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The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

The NLRP3 inflammasome, a caspase-1 activation platform, plays a key role in the modulation of liver inflammation and fibrosis. Here, we tested the hypothesis that interleukin 17 (IL-17) and tumor necrosis factor (TNF) are key cytokines involved in amplifying and perpetuating the liver damage and fibrosis resulting from NLRP3 activation. To address this hypothesis, gain-of-function Nlrp3A350V knock-in mice were bred onto il17a and Tnf knockout backgrounds allowing for constitutive Nlrp3 activation in myeloid derived cells in mice deficient in IL-17 or TNF. Livers of Nlrp3A350V knock-in mice exhibited severe liver inflammatory changes characterized by infiltration with neutrophils, increased expression of chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL2 chemokines, activated inflammatory macrophages, and elevated levels of IL-17 and TNF. Mutants with ablation of il17a signal showed fewer neutrophils when compared to intact Nlrp3A350V mutants, but still significant inflammatory changes when compared to the nonmutant il17a knockout littermates. The severe inflammatory changes associated with mutant Nlrp3 were almost completely rescued by Tnf knockout in association with a marked decrease in circulating IL-1β levels. Intact Nlrp3A350V mutants showed changes in liver fibrosis, as evidenced by morphometric quantitation of Sirius Red staining and increased mRNA levels of profibrotic genes, including connective tissue growth factor and tissue inhibitor of matrix metalloproteinase 1. Il17a lacking mutants exhibited amelioration of the aforementioned fibrosis, whereas Tnf-deficient mutants showed no signs of fibrosis when compared to littermate controls. Conclusion: Our study uncovers key roles for TNF and, to a lesser extent, IL-17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid-derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2018;67:736-749).

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3(A350V/+)CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.

Aim: Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. Materials & methods: RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. Results: The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. Conclusion: These findings uncover RNP as a novel potential NASH therapy.

© 2018 American Society for Biochemistry and Molecular Biology Inc. All Rights Reserved. Circulating oxidized linoleic acid (LA) metabolites (OXLAMs) are increased in patients with nonalcoholic steatohepatitis (NASH) and their levels correlate with disease severity. However, the mechanisms by which OXLAMs contribute to NASH development are incompletely understood. We tested the hypothesis that LA or OXLAMs provided directly through the diet are involved in the development of hepatic injury. C57BL/6 mice were fed an isocaloric high-fat diet containing low LA, high LA, or OXLAMs for 8 weeks. The livers of OXLAM-fed mice showed lower triglyceride concentrations, but higher FA oxidation and lipid peroxidation in association with increased oxidative stress. OXLAM-induced mitochondrial dysfunction was associated with reduced Complex I protein and hepatic ATP levels, as well as increased mitochondrial biogenesis and cytoplasmic mitochondrial DNA. Oxidative stress increased thioredoxin-interacting protein (TXNIP) in the liver and stimulated the activation of mitochondrial apoptosis signal-regulating kinase 1 (ASK1) leading to apoptosis. We also found increased levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and Caspase-1 activation in the livers of OXLAM-fed mice. In vitro, OXLAMs induced hepatocyte cell death, which was partly dependent on Caspase-1 activation. This study identified key mechanisms by which dietary OXLAMs contribute to NASH development, including mitochondrial dysfunction, hepatocyte cell death, and NLRP3 inflammasome activation.

Background & Aims Activation of Fas death receptor results in apoptosis in multiple organs, particularly liver, in a process dependent on Bid cleavage. Mice injected with an anti-Fas antibody die within hours of acute liver failure associated with massive apoptosis and hemorrhage. Our aim was to investigate the crosstalk of apoptotic and inflammatory pathways and the contribution of selective hepatocellular apoptosis during in vivo Fas activation. Methods We generated hepatocyte-specific Bid deficient mice ( hBid −/− ). Acute liver injury was induced by Fas-activating antibody (Jo2) in a time-course study. Results In contrast to controls, nearly all Jo2 injected hBid −/− survived. Their livers showed complete protection against hepatocellular apoptosis with minimal focal hemorrhagic changes and mainly non-parenchymal cell apoptosis. In agreement, the hepatocytes had no mitochondrial cytochrome c release in cytosol, or caspase 3 activation. hBid −/− livers showed marked increase in acute inflammatory foci composed of neutrophils and monocytes associated with the increased expression of proinflammatory chemokines and cytokines, in the manner dependent on non-canonical interleukin-1β activation and amplified in the absence of caspase-3 activation. In addition, hBid −/− mice were completely protected from hepatotoxicity and the infiltrated cells were cleared 2weeks post single Jo2 injection. Conclusions Hepatocyte Bid suppression is critical for the resistance to the lethal effects of Fas activation in vivo . Fas signaling induces differential activation of non-canonical interleukin-1β maturation, amplified in the absence of apoptotic Bid-mitochondrial loop, in hepatocytes. These findings may have important pathophysiological and therapeutic implications in a variety of liver disorders associated with Fas activation.

