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Lleo A.,Center for Autoimmune Liver Diseases | Lleo A.,University of Milan | Invernizzi P.,Center for Autoimmune Liver Diseases | Invernizzi P.,University of California at Davis
Digestive and Liver Disease | Year: 2013

Our understanding of primary biliary cirrhosis has been rapidly growing over the past decade and the disease is now regarded as a model for other female-predominant, organ-specific autoimmune conditions. Primary biliary cirrhosis ensues from a multi-lineage loss of tolerance to the E2 component of the pyruvate dehydrogenase complex. One of the major unanswered questions in the pathogenesis of primary biliary cirrhosis is the specificity of small intrahepatic bile ducts attack while PDC-E2 is present in mitochondria of all nucleated cells. Recent findings suggest that the uniqueness of the primary target tissue, biliary epithelium, may be of considerable importance for understanding primary biliary cirrhosis and that the biliary epithelial cell is more than an innocent victim. Rather, it attracts an immune attack by virtue of the unique apoptotic mechanisms and by the way it handles PDC-E2. Moreover, recent evidence suggests that apoptotic bodies of biliary epithelial cell are able to activate the innate immune system in the presence of anti-mitochondrial antibodies. This review article is intended to provide a critical overview of the role of apoptosis in biliary epithelial cells, the activation of the innate immune system, and its biological and clinical significance in primary biliary cirrhosis. © 2013 Editrice Gastroenterologica Italiana S.r.l.

Chen R.C.Y.,University of California at Davis | Naiyanetr P.,University of California at Davis | Naiyanetr P.,Mahidol University | Shu S.-A.,University of California at Davis | And 14 more authors.
Hepatology | Year: 2013

Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. Conclusion: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host. © 2012 American Association for the Study of Liver Diseases.

Lleo A.,Center for Autoimmune Liver Diseases | Oertelt-Prigione S.,Charité - Medical University of Berlin | Bianchi I.,Center for Autoimmune Liver Diseases | Caliari L.,Center for Autoimmune Liver Diseases | And 11 more authors.
Journal of Autoimmunity | Year: 2013

Sex chromosome abnormalities have been advocated to be involved in the striking female prevalence of primary biliary cirrhosis (PBC) and women with PBC manifest an increased X chromosome loss in peripheral blood mononuclear cells compared to age-matched healthy women. Our knowledge of the etiopathogenesis of autoimmunity in male patients remains, however, limited. Next to the possible role of androgens and their imbalances, the Y chromosome appears as a potential candidate for influence of the immune function in men. Herein we analyzed a population of male patients with primary biliary cirrhosis (n = 26) and healthy controls (n = 88) to define a potential association of disease and the loss of the Y chromosome. We demonstrate that Y chromosome loss indeed is higher in PBC males compared to healthy controls, and this phenomenon increases with aging. We were, thus, able to confirm the existence of an analogous mechanism in the male population to previously identified X haploinsufficiency in female patients with organ-specific autoimmune disease. We propose that this commonality might represent a relevant feature in the etiopathogenesis of autoimmune diseases that should be further investigated. © 2013 Elsevier Ltd.

Bogdanos D.P.,King's College London | Bogdanos D.P.,University of Thessaly | Smyk D.S.,King's College London | Invernizzi P.,Center for Autoimmune Liver Diseases | And 4 more authors.
Autoimmunity Reviews | Year: 2013

The "exposome" is a term recently used to describe all environmental factors, both exogenous and endogenous, which we are exposed to in a lifetime. It represents an important tool in the study of autoimmunity, complementing classical immunological research tools and cutting-edge genome wide association studies (GWAS). Recently, environmental wide association studies (EWAS) investigated the effect of environment in the development of diseases. Environmental triggers are largely subdivided into infectious and non-infectious agents. In this review, we introduce the concept of the "infectome", which is the part of the exposome referring to the collection of an individual's exposures to infectious agents. The infectome directly relates to geoepidemiological, serological and molecular evidence of the co-occurrence of several infectious agents associated with autoimmune diseases that may provide hints for the triggering factors responsible for the pathogenesis of autoimmunity. We discuss the implications that the investigation of the infectome may have for the understanding of microbial/host interactions in autoimmune diseases with long, pre-clinical phases. It may also contribute to the concept of the human body as a superorganism where the microbiome is part of the whole organism, as can be seen with mitochondria which existed as microbes prior to becoming organelles in eukaryotic cells of multicellular organisms over time. A similar argument can now be made in regard to normal intestinal flora, living in symbiosis within the host. We also provide practical examples as to how we can characterise and measure the totality of a disease-specific infectome, based on the experimental approaches employed from the "immunome" and "microbiome" projects. © 2012 Elsevier B.V.

