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Background & Aims Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. Methods Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3−/−) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. Results Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepa mice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3−/− mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. Conclusion These findings show that intervention against CASP8 in liver parenchymal cells – specifically in cholangiocytes – might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. Lay summary Loss of caspase 8 – a protein involved in programmed cell death – in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.

Background & Aims Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. Methods Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. Results HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3 hi effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. Conclusions The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.

A spatial and seasonal modeling approach for precipitation extremes is introduced and exemplified for the Berlin–Brandenburg region in Germany. Monthly maxima of daily precipitation sums are described with a generalized extreme value distribution (GEV) with spatially and seasonally varying parameters. This allows for a return-level prediction also at ungauged sites. The seasonality is captured with harmonic functions, spatial variations are modeled with Legendre polynomials for longitude, latitude and altitude. Interactions between season and space allow for a spatially varying seasonal cycle. Orders of the harmonic and Legendre series are determined using a step-wise forward regression approach with the Bayesian Information Criterion (BIC) as model selection criterion. The longest 80 series are used to verify the approach in a cross-validation experiment based on the Quantile Skill Score (QSS). The model presented describes the observations at all these stations more accurately than a GEV applied to each month and location separately. These improvements are due to the assumption of smoothly varying GEV parameters in time and space; information from neighboring observations in time and space are used to obtain parameters at a given location. Apart from robustness, this approach allows also a seasonally and spatially varying shape parameter and results are found to be more accurate.

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2−/− mice were exposed to short term application of CCl4, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl4 intoxication showed increased liver damage in LCN2−/− mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2−/− mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl4 application), LCN2−/− mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.

Background & Aims Factor VII activating protease (FSAP) is a circulating serine protease produced in the liver. A single nucleotide polymorphism (G534E, Marburg I, MI-SNP) in the gene encoding FSAP ( HABP2 ) leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. FSAP is activated by damaged cells and its substrates include growth factors and hemostasis proteins. Methods We have investigated the progression of liver fibrosis in FSAP deficient mice and FSAP expression in human liver fibrosis. Results Serum FSAP concentrations declined in patients with end-stage liver disease, and hepatic FSAP expression was decreased in patients with advanced liver fibrosis and liver inflammation. Moreover, there was an inverse correlation between hepatic FSAP expression and inflammatory chemokines, chemokine receptors as well as pro-fibrotic mediators. Upon experimental bile duct ligation, FSAP −/− mice showed enhanced liver fibrosis in comparison to wild type mice, alongside increased expression of α-smooth muscle actin, collagen type I and fibronectin that are markers of stellate cell activation. Microarray analyses indicated that FSAP modulates inflammatory pathways. Conclusions Lower FSAP expression is associated with enhanced liver fibrosis and inflammation in patients with chronic hepatic disorders and murine experimental liver injury. This strengthens the concept that FSAP is a "protective factor" in liver fibrosis and explains why carriers of the Marburg I SNP have more pronounced liver fibrosis.

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

Impaired activity of natural killer cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. Natural cytotoxicity is regulated by interactions of HLA-E with inhibitory CD94/NKG2A receptors on natural killer (NK) cells. Here, we studied whether HCV core encodes peptides that bind to HLA-E and inhibit natural cytotoxicity. We analyzed 30 HCV core-derived peptides. Peptide-induced stabilization of HLA-E expression was measured flow cytometrically after incubating HLA-E-transfected cells with peptides. NK cell function was studied with a 51 chromium-release-assay. Intrahepatic HLA-E expression was analyzed by an indirect immunoperoxidase technique and flow cytometry of isolated cells using a HLA-E-specific antibody. We identified peptide aa35–44, a well-characterized HLA-A2 restricted T cell epitope, as a peptide stabilizing HLA-E expression and thereby inhibiting NK cell-mediated lysis. Blocking experiments confirmed that this inhibitory effect of peptide aa35–44 on natural cytotoxicity was mediated via interactions between CD94/NKG2A receptors and enhanced HLA-E expression. In line with these in vitro data we found enhanced intrahepatic HLA-E expression on antigen-presenting cells in HCV-infected patients. Our data indicate the existence of T cell epitopes that can be recognized by HLA-A2 and HLA-E. This dual recognition may contribute to viral persistence in hepatitis C.

Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(FZY)/Cdc20 and Fizzy-related(FZR)/Hct1/Cdh1 proteins [1–5]. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis [6, 7]. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous FZR is not involved during exit from mitosis [3, 8]. However, the potential involvement of minor amounts of maternally derived FZR was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1 −/− cells [9] might be explained by the recent identification of multiple Cdh1 genes [10]. Here, we have carefully analyzed the FZR requirement during exit from mitosis in Drosophila , which, apart from fzr , has only one additional homolog. We find that this fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, we demonstrate that completion of mitosis including Cyclin B degradation does not require FZR. However, fzr is an essential gene corresponding to the rap locus, and FZR, which accumulates predominantly in the cytoplasm, is clearly required during G1.

Bone is a metabolically active tissue, and recent research advances have enabled us to detect disturbances of bone turnover by the quantification of specific bone-derived molecules in serum or urine. The central components of tissue homeostasis consist of three distinct types of bone cells: the bone-forming osteoblasts, the matrix-embedded osteocytes, and bone-resorbing osteoclasts. Important functions of these cells include the production of a variety of collagenous and noncollagenous bone matrix proteins and proteolytic enzymes. Some of these cell-derived components may be quantified in body fluids and may serve as more or less reliable estimates of the actual rate of bone turnover. Approximately 90% of the organic fraction of bone mass consists of collagen. The main component in bone is type I collagen, but a number of other collagens have also been identified, including type III and type V collagen. The noncollagenous matrix proteins present in bone are macromolecules such as osteocalcin (OC), osteopontin, and bone sialoprotein (BSP). The exact functions of the these proteins are unknown, but they likely play roles in the supramolecular organization of the bone matrix, in the adhesion of bone-resorbing cells to the matrix, and during the mineralization and degradation of bone tissue. Biochemical markers of bone turnover may be classified according to: •Origin (e.g., products of bone cells, molecules derived from the organic or anorganic bone matrix) •Biochemical properties (e.g., enzymatic markers of bone cells, precursors or degradation products of the collagenous or noncollagenous bone matrix) •Function (e.g., markers of bone formation or bone resorption, adhesion molecules). Physiologically, bone formation and bone resorption are coupled to keep a steady state of overall bone turnover. Disturbances in bone turnover may be detected by an increase or decrease in the concentration of markers of bone formation or bone resorption. Thus, from a clinical point of view, the most practicable classification of bone turnover markers, although not exclusive, remains the grouping according to parameters primarily reflecting bone formation or bone resorption processes. As a result of these considerations, we here follow the more functional classification to discuss some of the biochemical features and technical aspects of the most widely used biochemical markers of bone metabolism.

This article describes the biochemical background and functional properties of molecular markers derived from osteoblasts, the collagenous, or noncollagenous bone matrix. Clinical applications and the selection, timing, and monitoring of antiosteoporotic treatment are critically discussed. Special emphasis has been put on reviewing the value of bone resorption markers in the prediction of future bone loss and the risk assessment for osteoporotic fractures. The potential of these indices as independent risk factors for future fractures alone or in combination with bone mineral density measurements has recently been demonstrated in large cohort studies. More data are needed, however, to establish biochemical markers of bone resorption as useful tools in the assessment of individual fracture risk.

Hypotrichosis simplex of the scalp (HSS) is an autosomal dominant form of isolated alopecia causing almost complete loss of scalp hair, with onset in childhood. After exclusion of candidate regions previously associated with hair-loss disorders, we performed a genomewide linkage analysis in two Danish families and localized the gene to chromosome 6p21.3. This was confirmed in a Spanish family, with a total LOD score of 11.97 for marker D6S1701 in all families. The combined haplotype data identify a critical interval of 14.9 cM between markers D6S276 and D6S1607. Localization of the locus for HSS to 6p21.3 is a first step toward identification of the gene. The gene will give important insights into the molecular and cellular basis of hair growth on the scalp.