Hepatocyte cell death is a key feature of nonalcoholic steatohepatitis (NASH). As the contribution of specific caspases remains unclear, our aim was to ascertain the effect of caspase 3 suppression on liver injury and fibrogenesis.C57BL/6 wild-type (WT) and caspase 3 knock out (Casp3 (-/-)) mice were placed on a methionine- and choline-deficient (MCD) diet for 6 weeks to induce steatohepatitis and liver fibrosis. Thereafter, liver injury, liver fibrosis and hepatocellular apoptosis were quantified in liver sections. Additionally, expression of proteins associated with liver inflammation and fibrogenesis was analyzed.WT mice fed MCD diet showed marked activation of caspase 3 in hepatocytes, in conjunction with steatohepatitis and increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3 (-/-) mice fed the MCD diet showed similar serum aminotransferase levels and NAFLD activity scores (NAS) compared with WT MCD-fed mice. However, Casp3 (-/-) mice on the MCD diet showed a marked reduction in expression of transcripts for profibrogenic genes, which translated into reduced hepatic collagen deposition. These changes were associated with decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling. Casp3 (-/-) mice on the MCD showed a reduction in expression of chemokine receptor 2 (CCR2) leading to ameliorated infiltration of inflammatory lymphocyte antigen 6 complex, locus C1 (Ly6c) positive monocytes.These findings support a prominent role for hepatocyte caspase 3 activation in NASH-related apoptosis, fibrogenesis and fibrosis which in part is mediated via CCR2-dependent infiltration of Ly6c positive monocytes.

Autoimmune pancreatitis is highly responsive to steroid therapy, but because it mimics pancreatic cancer, it often precipitates unnecessary surgery. Adequate diagnostic tests are needed to permit appropriate medical therapy. Lymphocytic and obliterative phlebitis are reported in the majority of cases, as are elevated IgG4-positive plasma cells, indicating their high sensitivity. Their specificities, especially when used in conjunction, however, remain largely unknown. Movat pentachrome vascular and IgG4 immunohistochemical stains were performed on a total of 15 autoimmune pancreatitis cases (11 pancreatic resections and 4 biopsies), 39 usual-type alcoholic or idiopathic chronic pancreatitis cases, 35 pancreatic ductal adenocarcinoma-associated chronic pancreatitis cases, and 29 normal pancreata. Marked and diffuse lymphocytic and obliterative venulitis were detected in all 15 cases of autoimmune pancreatitis on Movat staining (100% sensitivity). Only a single carcinoma-associated chronic pancreatitis case among all of the controls showed diffuse benign venulitis that was nonobliterative (99% specificity). Nine of 13, 9 autoimmune pancreatitis cases showed marked IgG4 immunopositivity at >or=10 positive plasma cells per x 400 field (69% sensitivity). No increased IgG4 plasma cells were found in any of 103 controls (100% specificity). In combination, all of the autoimmune pancreatitis cases had at least one (13/13) and most had both markers (9/13), whereas none of the controls had both markers. Overall, these combined stains show very promising diagnostic utility and should be considered in combination with clinical and serologic analyses in the evaluation of chronic pancreatitis suspicious for malignancy. Future validating studies on preoperative biopsies with outcome data following steroid therapy will be essential.