PubMed | Humanitas Research Hospital, University of Milan Bicocca, Copenhagen University, University of the Basque Country and 8 more.
Type: Journal Article | Journal: Journal of hepatology | Year: 2016

A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche.CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Gentilini A.,University of Florence | Rombouts K.,University of Florence | Galastri S.,University of Florence | Caligiuri A.,University of Florence | And 9 more authors.
Journal of Hepatology | Year: 2012

Backgrounds & Aims: Cholangiocarcinoma (CCA) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. Migration, invasion, and metastasis of CCA cells are modulated by signals received from stromal cells. The SDF-1-CXCR4 axis emerges as a pivotal regulator of migration and survival of different tumor cells. The aim of the present study was to characterize the interaction between CCA cells and human hepatic stellate cells (hHSC) focusing on the role of SDF-1. Methods: The intrahepatic CCA cell line HuCCT-1 and primary hHSC were used for this study. RNA expression was examined by RTQ-PCR and protein expression by Western blotting. Immunofluorescence microscopy and immunohistochemistry were also employed. Migration of CCA cells was assessed using modified Boyden chambers. Results: CXCR4 was clearly expressed in CCA cells of human CCA liver specimens. SDF-1 and hHSC conditioned medium (CM) promoted HuCCT-1 cell migration, which was abrogated by pre-incubation with AMD3100, a non-peptide antagonist of the CXCR4 receptor. In addition, HuCCT-1 cells silenced for CXCR4 did not migrate in presence of SDF-1. Both P-ERK and p-AKT were implicated in HuCCT-1 migration and showed a biphasic trend under stimulation of SDF-1. Finally, SDF-1 induced apoptotic rescue of HuCCT-1 cells by binding to CXCR4. Conclusions: Our study demonstrates that CCA cells migration and survival are modulated by the crosstalk between SDF-1, released by hHSC, and HuCCT-1 cells bearing CXCR4. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Iborra M.,Polytechnic University of Valencia | Bernuzzi F.,Center for Autoimmune Liver Diseases | Correale C.,IBD Unit | Vetrano S.,IBD Unit | And 8 more authors.
Clinical and Experimental Immunology | Year: 2013

The altered expression of micro-RNA (miRNA) has been associated with Crohn's disease (CD) and ulcerative colitis (UC). The aim of this study was to establish specific miRNA expression patterns in the serum and mucosa of inflammatory bowel disease (IBD) patients (UC and CD with colonic involvement) at different stages of the disease. Serum and biopsies from nine active CD (aCD), nine inactive CD (iCD), nine active UC (aUC) and nine inactive UC (iUC) and serum from 33 healthy subjects were collected. Up to 700 miRNAs were evaluated by the TaqMan® human miRNA array. The ΔCt values were obtained using the mean expression values of all expressed miRNAs in a given sample as a normalization factor for miRNA real-time quantitative polymerase chain reaction data. The levels of serum miRNAs in CD and UC patients were different to healthy subjects. Thirteen serum miRNAs were expressed commonly in CD and UC patients. Two miRNAs were higher and four miRNAs were lower in the serum of aCD than iCD. No serum miRNA was regulated exclusively in aUC compared with iUC patients. Four miRNAs were higher and three miRNAs were lower in the mucosa of aCD than iCD. Two miRNAs were higher and three miRNAs were lower in the mucosa of aUC than iUC. No serum miRNAs coincided with tissue miRNAs in aCD and aUC patients. Our results suggest the existence of specific miRNA expression patterns associated with IBD and their different stages and support the utility of miRNA as possible biomarkers. This pilot study needs to be validated in a large prospective cohort. © 2013 British Society for Immunology.

Castronovo C.,IRCSS Instituto Auxologico Italiano | Rossetti R.,University of Milan | Rusconi D.,IRCSS Instituto Auxologico Italiano | Recalcati M.P.,IRCSS Instituto Auxologico Italiano | And 10 more authors.
Human Reproduction | Year: 2014

