Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury

The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.

Autophagic programmed cell death in the suspensor and endosperm of Vicia faba : An ultrastructural study

Programmed cell death (PCD) is a widely spread phenomenon among multi-cellular organisms. Without the deletion of cells no longer needed, the organism will not be able to develop in a predicted way. It is now belived that all cells have the capacity to self-destruct and that the survival of the cells is depending on the repression of this suicidal programme. PCD has turned out to show similarities in many different species and there are strong indications that the mechanisms running the programme might, at least in some parts, be evolutionarily conserced. PCD is a generic term for different programmes of cell destruction, such as apoptosis and autophagic PCD. An important tool to determine if a cell is undergoing PCD is the transmitting electron microscope. The aims of my study were to find out if, and in what way, the suspensor and endosperm in Vicia faba (Broad bean), which are short-lived structures, undergoes PCD. The endosperm degradation preceed the suspensor cell death and they differ to some extent ultrastructurally. The cell death occurs in both tissues about 13-14 days after pollination when the embryo proper is mature enough to support itself. It was found that both tissues are committed to autophagic PCD, a cell death characteristic of conspicuous formations of autophagic vacuoles. It was shown by histochemical staining that acid phosphatases are accumulated in these vacuoles but are also present in the cytoplasm. These vacuoles are similar to autophagic vacuoles formed in rat liver cells, indicating that autophagy is a widely spread phenomenon. DNA fragmentation is the first visible sign of PCD in both tissues and it is demonstrated by a labelling technique (TUNEL). In the endosperm nuclei the heterochromatin subsequently appears in the form of a network, while in the suspensor it is more conspicuous, with heterochromatin that forms large electron dense aggregates located close to the nuclear envelope. In the suspensor, the plastids develop into chromoplasts with lycopene crystals at the same time or shortly after DNA fragmentation. This is probably due to the fact that the suspensor plastids function as hormone producing organelles and support the embryo proper with indispensable growth factors. Later the embryo will be able to produce its own growth factors and the synthesis of these, in particular gibberelines, might be suppressed in the suspensor. The precursors can then be used for synthesis of lycopene instead. Both the suspensor and endosperm are going through autophagic PCD, but the process differs in some respects. This is probably due the the different function of the two tissues, and that the signals that trigger the process presumably are different. The embryo proper is probably the source of the death signal affecting the suspensor. The endosperm, which has a different origin and function, might be controlling the death signal within its own cell. The death might in this case be related to the age of the cell.

Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

The role of Interleukin-12 in liver inflammation

Chronic liver inflammation during viral hepatitis is a major health problem worldwide. The role of proinflammatory cytokines, like IL-12, in breaking hepatic immune tolerance, and inducing acute liver inflammation and virus clearance is not clear. Nor is clear its role in uncontrolled severe inflammatory response, leading to fulminant hepatitis and hepatic failure. This work, focused in the study of the role of endogenous produced IL-12 in inducing hepatic inflammatory responses, demonstrates: In vitro, using adenovirus coding for IL-12, that hepatocytes stimulate CD4+ T cells in a tolerogenic manner, and that endogenous IL-12 is able to switch the immune response into Th1; and in vivo, that endogenous IL-12 induces hepatocyte damage and virus elimination in mice infected with adenovirus. In addition, and in order to study in vivo the relevance of IL-12 in acute inflammation, conditional IL-12 transgenic mice expressing IL-12 in the liver after cre-recombinase mediated induction were generated. For this purpose, an IL-12 fusion protein was created, which demonstrated high levels of bioactivity. Induction of IL-12 expression during embryonic development was achieved by crossbreeding with Act-Cre transgenic mice; induction of IL-12 expression in adult mice was achieved by a plasmid coding for the cre-recombinase. This study demonstrates that after induction, IL-12 is expressed in the liver of the transgenic mice. It also demonstrates that hepatic expression of IL-12 induces splenomegaly and liver inflammation, characterized by large infiltrations in portal tracts and veins, associated with hepatic damage, necrosis areas and lethality. Furthermore, constitutive hepatic IL-12 expression does not lead to abortion, but to total lethality, short after delivery. In conclusion, in this study, a transgenic mouse model has been generated, in which the expression of active IL-12 in the liver can be induced at any time; this model will be very helpful for studying hepatic pathologies. This study has also demonstrated that hepatic produced IL-12 is able of breaking liver tolerance inducing inflammation, virus elimination, severe hepatocyte damage, and lethality. These findings suggest IL-12 as a key cytokine in acute liver inflammation and fulminant hepatic failure. 5.1 Future studies Once the importance of IL-12 in inducing hepatic inflammation and virus elimination was demonstrated in this study, understanding the mechanisms of the IL-12 induced liver damage, and more important, how to avoid it will be the main focus in the future. It is very important to achieve hepatic inflammation for a more effective and faster viral elimination, but avoiding the toxicity of IL-12, which leads to massive liver injury and lethality is obviously necessary to allow IL-12 as therapy. For that purpose, future studies will be mainly base on three different points: 1. The determination of different cell populations present in the hepatic infiltration, which of them are responsible for liver injury, and as well their state of activation. 2. The measure of other pro- and anti-inflammatory cytokines and chemokines, which can play a role in IL-12-induced liver inflammation and hepatocyte damage. For these purposes, specific blocking antibodies (anti TNF-alpha, anti IL-12, anti IFN-g) will be used. The study with different transgenic mice: TNF-alpha Receptor knockout, TGF-b, will also help in determining the role of those cytokines during IL-12-induced liver damage and lethality. 3. The establishing of liver pathology models (viral infection, tumours, auto-antigens) in mice. Induction of IL-12 at any time of the pathology development will help in clarifying the role of IL-12 in those models. Finally, the transgenic mice expressing IL-23 in the liver will be generated.

