Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo.

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor that regulates inflammation, cell migration, and cell fate. Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disease. To understand the function of RAGE in SLE, we generated RAGE-deficient (Ager-/-) lupus-prone mice by backcrossing MRL/MpJ-Faslpr/J (MRL-lpr) mice with Ager-/- C57BL/6 mice. In 18-week-old Ager-/- MRL-lpr, the weights of the spleen and lymph nodes, as well as the frequency of CD3+CD4-CD8- cells, were significantly decreased. Ager-/- MRL-lpr mice had significantly reduced urine albumin/creatinine ratios and markedly improved renal pathological scores. Moreover, neutrophil infiltration and neutrophil extracellular trap formation in the glomerulus were significantly reduced in Ager-/- MRL-lpr. Our study is the first to reveal that RAGE can have a pathologic role in immune cells, particularly neutrophils and T cells, in inflammatory tissues and suggests that the inhibition of RAGE may be a potential therapeutic strategy for SLE.

Allogeneic hematopoietic transplantation (allo-HCT) is a curative therapy for a variety of hematologic malignancies, primarily through immune-mediated clearance of malignant cells. This graft-versus-leukemia (GvL) effect is mediated by alloreactive donor T-cells against recipient malignant cells. Unfortunately, graft versus host disease is a potentially lethal complication of this procedure, also mediated by alloreactive donor T-cells against recipient normal tissues. Graft-versus-host disease (GVHD) remains a key contributor to nonrelapse mortality and long-term morbidity in patients undergoing allo-HCT. Reducing GVHD without interfering with - or ideally while enhancing - GvL, would improve outcomes and increase patient eligibility for allo-HCT. The JAK/STAT signaling pathway acts downstream of over 50 cytokines and is central to a wide variety of inflammatory pathways. These pathways play a role in the development and maintenance of GVHD throughout the disease process and within T-cells, B-cells, macrophages, neutrophils, and natural killer cells. Agents targeting JAK/STAT signaling pathways have shown clinical efficacy and gained US Food and Drug Administration approval for numerous diseases. Here, we review the preclinical and clinical evidence for the role of JAK/STAT signaling in the development and maintenance of GVHD and the utility of blocking agents at preventing and treating GVHD.

Resistance to conventional chemotherapeutic agents, a typical feature of cholangiocarcinoma, prevents the efficacy of the therapeutic arsenal usually used to combat malignancy in humans. Mechanisms of chemoresistance by neoplastic cholangiocytes include evasion of drug-induced apoptosis mediated by autocrine and paracrine cues released in the tumor microenvironment. Here, recent evidence regarding molecular mechanisms of chemoresistance is reviewed, as well as associations between well-developed chemoresistance and activation of the cancer stem cell compartment. It is concluded that improved understanding of the complex interplay between apoptosis signaling and the promotion of cell survival represent potentially productive areas for active investigation, with the ultimate aim of encouraging future studies to unveil new, effective strategies able to overcome current limitations on treatment.

The liver is an immunoregulatory organ in which a tolerogenic microenvironment mitigates the relative "strength" of local immune responses. Paradoxically, necro-inflammatory diseases create the need for most liver transplants. Treatment of hepatitis B virus, hepatitis C virus, and acute T cell-mediated rejection have redirected focus on long-term allograft structural integrity. Understanding of insults should enable decades of morbidity-free survival after liver replacement because of these tolerogenic properties. Studies of long-term survivors show low-grade chronic inflammatory, fibrotic, and microvascular lesions, likely related to some combination of environment insults (i.e. abnormal physiology), donor-specific antibodies, and T cell-mediated immunity. The resultant conundrum is familiar in transplantation: adequate immunosuppression produces chronic toxicities, while lightened immunosuppression leads to sensitization, immunological injury, and structural deterioration. The "balance" is more favorable for liver than other solid organ allografts. This occurs because of unique hepatic immune physiology and provides unintended benefits for allografts by modulating various afferent and efferent limbs of allogenic immune responses. This review is intended to provide a better understanding of liver immune microanatomy and physiology and thereby (a) the potential structural consequences of low-level, including allo-antibody-mediated injury; and (b) how liver allografts modulate immune reactions. Special attention is given to the microvasculature and hepatic mononuclear phagocytic system.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor (TRAIL-R) family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC), caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS) has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB) activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients.

Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets.

Liver is a primary organ involved in biotransformation of food and drugs. Hepatic diseases are a major worldwide problem. Hepatic disorders are mainly caused by toxic chemicals (alcohol), xenobiotics (carbon tetrachloride, chlorinated hydrocarbons and gases CO₂ and O₂) anticancer (azathioprine, doxorubicin, cisplatin), immunosuppressant (cyclosporine), analgesic anti-inflammatory (paracetamol, thioacetamide), anti-tubercular (isoniazid, rifampicin) drugs, biologicals (Bacillus-Calmette-Guerin vaccine), radiations (gamma radiations), heavy metals (cadmium, arsenic), mycotoxin (aflatoxin), galactosamine, lipopolysaccharides, etc. Various risk factors for hepatic injury include concomitant hepatic diseases, age, gender, alcoholism, nutrition and genetic polymorphisms of cytochrome P450 enzymes have also been emphasized. The present review enumerates various in vivo animal models and in vitro methods of hepatic injury using diverse toxicants, their probable metabolic pathways, and numerous biochemical changes viz. serum biomarkers enzymes, liver function, oxidative stress associated events like free radicals formation, lipid peroxidation, enzyme antioxidants and participation of cytokines (tumour necrosis factor-α, transforming growth factor-β, tumour necrosis factor-related apoptosis inducing ligand), and other biomolecules (Fas and C-jun N-terminal kinase) are also discussed. The underlying cellular, molecular, immunological, and biochemical mechanism(s) of action responsible for liver damage (toxicity) are also been discussed. This review should be immensely useful for researchers especially for phytochemists, pharmacologists and toxicologists working on hepatotoxicity, hepatotoxic chemicals and drugs, hepatoprotective agents and drug research organizations involved especially in phytopharmaceuticals and other natural products.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Primary Biliary Cirrhosis (PBC) is considered an autoimmune disease characterized by immune-mediated destruction of the intrahepatic bile ducts and its characteristic serologic marker, the anti-mitochondrial antibody (AMA). Several factors were proposed to clarify the pathological and immunological mechanisms of PBC. Immunological reaction with a bacterial or a viral association was identified in the previous report, and it seems probable that PBC was thought to have such an etiology. The majority of patients with PBC was reported to have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes or in 2008, the patient who developed PBC with high HIV viral load had an antiviral therapy and recovered. To understand the etiology of PBC associated with infection, several factors should be considered and especially animal models may be useful. In this paper, we introduce three typical animal models of PBC: the dominant-negative form of transforming growth factor-β receptor type II (dnTGFβRII) mouse, IL-2Rα(-/-) mouse and NOD.c3c4 mouse, are enumerated and described, and we discuss previous reports of viral infection associated with PBC and consider the etiology of PBC from our analysis of results in NOD.c3c4 mouse.

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.

Acute liver failure (ALF) is a syndrome of diverse aetiology, including hepatic encephalopathy, renal, cardiac and pulmonary failures, which result in a rapid loss of hepatic function. The mechanisms of liver injury contributing to ALF can be summarized into two categories: direct damage and immune-mediated liver injury. This review summarizes current concepts of immune-mediated liver injury from both clinical studies and animal models. We highlight immune responses of ALF from the liver injury perspective, which combines a variety of molecular and cellular mechanisms, particularly, the contribution of cytokines and the innate immune system. Hepatic and circulating inflammatory cytokines play a significant role in the pathophysiology of ALF including hepatocyte necrosis, extrahepatic complications and hepatocyte regeneration. Overproduction of cytokines, if unchecked, is hazardous to the host and may cause severe outcomes. Measuring pro-inflammatory cytokines in ALF may be of value for predictors of outcome. Innate and adaptive immune systems both involved in ALF contribute to immune-mediated liver injury. The innate immune response is activated much more rapidly compared with adaptive immunity, particularly in acute liver injury where the host has little time to trigger an effective adaptive immune response. From this point of view, the innate immune system may make a more profound contribution than the adaptive immune system. Furthermore, immune responses crosstalk with other physiological or pathophysiological factors, for example, coagulation factors which in turn determine the outcome of ALF and these are discussed.

