Inclusion Body Herpesvirus Hepatitis in Captive Falcons in the Middle East: A Review of Clinical and Pathologic Findings.

Inclusion body hepatitis in falcons is caused by a herpesvirus designated Falconid HV-1. This herpesvirus and other herpesviruses affecting birds of prey have not been assigned to a genus and include inclusion body herpesvirus hepatitis in eagles (Accipitrid HV-1) and inclusion body herpesvirus hepatitis in owls (Strigid HV-1). Herpesvirus infections have been diagnosed in both captive and free-living raptors across Europe, North America, and Asia in different species of the family Falconidae. Herpesviruses affecting owls and falcons have been found to be antigenically similar to pigeon herpesvirus (Columbid HV-1) and distinct from other avian herpesviruses. When the herpesvirus isolates from owls, falcons, and pigeons were compared by sequencing a fragment of the herpes viral DNA polymerase gene from those birds naturally infected with the virus, the sequences from these 3 sources were found to be nearly identical. The authors of this study concluded that the Falconid HV-1, Strigid HV-1, and Columbid HV-1 were the same virus. Furthermore, the authors also proposed that the virus therefore be referred to as Columbid HV-1 (CoHV-1), because pigeons may be responsible for the transmission of the virus to birds of prey. Pigeons are often carriers of the virus without showing any clinical signs. It has long been suspected that raptors may contract the infection by the ingestion of infected pigeons. Some studies have suggested that falcons may not contract the infection through the oral route by ingesting carrier pigeons, but through the ocular or nasal route. Inclusion body herpesvirus hepatitis is a frequently diagnosed disease in the captive falcon population used for falconry, racing, and breeding in the Middle East, and it seems to be associated with the extensive use of pigeons for training and as a food item. This paper reviews the clinical and pathological findings in falcons affected by inclusion body herpesvirus hepatitis in the Middle East.

Glt25d2 Knockout Directly Increases CD25+CD69- but Decreases CD25-CD69+ Subset Proliferation and is Involved in Concanavalin-Induced Hepatitis.

BACKGROUND/AIMS: The elaborate structure of the extracellular matrix (ECM) and the appropriate surface glycoforms upon it are indispensable to CD4+ T cell regulation. METHODS: To explore the effects of Glcα1,2Galß1 glycosylation mediated by GLT25D2 (Colgalt2) for CD4+ T cell regulation, we prepared C57BL/6J Glt25d2-/- mice. In the induction of hepatitis, after concanavalin A (Con A) challenge for 6, 12, and 24 h, more extensive parenchymal injury was noted in Glt25d2-/- mice than in wild-type (WT) mice at 12 h. Immunohistochemistry and laser scanning confocal microscopy were used to detect GLT25D2 expression, and subsets of CD4+T cells was analyzed by flow cytometry. A total of 26 cytokines in serum samples were determined using Luminex technology. RESULTS: The trend in liver injury score variation was consistent with serum alanine aminotransferase and aspartate aminotransferase levels. The levels of interleukin 4 (IL-4), IL-1ß, IL-9, and several chemokines such as macrophage inflammatory protein-2, eotaxin, and growth-related oncogene α were significantly increased in Glt25d2-/- mice compared with WT mice after Con A challenge. A further phenotype analysis of primary Glt25d2-/- CD4+ T cells showed that Glt25d2 knockout increased the frequency of the CD25+CD69- subset but decreased the frequency of the CD25-CD69+ subset after Con A challenge for 6, 12, and 24 h compared with those of WT CD4+ T cells. Activation-induced apoptosis was also significantly increased in Glt25d2-/- CD4+ T cells after Con A challenge compared with WT CD4+ T cells. Lectin microarray hybridization showed that Glt25d2 knockout increased the binding activity of Narcissus pseudonarcissus lectin to CD4+ T cells but Amaranthus caudatus lectin-binding activity was lost during Con A challenge. CONCLUSION: The present results suggest that collagen glycosylation mediated by GLT25D2 may regulate a subset of CD4+ T cells and be involved in the pathogenesis of Con A-induced hepatitis.

RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis.

BACKGROUND & AIMS: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. METHODS: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1LPC-KO). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-α during LPS-mediated liver injury. RESULTS: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. CONCLUSIONS: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. LAY SUMMARY: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure.

Spatiotemporal Characterization of the Cellular and Molecular Contributors to Liver Fibrosis in a Murine Hepatotoxic-Injury Model.

