and has many etiologies. Gastritis can be classified as being acute or chronic. For the purpose of this chapter, we will focus only on chronic gastritis due to its relevance to gastric cancer. Among the causes of chronic gastritis are chronic bile reflux, stress, certain autoimmune disorders and bacterial infection, primarily Helicobacter pylori. Since 1870, both human and veterinary pathologists have described the presence of tiny curved bacteria within gastric mucosa, but the organisms were dismissed as irrelevant contaminants (1, 2). In 1947, when gastroscopy was first being used, Rudolf Schindler deemed gastritis as “one of the most debated diseases of the human body” and predicted that its significance would be discussed “for some time to come” (3). Schindler himself claimed that the “bacteriological etiology of chronic gastritis has not been convincingly proved in a single case” (3). In 1984, Warren and Marshall proposed that chronic “idiopathic” gastritis had a bacterial cause (i.e., H. pylori) (4). Their hypothesis was met with great skepticism. However, within a few years, the association between H. pylori gastritis, peptic ulcer, and gastric cancer came to be acknowledged and ultimately accepted (4). Subsequently, accurate morphological data were gathered by pathologic examination of autopsy material (5) and, later, of endoscopic biopsy specimens. As a result, distinct types and patterns of gastritis were recognized, which led to the conception, presentation, dismissal, and replacement of many different classification systems.

El imaginario de la urgencia: ¿es tan urgente lo urgente?

espanolEl imaginario de la urgencia se ha apoderado del discurso arquitectonico con una velocidad coherente con la rapidez de las respuestas que supone. La propia naturaleza de este concepto, sin embargo, ha imposibilitado su analisis mas detenido. Por eso en esta edicion de ARQ nos tomamos el tiempo para preguntar: ?es tan urgente lo urgente? ?Estamos ante una nueva conciencia social o es solo un cambio retorico? ?Es el reflejo de una necesidad o es simplemente un nuevo imaginario? EnglishThe imaginary of urgency has taken over architectural discourse at a speed consistent with the promptness of the answers it supposes. The very nature of the concept, still, has made it impossible to analyze more closely. That is why in this issue of ARQ we take the time for asking: is the urgent so urgent? Are we facing a new social consciousness or is it just a rhetorical change? Does it reflect an actual need or is it simply a new imaginary?

Abstract 1992: In silico transciptome analysis and experimental validation of epigenetically regulated genes in gastric carcinoma

Gastric Cancer is one of the most common cancers worldwide and no biomarkers are available for early detection. To search for potential biomarkers we analysed in silico transcriptome followed by experimental validation of epigenetically regulated genes. Methods. In silico transcriptome analysis was performed after download 4 normal and 10 tumor gastric Serial Gene Expression (SAGE) libraries from CGAP/GEO domains to create a single file containing 121,409 tags. Clustering libraries were identified by PCA, COA and Support Clustering. Significance Analysis for Microarray (SAM) was performed to select differentially expressed tags and SAGE Genie/TAGmapper was applied for tag-gene association. CpG island in the promoter region of selected genes was interrogated by UCSC or EMBOSS CpGPlot browsers. Functional annotation was performed by Gene Ontology (GO) using Babelomics. Experimental validation of epigenetically regulated genes was performed by RT-PCR in five gastric cancer cell lines (NCI-N87, MKN-45, KATO-III, AGS, HSC-38) and other cell lines (MCF7, ZR-75-1, A549, Hep2, JURKAT, CASKI, FS and Hek293T]) after 5 uM of 5-azaC incubation for 72 hrs. Genes with restored expression were further analyzed by bisulfite sequencing and clinico-pathological significance was evaluated by in 91 early gastric cancers and 148 chronic gastritis cases by Tissue Microarray (TMA) methodology. Results. Two thousand and four hundred and thirty seven (2%) tags were expressed in all normal libraries and all tumor libraries. PCA, COA and Support Cluster analyses clearly identified normal and tumor libraries and were confirmed by Support clustering analysis. SAM revealed 90 tags differentially expressed between these two libraries with 78 down-regulated and 12 up-regulated tags in tumor libraries. Tag to gene association identified 59 genes. Functional annotation revealed that most of downregulated genes were negative regulators of apoptosis (GO:0016265) and overexpressed genes were cell cycle regulators (GO:0007049) or angiogenesis inducers (GO:0001525). Interestingly, 16 genes did not have any prior investigation in gastric carcinoma. Finally by bona-fide CpG island analysis, 38 genes contained CpG in their promoter regions, and 12 demonstrated epigenetic regulation after 5-azaC incubation including FLT1 and STAT1. Bisulfite sequence confirmed CpG methylation changes for FLT1 gene and IHC staining of STAT1 on TMAs revealed almost significant overexpression in cancer-associated gastritis vs chronic gastritis control cases (p=0.057). Discussion. Experimental validation of in silico analysis of gastric carcinoma transcriptomes reveal epigenetic regulation of several genes. Among these, STAT1 is a promissory candidate to distinguish chronic gastritis from cancer-associated gastritis. Supported by FONDECYT 1080563 from Government of Chile. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1992.