The FS7-associated cell surface antigen (Fas, also named CD95, APO-1 or TNFRSF6) attracted considerable interest in the field of apoptosis research since its discovery in 1989. The groups of Shin Yonehara and Peter Krammer were the first reporting extensive apoptotic cell death induction upon treating cells with Fas-specific monoclonal antibodies.1,2 Cloning of Fas3 and its ligand,4,5 FasL (also known as CD178, CD95L or TNFSF6), laid the cornerstone in establishing this receptor-ligand system as a central regulator of apoptosis in mammals. Therapeutic exploitation of FasL-Fas-mediated cytotoxicity was soon an ambitous goal and during the last decade numerous strategies have been developed for its realization. In this chapter, we will briefly introduce essential general aspects of the FasL-Fas system before reviewing its physiological and pathophysiological relevance. Finally, FasL-Fas-related therapeutic tools and concepts will be addressed.

Derangements in apoptosis of liver cells are mechanistically important in the pathogenesis of end-stage liver disease. Vulnerable hepatocytes can undergo apoptosis via an extrinsic, death receptor-mediated pathway, or alternatively intracellular stress can activate the intrinsic pathway of apoptosis. Both pathways converge on mitochondria, and mitochondrial dysfunction is a prerequisite for hepatocyte apoptosis. Persistent apoptosis is a feature of chronic liver diseases, and massive apoptosis is a feature of acute liver diseases. Fibrogenesis is stimulated by ongoing hepatocyte apoptosis, eventually resulting in cirrhosis of the liver in chronic liver diseases. Endothelial cell apoptosis occurs in ischemia-reperfusion injury. Natural killer and natural killer T cells remove virus-infected hepatocytes by death receptor-mediated fibrosis. Lastly, activated stellate cell apoptosis leads to slowing and resolution of apoptosis. This review summarizes recent cellular and molecular advances in the understanding of the injury mechanisms leading to end-stage liver disease.

The heterogeneous functions of cholangiocytes regulate the physiology of the biliary epithelium regarding secretory, proliferative and apoptotic activities. However, due to their technical difficulties for isolation and culture, the pathophysiology of cholangiocytes was poorly understood. Recently, cutting-edge technology, such as the microarray, has given the opportunity for investigating cholangiocytes. We have found the distinct expression profiles of two murine cholangiocytes lines, termed small and large, revealed by microarray analysis. The features of the two cholangiocyte cell lines, categorized partly according to gene ontology, indicate the specific physiological role of each cell line. Namely, large cholangiocytes are characterized as "transport" and "immune/inflammatory responses". In contrast to large, small cholangiocytes are associated with properties of limited physiological functional ability and proliferating/migratingpotential with specific molecules like Eph receptors, comparable to mesenchymal cells. Further analysis using other modern technology, such as proteomics, will provide more information to understand the pathophysiology of cholangiocytes. These novel methods enable us to investigate the key molecules and their mutual relationship. Although the evaluation of some important biological regulatory processes like protein modification requires methodologies other than microarray, microarray technology is anticipated to grow with the development of data-analysis theory for the comprehension of cellular cross-talk in the liver.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and nonmalignant hematologic diseases. Donor T cells from the allografts are critical for the success of this effective therapy. Unfortunately these T cells not only recognize and attack the disease cells/tissues but also the other normal tissues of the recipient as "foreign" or "nonself" and cause severe, immune-mediated toxicity, graft-versus-host disease (GVHD). Several insights into the complex pathophysiology of GVHD have been gained from recent experimental observations, which show that acute GVHD is a consequence of interactions between both the donor and the host innate and adaptive immune systems. These insights have identified a role for a variety of cytokines, chemokines, novel T-cell subsets (naĩve, memory, regulatory, and NKT cells) and for non-T cells of both the donor and the host (antigen presenting cells, delta T cells, B cells, and NK cells) in modulating the induction, severity, and maintenance of acute GVHD. This review will focus on the immunobiology of experimental acute GVHD with an emphasis on the recent observations.