The interest in and the need for effective measures to be used in the screening, diagnosis, and follow-up of disorders of connective tissue, bone, and mineral metabolism has markedly grown. Next to clinical and imaging techniques, indices of bone turnover have come to play an important role in the assessment of metabolic bone disease. In osteoporosis, recent research has shown that bone markers may also be used to predict future bone loss and hip fractures (in larger cohorts of older patients), identify individuals at risk for osteoporosis, select therapy, and predict and monitor the therapeutic response in individual patients. The development of new markers of bone metabolism has greatly enriched the spectrum of serum and urine analytes used in the assessment of skeletal pathologies. Besides total alkaline phosphatase, other markers such as bone-specific alkaline phosphatase, osteocalcin, or the collagen propeptides are being used to measure bone formation. Bone resorption, previously assessed only by the measurement of urinary calcium and hydroxyproline, may now be detected more precisely by a number of new serum and urine markers. Among these, the pyridinium crosslinks and the telopeptides of collagen type I are presently considered the most specific markers of bone resorption. More recently, bone sialoprotein has also been suggested as a marker of bone resorption in serum. Tartrate-resistant acid phosphatase is now measurable by immunoassay. This article surveys the biochemistry and relevant technical aspects of the currently available markers of bone metabolism.

The formation of 1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecanes (dispiroketals) of various D-glucopyranosyl substrates is described.

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.

Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital, Aachen, Germany; Department of Medicine III, RWTH University Hospital, Aachen, Germany; Institute of Pathology, RWTH University Hospital, Aachen, Germany; Miltenyi Biotec GmbH, Friedrich-Ebert-Strase 68, 51429 Bergisch Gladbach, Germany; Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway; Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany

The subset of gd T cells, especially Vd2 gd T cells, plays a critical role in protective immune responses against cancer and viral infections; however, their role in chronic hepatitis B virus infection remains unclear. Here we characterized peripheral and intrahepatic gd T cells, and analyzed their association with liver damage in a cohort of HBV-infected patients, including 64 immune activated (IA) patients, 22 immune tolerant (IT) carriers and 30 healthy controls (HCs). We demonstrated that the numbers and percentages of peripheral and hepatic Vd2 T cells were significantly lower in IA patients than that in HC and IT subjects, and the frequency of Vd2 T cells was reversely correlated with liver damage, but not with HBVDNA load. Vd2 T cells in IA patients exhibited an impaired proliferative capacity, due to abnormalities in the G2/M cell cycle phases and a skewed chemotactic ability, which contributed to the lower number of these cells in the liver. Finally, an in vitro co-culture assay showed that Vd2 T cells significantly suppressed the production of the interleukin-17 producing CD4 T cells (Th17)-associated cytokines, IL-17 and IL-22, in both cell contact-dependent and interferon (IFN)-g-dependent mechanisms. In summary, inflammatory microenvironment in IA patients results in decreased numbers of Vd2 T cells in peripheral blood and liver, which in turn impairs the suppressive function of Vd2 T cells to Th17 cells. Therefore, adoptive transfer of Vd2 T cells may provide a novel therapeutic approach for IA patients.

1020 POOR AGREEMENT OF DIFFERENT APPROACHES TO ASSESS BACTERIAL TRANSLOCATION IN PATIENTS WITH DECOMPENSATED CIRRHOSIS T. Bruns, P.A. Reuken, A. Vogt, H.W. Zimmermann, A. Stallmach. Division of Gastroenterology, Hepatology and Infectious Diseases, Department of Internal Medicine II, Jena University Hospital, Integrated Research and Treatment Center – Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany; NIHR Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK; Department of Medicine III, University Hospital Aachen, Aachen, Germany E-mail: t.bruns@bham.ac.uk