Background & Aims Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The trigger for HSC activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype. Methods EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSC activation, migration, and proliferation were assessed. Loss- and gain-of-function studies were performed to explore the potential role of peroxisome proliferator-activated receptor-γ (PPAR-γ)-targeting microRNAs (miRNAs) carried by EVs into HSC. Results Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of profibrogenic genes (collagen-I, α-smooth muscle actin, and tissue inhibitor of metalloproteinases-2), proliferation, chemotaxis, and wound-healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSCs. The hepatocyte-derived EV miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression, with miR-128-3p being the most efficiently transferred. Furthermore, loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation. Conclusions Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel antifibrotic targets for NAFLD and other fibrotic diseases.

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of many tumors. To analyze the expression of EGFR and activated EGFR in well-differentiated neuroendocrine carcinomas including primary and metastatic gastrointestinal carcinoid tumors and pancreatic endocrine tumors (PET), we examined 58 gastrointestinal carcinoid tumors and 48 PET using immunohistochemistry, Western blotting, and RT-PCR. EGFR and activated EGFR (P-EGFR) were expressed by both gastrointestinal carcinoids and PET in primary and metastatic tumors, although a higher percentage of gastrointestinal carcinoid tumors expressed EGFR and activated EGFR. Western blotting detected a 170 kDa band for both EGFR and activated EGFR in three primary carcinoid tumors and two metastatic carcinoid tumors to the liver. RT-PCR analysis confirmed the expression of EGFR mRNA in both primary and metastatic carcinoid tumors. Patients with activated EGFR expression in their primary PET had a significantly worse prognosis compared to those who did not express activated-EGFR (P = 0.043). These results indicate that gastrointestinal carcinoid tumors as well as PET express EGFR and activated EGFR, and that expression is more common in gastrointestinal carcinoid tumors compared to pancreatic endocrine tumors. These findings implicate the EGFR and P-EGFR signal transduction pathway in the pathogenesis of these neuroendocrine tumors and suggest that targeted therapy directed against the EGFR tyrosine kinase domain may be a useful therapeutic approach in patients with unresectable metastatic gastrointestinal carcinoid tumors and pancreatic endocrine tumors.

Only a subset of hepatitis C virus (HCV)-infected patients develop progressive hepatic fibrosis after liver transplantation (LT). Hepatic stellate cell (HSC) activation is a pivotal step in hepatic fibrosis and precedes clinically apparent fibrosis. We determined whether early HSC activation, measured in 4-month protocol post-LT biopsies, is predictive of subsequent development of more histologically severe recurrence of HCV. Early (4 month) post-LT HSC activation, as measured by α-smooth muscle actin (α-SMA) staining, was determined in liver biopsies from recipients with severe (fibrosis score ≥ 2, n = 13) and with mild (fibrosis score of 0, n = 13) recurrence of HCV at one-year post-LT. Immunohistochemical staining for α-smooth muscle actin (α-SMA) was used to generate HSC activation scores (regional and total). Total HSC activation scores at 4 months were similar in patients with severe and mild HCV recurrence (3.9 ± 2.0 vs. 2.7 ± 2.2, P = 0.2). Regional HSC activation, assessed as parenchymal (zones 1, 2, and 3) or mesenchymal (portal tracts and fibrous septa), was different between the study groups, with higher mesenchymal scores predictive of progression. No patients in the mild recurrence group had detectable mesenchymal α-SMA staining vs. 46% (6/13) of patients with severe recurrence (P < 0.01). Mesenchymal activation of HSC had a specificity and positive predictive value of 100% for development of progressive fibrosis in liver allografts of patients with hepatitis C. In conclusion, early activation of mesenchymal HSCs is a marker for progressive fibrosis in patients with hepatitis C post-LT and may help select patients who would benefit from HCV or HSC-targeted therapy. (Liver Transpl 2005;11:1207–1213.)