A major handicap in understanding the pathogenesis of autoimmune cholangitis has been the absence of an informative mouse model. Recently, autoimmune cholangitis, with several features similar to PBC, has been described in NOD.c3c4 mice, including anti-mitochondrial antibodies, lymphocytic portal tract infiltrates, biliary destruction and the adoptive transfer of disease to naïve recipients using liver-derived lymphocytes. A unique feature, and a characteristic quite distinct from human PBC, is the presence of bile cyst formation. We have addressed the issue of cysts in NOD.c3c4 mice by performing comprehensive microarray analysis using cholangiocytes from NOD.c3c4 mice compared to NOD controls. Several key differences in gene expression were noted in NOD.c3c4 cholangiocytes. First, there was consistent impairment in the expression of Fas antigen (CD95). Second, cholangiocytes were PCNA positive but TUNEL negative, suggesting an absence of apoptosis despite abnormal proliferation. In conclusion, we propose that autoimmune cholangitis develops in NOD.c3c4 mice secondary to impaired biliary cell apoptosis with exposure of mitochondrial antigens, loss of tolerance and subsequent development of multi-lineage anti-mitochondrial responses.

Primary biliary cirrhosis (PBC) is a chronic progressive cholestatic disease of unknown pathogenesis. However, several reports have demonstrated the involvement of genetic backgrounds in this syndrome. The aim of this study is to examine the genetic disequilibrium in the HLA region in Japanese patients using six microsatellite markers.

Genomic DNAs were obtained from 73 patients with PBC (patient cohort) and 186 healthy volunteers (control cohort). Genetic polymorphisms at six microsatellite markers (D6S1568, DQ.CAR, D6S273, TNF-d, C1-2-A, C3-2-11) were determined using fluorescence-labeled polymerase chain reaction (PCR) genetic analyzer. Allele frequencies were estimated by direct counting and the genotypic differentiation test was performed by the Markov chain method using Genepop software.

Among these six microsatellite markers, four markers in the patients significantly (P < 0.05) deviated from the Hardy-Weinberg equilibrium: DQ.CAR (P = 0.0278), D6S273 (P = 0.0168), TNF-d (P = 0.0089) and C1-2-A (P = 0.0005). Genotypic differentiation test between the patients and controls demonstrated that DQ.CAR (P = 0.0111), TNF-d (P = 0.0051) and C1-2-A (P = 0.0371) were significant. Finally, allelic association test revealed before correction for multiple testing demonstrated allele125 of TNF-d (P = 0.00065, Pc = 0.0052) and allele246 of C1-2-A (P = 0.0026 Pc = 0.033) had significant association after Bonferroni's correction.

Disequilibrium mapping using microsatellite markers was a useful method to narrow a disease susceptibility locus. The possible susceptibility gene in the HLA region is thought to be localized around or in the TNF gene. Further studies seem feasible using more closely distributed microsatellite markers or single nucleotide polymorphisms (SNPs) to narrow the susceptibility locus in PBC in Japanese populations.

Bile duct paucity, ductopenia, is a feature of end-stage chronic cholangiopathies such as primary biliary cirrhosis. The limited proliferative ability of cholangiocytes after specific injury is thought to be the principal cause of ductopenia, although the detailed mechanisms involved are unclear. It has been reported that human amniotic epithelial cells (AEC) express differentiation markers of hepatic parenchymal cells, suggesting a resemblance of AEC to hepatic progenitor cells. The aim of the present study was to develop a mouse model of experimental cholestasis to assess the capability of mouse AEC to trans-differentiate into cholangiocytes.

Enhanced green fluorescent protein (EGFP)-transgenic C57BL/6 pregnant female mice were used as the source of AEC. At 11.5 gestational days, 1 x 10(5) AEC were isolated from EGFP-transgenic mouse embryos and transferred into C57BL/6 mice. Chronic cholestasis was induced by 0.1%alpha-naphthylisothiocyanate (ANIT) feeding immediately after the transfer of AEC. The proliferation of cholangiocytes in the livers was assessed morphologically and immunohistochemically (cytokeratin 7; CK7). The proliferative activity was also quantified immunohistochemically by proliferating cell nuclear antigen (PCNA) protein expression. EGFP of transferred AEC was confirmed by fluorescent laser microscopy and immunofluorescent staining for EGFP. Also, Notch2 and Hes1 expression was evaluated to examine the roles of the differentiation markers in this process.