The interplay between the inflammatory infiltrate and tissue resident cell populations invokes fibrogenesis. However, the temporal and mechanistic contributions of these cells to fibrosis are obscure. To address this issue, liver inflammation, ductular reaction (DR), and fibrosis were induced in C57BL/6 mice by thioacetamide administration for up to 12 weeks. Thioacetamide treatment induced two phases of liver fibrosis. A rapid pericentral inflammatory infiltrate enriched in F4/80(+) monocytes co-localized with SMA(+) myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1 to 6 weeks). An expansion of bone marrow-derived macrophages preceded a second phase, characterized by accelerated progression of fibrosis (>6 weeks) after DR migration from the portal tracts to the centrilobular site of injury, in association with an increase in DR/macrophage interactions. Although chemokine (C-C motif) ligand 2 (CCL2) mRNA was induced rapidly in response to thioacetamide, CCL2 deficiency only partially abrogated fibrosis. In contrast, colony-stimulating factor 1 receptor blockade diminished C-C chemokine receptor type 2 [CCR2(neg) (Ly6C(lo))] monocytes, attenuated the DR, and significantly reduced fibrosis, illustrating the critical role of colony-stimulating factor 1-dependent monocyte/macrophage differentiation and linking the two phases of injury. In response to liver injury, colony-stimulating factor 1 drives early monocyte-mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation, and their association with the advancing DR, the formation of fibrotic septa, and the progression of liver fibrosis to cirrhosis.

Identification of Glyceraldehyde-3-Phosphate and Alcohol Dehydrogenases as Autoantigens in Doberman Hepatitis.

An autoimmune background is suspected for Doberman hepatitis (DH). It is based on the finding of mononuclear cell infiltrates in the liver, strong female bias, association to the homozygous risk factor dog leucocyte antigen (DLA) allele DRB1*00601 and aberrant major histocompatibility complex (MHC) class II expression on hepatocytes that correlates with the degree of inflammation in the liver. The aim of this study was to search for autoantibodies against liver-related antigens associated with DH. Twenty-five Dobermans with subclinical DH (SDH), 13 that clinically manifest DH (CDH) and 17 healthy controls were studied. Immunoblotting analysis detected specific antibodies in the DH sera. By mass spectrometry the targets were identified as liver-related enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). Using ELISA, anti-GAPDH IgG was detected in 36% (9/25) of SDH dogs and 69.2% (9/13) of the CDH dogs compared to healthy controls (0/17) (P < 0.0005). Anti-ADH IgG was detected in 72% (18/25) of SDH dogs and 76.9% (10/13) of CDH dogs and only in one (1/17) control (P < 0.0005). The finding of novel autoantigens, GAPDH and ADH strengthen the hypothesis that DH is an autoimmune disease of the liver. These findings suggest that DH could be diagnosed by screening for autoantibodies against the defined antigens.

Necrotizing and haemorrhagic hepatitis and enteritis in commercial layer pullets.

This case report describes an episode of recurring severe necrotizing and haemorrhagic hepatitis and enteritis experienced in a flock of commercial layer pullets at 12 weeks of age and again at 18 weeks of age in Indiana. Pullets had been vaccinated at 10 weeks old using a trivalent Salmonella Enteritidis (SE)/Newcastle disease/infectious bronchitis oil-emulsion-inactivated vaccine. The pullets were found dead at 12 weeks with firm but friable, enlarged, haemorrhagic livers, enlarged spleens, and necrohaemorrhagic intestines. Histopathologic findings were consistent with a necrotizing and haemorrhagic enteritis and hepatitis. Livers had multiple intra-sinusoidal thrombi, intestines contained Gram-positive bacterial colonies, and spleens had marked lymphoid depletion. The pullets seemed to improve after antibiotic treatment. Pullets were vaccinated with an inactivated SE vaccine at 14 weeks of age. A second spike of mortality occurred at 18 weeks of age. Although clostridial enteritis and hepatitis were highly suspected in the two cases based on macroscopic and microscopic findings, no significant bacterial or viral agents were isolated from the livers and intestines. In summary, lesions in the liver and intestines are speculated to be due to repetitive vaccination, leading to an anamnestic response by the immune system, and resulting in an immune-mediated response. However, much of the pathogenesis is still unclear, and other causes such as unidentified infectious aetiology, transmissible amyloidosis, and hypersensitivity may need further investigation.

The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD⁺ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice.

Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, exhibits beneficial effects on metabolic syndrome. Sustained inflammation plays a crucial role in the pathogenesis of metabolic syndrome. Here we explored the effects of PSPC on high-fat diet (HFD)-induced hepatic inflammation and the mechanisms underlying these effects. Mice were divided into four groups: Control group, HFD group, HFD + PSPC group, and PSPC group. PSPC was administered by daily oral gavage at doses of 700 mg/kg/day for 20 weeks. Nicotinamide riboside (NR) was used to increase NAD⁺ levels. Our results showed that PSPC effectively ameliorated obesity and liver injuries in HFD-fed mice. Moreover, PSPC notably blocked hepatic oxidative stress in HFD-treated mice. Furthermore, PSPC dramatically restored NAD⁺ level to abate endoplasmic reticulum stress (ER stress) in HFD-treated mouse livers, which was confirmed by NR treatment. Consequently, PSPC remarkably suppressed the nuclear factor-κB (NF-κB) p65 nuclear translocation and nucleotide oligomerization domain protein1/2 (NOD1/2) signaling in HFD-treated mouse livers. Thereby, PSPC markedly diminished the NLR family, pyrin domain containing 3 (NLRP3) inflammasome activation, ultimately lowering the expressions of inflammation-related genes in HFD-treated mouse livers. In summary, PSPC protected against HFD-induced hepatic inflammation by boosting NAD⁺ level to inhibit NLRP3 inflammasome activation.

Treatment effect of a flavonoid prescription on duck virus hepatitis by its hepatoprotective and antioxidative ability.

CONTEXT: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. OBJECTIVE: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. MATERIALS AND METHODS: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. RESULTS: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 µg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p < 0.05) existed between the hepatic injury indices and the oxidative stress indices. CONCLUSIONS: BLIN showed a significant curative effect on DVH. The antioxidative and hepatoprotective effects of BLIN made great contributions to the treatment of DVH. Furthermore, BLIN is expected to be exploited as a new drug for the clinical treatment of DVH.

SIV-induced Translocation of Bacterial Products in the Liver Mobilizes Myeloid Dendritic and Natural Killer Cells Associated With Liver Damage.

Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)-infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Survival and inflammation promotion effect of PTPRO in fulminant hepatitis is associated with NF-κB activation.

Previous investigations demonstrated that protein tyrosine phosphatase, receptor type, O (PTPRO) acts as a tumor suppressor in liver cancer; however, little is known about its role in liver inflammation. Thus, we investigated the role of PTPRO in fulminant hepatitis (FH) using a Con A-induced mouse model. Significantly more severe liver damage, but attenuated inflammation, was detected in PTPRO-knockout (KO) mice, and PTPRO deficiency could confer this phenotype to wild-type mice in bone marrow transplantation. Moreover, hepatocytes with PTPRO depletion were more sensitive to TNF-α-induced apoptosis, and secretion of cytokines was significantly decreased in both T and NK/NKT cells and led to marked impairment of NF-κB activation. Intriguingly, wild-type and PTPRO-KO cells responded equally to TNF-α in activation of IKK, but NF-κB activation was clearly decreased in PTPRO-KO cells. PTPRO associated with ErbB2, and loss of PTPRO potentiated activation of the ErbB2/Akt/GSK-3ß/ß-catenin cascade. Increased ß-catenin formed a complex with NF-κB and attenuated its nuclear translocation and activation. Importantly, in humans, PTPRO was much decreased in FH, and this was associated with enhanced ß-catenin accumulation but reduced IFN-γ secretion. Taken together, our study identified a novel PTPRO/ErbB2/Akt/GSK-3ß/ß-catenin/NF-κB axis in FH, which suggests that PTPRO may have therapeutic potential in this liver disease.

1,25-(OH)2-vitamin D3 prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4(-/-) model.

BACKGROUND/PURPOSE OF THE STUDY: Vitamin D3-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D3-administration has thus been proposed as a therapeutic approach. Vitamin D3 has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH)2-vitamin D3 inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. METHODS: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen(®)-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4(-/-) (Abcb4(-/-))-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. RESULTS: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-ß-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4(-/-)-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. CONCLUSION: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4(-/-)-mice, administration of calcitriol ameliorates inflammatory liver-damage but has no effect on biliary fibrosis after 4 weeks of treatment.

Juglone prevents metabolic endotoxemia-induced hepatitis and neuroinflammation via suppressing TLR4/NF-κB signaling pathway in high-fat diet rats.