Boron determination in biological samples - Intercomparison of three analytical methods to assist development of a treatment protocol for neoplastic diseases of the liver with Boron Neutron Capture Therapy

Die Bor-Neuroneneinfang-Therapie (engl.: Boron Neutron Capture Therapy, BNCT) ist eine indirekte Strahlentherapie, welche durch die gezielte Freisetzung von dicht ionisierender Strahlung Tumorzellen zerstört. Die freigesetzten Ionen sind Spaltfragmente einer Kernreaktion, bei welcher das Isotop 10B ein niederenergetisches (thermisches) Neutron einfängt. Das 10B wird durch ein spezielles Borpräparat in den Tumorzellen angereichert, welches selbst nicht radioaktiv ist. rnAn der Johannes Gutenberg-Universität Mainz wurde die Forschung für die Anwendung eines klinischen Behandlungsprotokolls durch zwei Heilversuche bei Patienten mit kolorektalen Lebermetastasen an der Universität Pavia, Italien, angeregt, bei denen die Leber außerhalb des Körpers in einem Forschungsreaktor bestrahlt wurde. Als erster Schritt wurde in Kooperation verschiedener universitärer Institute eine klinische Studie zur Bestimmung klinisch relevanter Parameter wie der Borverteilung in verschiedenen Geweben und dem pharmakokinetischen Aufnahmeverhalten des Borpräparates initiiert.rnDie Borkonzentration in den Gewebeproben wurde hinsichtlich ihrer räumlichen Verteilung in verschiedenen Zellarealen bestimmt, um mehr über das Aufnahmeverhalten der Zellen für das BPA im Hinblick auf ihre biologischen Charakteristika zu erfahren. Die Borbestimung wurde per Quantitative Neutron Capture Radiography, Prompt Gamma Activation Analysis und Inductively Coupled Plasma Mass Spectroscopy parallel zur histologischen Analyse des Gewebes durchgeführt. Es war möglich zu zeigen, dass in Proben aus Tumorgewebe und aus tumorfreiem Gewebe mit unterschiedlichen morphologischen Eigenschaften eine sehr heterogene Borverteilung vorliegt. Die Ergebnisse der Blutproben werden für die Erstellung eines pharmakokinetischen Modells verwendet und sind in Übereinstimmung mit existierenden pharmakokinetische Modellen. Zusätzlich wurden die Methoden zur Borbestimmung über speziell hergestellte Referenzstandards untereinander verglichen. Dabei wurde eine gute Übereinstimmung der Ergebnisse festgestellt, ferner wurde für alle biologischen Proben Standardanalyseprotokolle erstellt.rnDie bisher erhaltenen Ergebnisse der klinischen Studie sind vielversprechend, lassen aber noch keine endgültigen Schlussfolgerungen hinsichtlich der Wirksamkeit von BNCT für maligne Lebererkrankungen zu. rn

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

Extensive extranodal metastases of basal-like breast cancer with predominant myoepithelial spindle cell differentiation

A differentiation towards myoepithelial cells has been demonstrated in several types of lesions in the breast. These include multifocal myoepitheliomatosis, the rare mixed tumor or pleomorphic adenoma, adenoid cystic carcinoma, adenomyoepithelioma and myoepithelial carcinoma (malignant myoepithelioma). Myoepithelial carcinoma is the only lesion purely composed of myoepithelial cells. All these tumors are benign and/or of low-grade malignancy, with the exception of malignant myoepithelioma. In contrast to the statement of the current World Health Organization (WHO), recent studies have reported that regional and distant metastases may occur in about 50% of pure myoepithelial carcinomas. The presented case of a breast carcinoma with dominant myoepithelial/spindle cell differentiation in a 58-year-old woman is an excellent example to document the highly aggressive biological behavior of this tumor phenotype. Despite an extensive chemotherapy and radiotherapy, the tumor was rapidly progressive, forming a finally exulcerating local tumor relapse and widespread metastases to the myocardium, lungs, liver, kidneys and skin. Similarities in morphology and biological behavior compared to patients with "triple-negative" (hormone receptor and Her2) monophasic sarcomatoid carcinomas and pure spindle cell sarcomas are discussed.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.