Marked proliferation of cholangiocytes was observed in ANIT-fed mice confirmed by quantitative CK7 (3-4 fold vs control) and PCNA (11-20 fold vs control) staining. EGFP and CK7 double positive cells in interlobular bile ducts were confirmed in the livers of AEC-transferred recipients. Positivity of EGFP was further confirmed by the immunofluorescent staining for EGFP. Moreover, both Notch2 and Hes1 expression was confirmed in the proliferative bile duct in this model.

Significant ductular proliferation was observed in ANIT-fed mice. EGFP-positive cholangiocytes were confirmed in this chronic cholestasis model. AEC transfer was able to contribute to the repopulating of proliferating cholangiocytes under cholestasis, suggesting AEC might be a candidate cell source for stem cell administration in future clinical applications to re-model interlobular bile ducts.

Despite optimal supportive care and high-resolution HLA matching, complications such as GVHD and infection remain major barriers to the success of allogeneic HCT (allo-HCT). This has led to growing interest in the non-HLA genetic determinants of complications after allo-HCT. Most studies have examined genetic predictors of GVHD, relapse, and mortality and have focused on 3 main areas: minor histocompatibility antigen (miHAs), inflammatory mediators of GVHD, and more recently NK cell-mediated allorecognition. The genetic basis of other outcomes such as infection and drug toxicity are less well studied but are being actively investigated. High-throughput methodologies such as single nucleotide polymorphism arrays are enabling the study of hundreds of thousands of genetic markers throughout the genome and the interrogation of novel genetic variants such as copy number variations. These data offer the opportunity to better predict those at risk of complications and to identify novel targets for therapeutic intervention. This review examines the current data regarding the non-HLA genomics of allo-HCT and appraises the promises and pitfalls for integration of this new genetic information into clinical transplantation practice.

Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear.

Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated.

Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7.

In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

The difficult separation of clinical graft-versus-tumor (GVT) effects from graft-versus-host disease (GVHD) reflects their shared biology. Experimental approaches to mediate GVT effects while limiting GVHD include: (1) allograft T cell depletion followed by immune enhancement; (2) modulation of T cell dose or T cell subset composition; (3) donor lymphocyte infusion; (4) reduced-intensity host preparation; (5) modulation of Th1/Th2 and Tc1/Tc2 cell balance; (6) cytokine therapy or neutralization; (7) T regulatory cell therapy; (8) co-stimulatory pathway modulation; (9) chemokine pathway modulation; (10) induction of antigen-specific T cells; (11) alloreactive NK cell therapy; and (12) targeted pharmaceutical inhibition of proteosome, mammalian target of rapamycin, and histone deacetylase pathways. Clearly, a multitude of approaches exist that hold promise for separating GVT effects from GVHD. Future success in this endeavor will require a strong commitment towards translational research and continued advances in cell, vaccine, cytokine, monoclonal antibody, and targeted molecular therapy.

Death of hepatocytes and other hepatic cell types is a characteristic feature of liver diseases as diverse as cholestasis, viral hepatitis, ischemia/reperfusion, liver preservation for transplantation and drug/toxicant-induced injury. Cell death typically follows one of two patterns: oncotic necrosis and apoptosis. Necrosis is typically the consequence of acute metabolic perturbation with ATP depletion as occurs in ischemia/reperfusion and acute drug-induced hepatotoxicity. Apoptosis, in contrast, represents the execution of an ATP-dependent death program often initiated by death ligand/death receptor interactions, such as Fas ligand with Fas, which leads to a caspase activation cascade. A common event leading to both apoptosis and necrosis is mitochondrial permeabilization and dysfunction, although the mechanistic basis of mitochondrial injury may vary in different settings. Prevention of these modes of cell death is an important target of therapy, but controversies still exist regarding which mode of cell death predominates in various forms of liver disease and injury. Resolution of these controversies may come with the recognition that apoptosis and necrosis frequently represent alternate outcomes of the same cellular pathways to cell death, especially for cell death mediated by mitochondrial permeabilization. An understanding of processes leading to liver cell death will be important for development of effective interventions to prevent hepatocellular death leading to liver failure and to promote cancer and stellate cell death in malignancy and fibrotic disease.