STUDY QUESTIONWhat is the burden of X chromosome mosaicism in the occurrence of spontaneous menarche (SM) in Turner syndrome (TS)?SUMMARY ANSWERSM was significantly associated with X chromosome mosaicism in the TS patients; a mosaicism with around 10% euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques on uncultivated tissues.WHAT IS KNOWN ALREADYSpontaneous puberty can be observed in a minority of patients with TS, more frequently, but not exclusively, in those with a high level of 46,XX/45,X mosaicism at standard karyotype. The genetic mechanisms contributing to ovarian function in TS patients are still not determined. However, submicroscopic X-linked and autosomal copy number variations (CNVs) have recently emerged as an important genetic risk category for premature ovarian insufficiency and may be involved in modulating the TS ovarian phenotype.STUDY DESIGN, SIZE, DURATIONA group of 40 patients with a diagnosis of TS at conventional karyotyping participated in the study; 6 patients had SM and 34 patients had primary amenorrhoea (PA). All clinical data and the patients' DNA samples were collected over the years at a single paediatric clinic.PARTICIPANTS/MATERIALS, SETTING, METHODSThe patients' samples were used to perform both genetic (Copy Number Assay) and molecular-cytogenetic (array-CGH and iFISH, interphase-FISH) analyses in order to evaluate the X chromosome mosaicism rate and to detect possible rare CNVs of genes with a known or predicted role in female fertility.MAIN RESULTS AND THE ROLE OF CHANCEAll TS patients showed variable percentages of the 46,XX lineage, but these percentages were higher in the SM group (P < 0.01). A mosaicism around 10% for the euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques performed in uncultivated tissues. A few CNVs involving autosomal and X-linked ovary-related loci were identified by array-CGH analysis and confirmed by real-time quantitative PCR, including a BMP15 gene duplication at Xp11.22, a deletion interrupting the PAPPA gene at 9q33.1, and an intragenic duplication involving the PDE8A gene at 15q25.3.LIMITATIONS, REASONS FOR CAUTIONThis is a pilot study on a relatively small sample size and confirmation in larger TS cohorts may be required. The ovarian tissue could not be studied in any patients and in a subgroup of patients, the mosaicism was estimated in tissues of different embryonic origin.WIDER IMPLICATIONS OF THE FINDINGSThe combined determination of X chromosome mosaicism by molecular and molecular-cytogenetic techniques may become useful for the prediction of SM in TS. The detection of CNVs in both X-linked and autosomal ovary-related genes further suggests gene dosage as a relevant mechanism contributing to the ovarian phenotype of TS patients. These CNVs may pinpoint novel candidates relevant to female fertility and generate further insights into the mechanisms contributing to ovarian function.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by Telethon Foundation (grant no: GGP09126 to L.P.), the Italian Ministry of the University and Research (grant number: 2006065999 to P.F.) and a Ministry of Health grant 'Ricerca Corrente' to IRCCS Istituto Auxologico Italiano (grant number: 08C704-2006). The authors have no conflict of interest to declare. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

PubMed | Scott & White Medical Center, Center for Autoimmune Liver Diseases, Baylor Scott & White Digestive Disease Research Center, Central Texas Veterans Health Care System and Texas A&M University
Type: | Journal: Cancer letters | Year: 2016

Cholangiocarcinoma (CCA) is a malignancy of the biliary epithelium that is associated with low five-year survival. The apelin receptor (APLNR), which is activated by the apelin peptide, has not been studied in CCA. The purpose of this study is to determine if inhibition of the apelin/APLNR axis can inhibit CCA growth.Immunohistochemistry, rtPCR, immunofluorescence, flow cytometry, and ELISA was used to measure APLNR expression in human CCA cells and tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative and angiogenic genes were measured via rtPCR. Invivo, Mz-ChA-1 cells were injected into the flanks of nu/nu mice, which were treated with ML221 (150g/kg) via tail vein injection.Expression of the apelin/APLNR axis was increased in CCA. Invitro, CCA proliferation and angiogenesis was inhibited by ML221 treatment. ML221 treatment significantly decreased tumor growth in nu/nu mice.The apelin/APLNR axis regulates CCA proliferation and angiogenesis. Inhibition of the apelin/APLNR axis decreases tumor growth in our xenograft model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA.

PubMed | Center for Autoimmune Liver Diseases
Type: | Journal: Oncotarget | Year: 2016

Poor prognosis and high recurrence remain leading causes of primary liver cancer-associated mortality. The spread of circulating tumor cells (CTCs) in the blood plays a major role in the initiation of metastasis and tumor recurrence after surgery. Nevertheless, only a subset of CTCs can survive, migrate to distant sites and establish secondary tumors. Consistent with cancer stem cell (CSC) hypothesis, stem-like CTCs might represent a potential source for cancer relapse and distant metastasis. Thus, identification of stem-like metastasis-initiating CTC-subset may provide useful clinically prognostic information. This review will emphasize the most relevant findings of CTCs in the context of stem-like biology associated to liver carcinogenesis. In this view, the emerging field of stem-like CTCs may deliver substantial contribution in liver cancer field in order to move to personalized approaches for diagnosis, prognosis and therapy.

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