Role of TRAIL and the pro-apoptotic Bcl-2 homolog Bim in acetaminophen-induced liver damage

Acetaminophen (N-acetyl-para-aminophenol (APAP), paracetamol) is a commonly used analgesic and antipyretic agent. Although considered safe at therapeutic doses, accidental or intentional overdose causes acute liver failure characterized by centrilobular hepatic necrosis with high morbidity and mortality. Although many molecular aspects of APAP-induced cell death have been described, no conclusive mechanism has been proposed. We recently identified TNF-related apoptosis-inducing ligand (TRAIL) and c-Jun kinase (JNK)-dependent activation of the pro-apoptotic Bcl-2 homolog Bim as an important apoptosis amplification pathway in hepatocytes. In this study, we, thus, investigated the role of TRAIL, c-JNK and Bim in APAP-induced liver damage. Our results demonstrate that TRAIL strongly synergizes with APAP in inducing cell death in hepatocyte-like cells lines and primary hepatocyte. Furthermore, we found that APAP strongly induces the expression of Bim in a c-JNK-dependent manner. Consequently, TRAIL- or Bim-deficient mice were substantially protected from APAP-induced liver damage. This study identifies the TRAIL-JNK-Bim axis as a novel target in the treatment of APAP-induced liver damage and substantiates its general role in hepatocyte death.

Role of integrins in fibrosing liver diseases

Integrins and other cell adhesion molecules regulate numerous physiological and pathological mechanisms by mediating the interaction between cells and their extracellular environment. Although the significance of integrins in the evolution and progression of certain cancers is well recognized, their involvement in nonmalignant processes, such as organ fibrosis or inflammation, is only beginning to emerge. However, accumulating evidence points to an instrumental role of integrin-mediated signaling in a variety of chronic and acute noncancerous diseases, particularly of the liver.

Organelle segregation into Plasmodium liver stage merozoites

The liver stage of the Plasmodium parasite remains one of the most promising targets for intervention against malaria as it is clinically silent, precedes the symptomatic blood stage and represents a bottleneck in the parasite life cycle. However, many aspects of the development of the parasite during this stage are far from understood. During the liver stage, the parasite undergoes extensive replication, forming tens of thousands of infectious merozoites from each invading sporozoite. This implies a very efficient and accurate process of cytokinesis and thus also of organelle development and segregation. We have generated for the first time Plasmodium berghei double-fluorescent parasite lines, allowing visualization of the apicoplast, mitochondria and nuclei in live liver stage parasites. Using these we have seen that in parallel with nuclear division, the apicoplast and mitochondrion become two extensively branched and intertwining structures. The organelles then undergo impressive morphological and positional changes prior to cell division. To form merozoites, the parasite undergoes cytokinesis and the complex process of organelle development and segregation into the forming daughter merozoites could be analysed in detail using the newly generated transgenic parasites.

Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients

Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed monospecific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.

Unhealthy alcohol use, HIV infection and risk of liver fibrosis in drug users with hepatitis C

Aim To analyze alcohol use, clinical data and laboratory parameters that may affect FIB-4, an index for measuring liver fibrosis, in HCV-monoinfected and HCV/HIV-coinfected drug users. Patients and Methods Patients admitted for substance abuse treatment between 1994 and 2006 were studied. Socio-demographic data, alcohol and drug use characteristics and clinical variables were obtained through hospital records. Blood samples for biochemistry, liver function tests, CD4 cell count, and serology of HIV and HCV infection were collected at admission. Multivariate linear regression was used to analyze the predictors of FIB-4 increase. Results A total of 472 (83% M, 17% F) patients were eligible. The median age at admission was 31 years (Interquartile range (IQR) 27–35 years), and the median duration of drug use was 10 years (IQR 5.5–15 years). Unhealthy drinking (>50 grams/day) was reported in 32% of the patients. The FIB-4 scores were significantly greater in the HCV/HIV-coinfected patients (1.14, IQR 0.76–1.87) than in the HCV-monoinfected patients (0.75, IQR 0.56–1.11) (p<0.001). In the multivariate analysis, unhealthy drinking (p = 0.034), lower total cholesterol (p = 0.042), serum albumin (p<0.001), higher GGT (p<0.001) and a longer duration of addiction (p = 0.005) were independently associated with higher FIB-4 scores in the HCV-monoinfected drug users. The effect of unhealthy drinking on FIB-4 scores disappeared in the HCV/HIV-coinfected patients, whereas lower serum albumin (p<0.001), a lower CD4 cell count (p = 0.006), higher total bilirubin (p<0.001) and a longer drug addiction duration (p<0.001) were significantly associated with higher FIB-4 values. Conclusions Unhealthy alcohol use in the HCV-monoinfected patients and HIV-related immunodeficiency in the HCV/HIV-coinfected patients are important risk factors associated with liver fibrosis in the respective populations.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.