Graft-versus-host disease (GVHD), induced by the reaction of donor T cells to recipient histoincompatible antigens, is a serious complication of allogeneic bone marrow transplantation (BMT), resulting in considerable morbidity and mortality. In MHC-disparate BMT, donor T cells directly react with major histocompatibility complex (MHC) antigens, while in MHC-matched BMT, T cells react with minor histocompatibility antigens (miHA) presented by shared MHC molecules. Clinically, acute and chronic GVHD can be distinguished on the basis of the time of onset, clinical manifestations and distinct pathobiological mechanisms. Acute GVHD usually occurs within 2 to 6 weeks following allogeneic BMT and primarily affects the skin, liver and the gastrointestinal tract with T cell infiltration of the epithelia of the skin, mucous membranes, bile ducts and gut. In addition, hair follicle cells, airways, bone marrow, and a variety of other tissue systems can be involved. Acute GVHD occurs in up to 50% of allogeneic HLA-matched and 70% of HLA-disparate BMT recipients despite prophylactic immunosuppressive drugs. Chronic GVHD involves a wider range of organs and clinical manifestations include scleroderma, liver failure, immune complex disease, glomerulonephritis, and autoantibody formation.

Graft-versus-host disease (GVHD) has been the primary limitation to the wider application of allogeneic bone marrow transplantation (BMT). The immunobiology of acute GVHD is complex and can be conceptualized to be a three-step process. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to the damage and activation of host tissues and induces the secretion of inflammatory cytokines TNF-alpha and IL-1. As a consequence expression of MHC antigens and adhesion molecules is increased, thus enhancing the recognition of host alloantigens by donor T cells. Donor T-cell activation in step 2 is characterized by donor T-cell interaction with host APCs and subsequent proliferation, differentiation, and secretion of cytokines. Cytokines such as IL-2 and IFN-gamma enhance T-cell expansion, induce cytotoxic T cells (CTL) and natural killer (NK) cell responses, and prime additional mononuclear phagocytes to produce TNF-alpha and IL-1. These inflammatory cytokines in turn stimulate production of inflammatory chemokines, thus recruiting effector cells into target organs. In step 3, effector functions of mononuclear phagocytes are triggered via a secondary signal provided by lipopolysaccharide (LPS) that leaks through the intestinal mucosa damaged during step 1. This mechanism may result in the amplification of local tissue injury and further promotion of an inflammatory response, which, together with the CTL and NK components, leads to target tissue destruction in the transplant host.

Existing data indicate that non-human leukocyte antigen (HLA) immunogenetic polymorphisms influence the risk of complications after allogeneic hemopoietic stem-cell transplantation. However, prior studies have been limited by small sample size and limited genotyping.

We examined 22 polymorphisms in 11 immunoregulatory genes including cytokines, mediators of apoptosis, and host-defense molecules by polymerase chain reaction using sequence-specific primers in 160 related myeloablative transplants. Associations were confirmed in two independent cohorts.

An intronic polymorphism in the tumor necrosis factor gene (TNF 488A) was associated with the risk of acute graft-versus-host disease (GVHD) (odds ratio [OR] 16.9), grades II to IV acute GVHD (OR 3.3), chronic GVHD (OR 12.5), and early death posttransplant (OR 3.4). Recipient Fas -670G and donor interleukin (IL)-6 -174G were independent risk factors for acute GVHD. Recipient IL-10 ATA and Fas -670 genotype were independent risk factors for chronic GVHD. Recipient IL-1beta +3953T was associated with hepatic acute GVHD, and Fas -670G was associated with major infection.