Juglone as a natural production mainly extracted from green walnut husks of Juglans mandshurica has been defined as the functional composition among a series of compounds. It showed powerful protective effect in various diseases by inhibiting inflammation and tumor cells growth. However, studies on its anti-inflammatory effect based on high-fat diet-induced hepatitis and neuroinflammation are still not available. In this regard, we first investigated whether juglone suppresses high-fat diet-stimulated liver injury, hypothalamus inflammation and underlying mechanisms by which they may recover them. SD rats were orally treated with or without high-fat diet, 0.25 mg/kg or 1 mg/kg juglone for 70 days. Subsequently, blood, hypothalamus and liver tissue were collected for different analysis. Also, the primary astrocytes were isolated and used to analyze the inhibitory effect of juglone in vitro. Analysis of inflammatory cytokines declared that the inhibition of TNF-α, IL-1ß and IL-6 could be carried by juglone in response to high-fat diet rats. Meanwhile, TLR4 expression and NF-kappa activity also have been confirmed to be the key link in the development of hepatitis and nerve inflammation. The activation was significantly suppressed in treatment group as compared with model. These results indicated that juglone prevents high-fat diet-induced liver injury and nerve inflammation in mice through inhibition of inflammatory cytokine secretion, NF-kappa B activation and endotoxin production.

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris).

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 µm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was "type 9", 1-2 µm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7-9 µm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were "type 1", thin walled (< 0.5 µm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin-fixed paraffin-embedded sections. Dogs were either singly infected with S. caninum or multiply co-infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).

Unconventional RORγt+ T cells drive hepatic ischemia reperfusion injury.

An emerging body of evidence suggests a pivotal role of CD3(+) T cells in mediating early ischemia reperfusion injury (IRI). However, the precise phenotype of T cells involved and the mechanisms underlying such T cell-mediated immune responses in IRI, as well as their clinical relevance, are poorly understood. In this study, we investigated early immunological events in a model of partial warm hepatic IRI in genetically targeted mice to study the precise pathomechanistic role of RORγt(+) T cells. We found that unconventional CD27(-)γδTCR(+) and CD4(-)CD8(-) double-negative T cells are the major RORγt-expressing effector cells in hepatic IRI that play a mechanistic role by being the main source of IRI-mediating IL-17A. We further show that unconventional IRI-mediating T cells are contingent on RORγt, as highlighted by the fact that a genetic deficiency for RORγt, or its therapeutic antagonization via digoxin, is protective against hepatic IRI. Therefore, identification of CD27(-)γδTCR(+) and CD4(-)CD8(-) double-negative T cells as the major source of IL-17A via RORγt in hepatic IRI opens new therapeutic options to improve liver transplantation outcomes.

IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation.

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride-induced liver fibrosis of IL-17RA-deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage-specific transcription factor Retinoic acid receptor-related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-ß mRNA expression, suggesting an IL-17A-driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A-induced HSC activation and collagen expression. In conclusion, IL-17A(+) Retinoic acid receptor-related orphan receptor γt(+) neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A-dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.

Beyond Parasitism: Hepatic Lesions in Stranded Harbor Porpoises (Phocoena phocoena) Without Trematode (Campula oblonga) Infections.

The liver can be an indicator of the health of an individual or of a group, which can be especially important to identify agents that can cause disease in multiple species. To better characterize hepatic lesions in stranded harbor porpoises (Phocoena phocoena), we analyzed the livers from 39 porpoises that stranded along the Dutch coast between December 2008 and December 2012. The animals were selected because they had either gross or histologic liver lesions with minimal autolysis and no evidence of trematode (Campula oblonga) infection. The most common finding was a chronic hepatitis (22/39, 56.4%) that was often associated with significant disease reported in another organ system (18/22, 81.8%), of which 14 had chronic systemic disease. One case of chronic hepatitis was so severe as to mimic lymphoma, which could only be differentiated with immunohistochemistry. The other common lesions were lipidosis (11/39, 28.2%) and acute hepatitis (6/39, 15.4%), often in combination with mild chronic changes. Overall, although there were no consistent trends in etiology for the hepatic lesions, lipidosis was associated with starvation (8/11, 72.7%) and acute disease, and acute hepatitis was associated with bacterial infections and sepsis (6/6, 100%).

Deletion of interleukin (IL)-12p35 induces liver fibrosis in dominant-negative TGFß receptor type II mice.

Mice with a dominant-negative transforming growth factor ß receptor restricted to T cells (dnTGFßRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40(-/-) dnTGFßRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFßRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFßRII mice, resulting in an IL-12p35(-/-) dnTGFßRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40(-/-) mice, the IL-12p35(-/-) mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFßRII mice. The p35(-/-) mice also demonstrated a distinct cytokine profile characterized by a shift from a T-helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35(-/-) mice. In conclusion, IL-12p35(-/-) dnTGFßRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC.

Interferon-gamma-mediated tissue factor expression contributes to T-cell-mediated hepatitis through induction of hypercoagulation in mice.

UNLABELLED: Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF. CONCLUSIONS: Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Metabolite profiling and pathway analysis of acute hepatitis rats by UPLC-ESI MS combined with pattern recognition methods.

BACKGROUND & AIMS: Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS: The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS: Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION: This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.