These results highlight the potential importance of cytokine and apoptosis gene polymorphisms in stem-cell transplantation, and indicate that non-HLA genotyping may be useful to identify individuals at the highest risk of complications and new targets for therapeutic intervention.

Graft-versus-host disease (GVHD) has been the primary limitation to the wider application of allogeneic bone marrow transplantation (BMT). The pathophysiology of acute GVHD is complex and can be conceptualized to be a three-step process based on murine studies. In step 1, the conditioning regimen leads to the damage and activation of host tissues and induces the secretion of inflammatory cytokines. As a consequence, the expression of MHC antigens and adhesion molecules is increased enhancing the recognition of host alloantigens by donor T cells. Donor T-cell activation in step 2 is characterized by donor T cell interaction with host APCs and subsequent proliferation, differentiation and secretion of cytokines. Cytokines such as IL-2 and IFN-gamma enhance T-cell expansion, induce cytotoxic T cells (CTL) and natural killer (NK) cell responses and prime additional mononuclear phagocytes to produce TNF-alpha and IL-1. These inflammatory cytokines in turn stimulate production of inflammatory chemokines, thus recruiting effector cells into target organs. In step 3, effector functions of mononuclear phagocytes are triggered via a secondary signal provided by lipopolysaccharide (LPS) that leaks through the intestinal mucosa damaged during step 1. This mechanism may result in the amplification of local tissue injury and further promotion of an inflammatory response, which, together with the CTL and NK components, leads to target tissue destruction in the transplant host. The following review discusses the three-step process of the pathophysiology of experimental acute GVHD.

We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed.

Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by a GenePix fluorescent scanner and analyzed using ArrayGauge software.

EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta chain and caspase 9 were more strongly expressed by large cholangiocytes.

Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information, which further supports our hypothesis that small and large bile ducts have different functions.

The pathophysiology of acute graft-versus-host disease (GVHD) is a complex process that can be conceptualized in three phases. In the first phase, high-dose chemoradiotherapy causes damage to host tissues, including a self-limited burst of inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin 1. These cytokines activate host antigen-presenting cells (APCs). In the second phase, donor T-cells recognize alloantigens on host APCs. These activated T-cells then proliferate, differentiate into effector cells, and secrete cytokines, particularly interferon (IFN)-gamma. In the third phase, target cells undergo apoptosis mediated by cellular effectors (eg, donor cytotoxic T-lymphocytes) and inflammatory cytokines such as TNF-alpha. TNF-alpha secretion is amplified by stimuli such as endotoxin that leaks across damaged gastrointestinal mucosa injured by the chemoradiotherapy in the first phase. TNF-alpha and IFN-gamma cause further injury to gastrointestinal epithelium, causing more endotoxin leakage and establishing a positive inflammatory feedback loop. These events are examined in detail in the following review.

M50054, 2,2'-methylenebis (1,3-cyclohexanedione), was identified as a novel inhibitor of apoptosis (programmed cell death) using an in vitro cell death assay system induced in human Fas-expressing WC8 cells by soluble human Fas ligand. Furthermore, M50054 inhibited the apoptotic cell death of U937, a human monocytic leukemic cell line, induced by anticancer agents such as etoposide; it was also confirmed that M50054 inhibited apoptotic features such as DNA fragmentation and phosphatidylserine exposure in these cells. These anti-apoptotic effects were attributable to inhibition of caspase-3 activation. Additionally, M50054 significantly inhibited anti-Fas-antibody-induced elevation of plasma alanine aminotransferase and aspartate aminotransferase. Alopecia (hair loss) symptoms were also significantly improved with topical treatment with M50054. In conclusion, M50054 inhibits apoptosis induced by a variety of stimuli via inhibition of caspase-3 activation, and may thus be effective for hepatitis and chemotherapy-induced alopecia.

Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas-mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40-induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF-kappaB and AP-1 were both activated after CD40 ligation. Increased functional NF-kappaB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up-regulation of AP-1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL-dependent apoptosis of liver epithelial cells supporting the importance of cross-talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP-1 in the absence of NF-kappaB signaling may be a critical factor in determining the outcome of CD40 engagement.

New insights into the regulation of hepatobiliary transport proteins have provided the basis for a better understanding of the pathogenesis of cholestatic liver diseases. Mutations of transporter genes can cause hereditary cholestatic syndromes, the study of which has shed much light on the basic mechanisms of bile secretion and cholestasis. Important new studies have been published about the pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, cholestasis of pregnancy, total parenteral nutrition-induced cholestasis, and drug-induced cholestasis.

Early chronic liver allograft rejection (CR) is characterized by distinctive cytological changes in biliary epithelial cells (BECs) that resemble cellular senescence, in vitro, and precede bile duct loss. If patients suffering from early CR are treated aggressively, the clinical and histopathological manifestations of CR can be completely reversed and bile duct loss can be prevented. We first tested whether the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR, and whether treatment reversed the expression. The percentage of p21+ BECs and the number of p21+ BECs per portal tract is significantly increased in early CR (26 +/- 17% and 3.6 +/- 3.1) compared to BECs in normal liver allograft biopsies or those with nonspecific changes (1 +/- 1% and 0.1 +/- 0.3; P: < 0.0001 and P: < 0.02), chronic hepatitis C (2 +/- 3% and 0.7 +/- 1; P: < 0.0001 and P: < 0.04) or obstructive cholangiopathy (7 +/- 7% and 0.7 +/- 0.6; P: < 0.006 and P: = 0.04). Successful treatment of early CR is associated with a decrease in the percentage of p21+ BECs and the number of p21+ BECs per portal tract. In vitro, nuclear p21(WAF1/Cip1) expression is increased in large and multinucleated BECs, and is induced by transforming growth factor (TGF)-beta. TGF-beta1 also increases expression of TGF-beta receptor II, causes phosphorylation of SMAD-2 and nuclear translocation of p21(WAF1/Cip1), which inhibits BEC growth. Because conversion from cyclosporine to tacrolimus is an effective treatment for early CR, we next tested whether these two immunosuppressive drugs directly influenced BEC growth in vitro. The results show that cyclosporine, but not tacrolimus, stimulates BEC TGF-beta1 production, which in turn, causes BEC mito-inhibition and up-regulation of nuclear p21(WAF1/Cip1). In conclusion, expression of the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR and decreases with successful recovery. Replicative senescence accounts for the characteristic BEC cytological alterations used for the diagnosis of early CR and lack of a proliferative response to injury. The ability of cyclosporine to inhibit the growth of damaged BECs likely accounts for the relative duct sparing properties of tacrolimus.

Chronic graft-versus-host disease (GVHD) of the liver usually presents as an indolent cholestatic disease in patients with skin, mouth, and eye involvement. We observed 14 patients in whom chronic GVHD of the liver presented with marked elevations of serum aminotransferases, clinically resembling acute viral hepatitis. Onset of liver dysfunction was at 294 days (range, 74-747 days) after allogeneic hematopoietic cell transplantation and coincided with a recent cessation or taper of immunosuppressive drugs. Median peak serum alanine transaminase (ALT) was 1,640 U/L (698-2,565 U/L), and median bilirubin was 12.3 mg/dL (0.9-55.9 mg/dL). All biopsies showed characteristic features of GVHD with damaged and degenerative small bile ducts. Other features included a marked lobular hepatitis, moderate to marked amounts of hepatocyte unrest, sinusoidal inflammation with perivenular necroinflammatory foci, and many acidophilic bodies scattered throughout the lobule. When high-dose immunosuppressive therapy was instituted soon after presentation, progressive improvement and eventual normalization of liver enzymes and bilirubin levels were observed. However, in cases in which the diagnosis was not made and therapy was delayed, a progressive cholestatic picture emerged with histologic evidence of loss of small bile ducts and portal fibrosis. We conclude that a distinct syndrome of chronic liver GVHD presenting as an acute hepatitis can be recognized in a patient at risk who is receiving no, or minimal, immunosuppressive medications. Liver biopsy is necessary to exclude viral causes of liver dysfunction and to confirm characteristic abnormalities of small bile ducts. Institution of high-dose immunosuppression can prevent progressive bile duct destruction and effect resolution of jaundice if given early.