The transcription factors CDX1 and CDX2 are homeobox genes that regulate development of the epithelium of the small and large intestine. A few studies have shown that Cdx2 protein expression is useful in the diagnosis of adenocarcinomas as well as neuroendocrine tumors of the small and large intestine. To examine the utility of Cdx2 in recognizing neuroendocrine tumors of unknown primary sites, we analyzed 224 primary and metastatic neuroendocrine tumors by immunohistochemistry. The specificity of the antibody reaction was confirmed by Western blotting. Cdx2 antibody stained all primary and most metastatic midgut carcinoid tumors. A few rectal and pulmonary carcinoids were positive, while gastric carcinoids were negative for Cdx2. One of five small cell carcinomas (20%) of the colon was positive for Cdx2, while all pulmonary small cell carcinomas were negative. Neuroendocrine tumors of the pituitary, parathyroid, medullary thyroid carcinomas, paragangliomas, pheochromocytomas and Merkel cell carcinomas were all negative for Cdx2. Western blot analysis of seven cases showed a 40 kDa band in both primary and metastatic midgut carcinoid tumors. These results indicate that Cdx2 can be very useful in recognizing metastatic neuroendocrine carcinomas of unknown primary sites, especially when they are derived from the small intestine.

Airway eosinophilic inflammation is a characteristic feature of allergic asthma. Exposure to allergens produced by the German cockroach (Blattella germanica) is a risk factor for allergic disease in genetically predisposed individuals, and has been linked to an increase in asthma morbidity among cockroach-sensitive inner city children. To determine the role and contribution of specific HLA class II in the pathogenesis of allergic airway inflammation in cockroach-induced asthma, we generated double-transgenic, double-knockout mice expressing human HLA-DQ8, HLA-DQ6, and CD4 molecules in the absence of mouse class II and mouse CD4. Mice were actively immunized and later challenged intranasally with cockroach allergen extract. These mice developed bronchoalveolar lavage fluid (BALF) eosinophilia and pulmonary eosinophilia. This was accompanied by an increase in total protein levels, IL-5, and IL-13 in BALF. There were also elevated levels of cockroach-specific serum IgG1 and total serum IgE. Histological analysis revealed peribronchial and perivascular eosinophilic inflammation in cockroach-treated mice. Other pathologic changes in the airways were epithelial cell hypertrophy and mucus production. Treatment with anti-DQ mAb significantly reduced pulmonary and BALF eosinophilia in cockroach allergen-sensitized mice. Aβ0 mice and transgenic mice expressing human CD4 molecule alone (without class II) or human HLA-DQ8 molecule (without CD4) treated in the same fashion showed no eosinophilia in bronchoalveolar fluid and no pulmonary parenchymal inflammation. Our results provide direct evidence that HLA-DQ molecules and CD4 T cells mediate cockroach-induced eosinophilic inflammation in the airways.

We investigated the immune response to the German cockroach (Blattella germanica), and one of its major antigens, Blattella germanica group 5 (Bla g 5), in a double-transgenic, double-knockout mouse expressing human HLA-DQ8, HLA-DQ6 and CD4 molecules in the absence of mouse class II and mouse CD4. Transgenic mice were primed and challenged with CR extract or individual synthetic peptides representing Bla g 5. Strong T-cell responses to CR extract were detected in both HLA-DQ/hCD4+ transgenic mice. The responses were two times lower in mice expressing HLA-DQ molecule in the context of mouse CD4. Under similar treatment, no responses were found in the double-knockout Abetadegrees/mCD4degrees mice and in mice expressing human CD4 molecule alone. HLA-DQ/hCD4+ mice produced primarily interleukin (IL)-5, IL-10, and IL-13. Minimal amounts of IL-4 were detected only in HLA-DQ6/ hCD4+ mice. Interferon (IFN)-gamma production was low in both transgenic mouse, suggesting a predominantly T-helper 2 (Th2)-type response. Cockroach allergen extract immunized HLA-DQ8/hCD4+ mice recognized only one of the 20 peptides of Bla g 5 while HLA-DQ6/hCD4+ mice responded primarily to three peptides. Primed with individual peptides, both HLA-DQ/hCD4+ mice responded maximally to peptides 10 (residues 91-110) and 17 (residues 161-180). In addition, HLA-DQ6/hCD4+ mice responded to peptide 16 (residues 151-170). Thus, peptides 10 and 17 contained the major HLA-DQ-restricted hCD4+ T-cell epitopes and could be recognized by both HLA-DQ8 and HLA-DQ6 transgenic mice. Transgenic mice represent a new tool for investigating the immune responses to cockroach allergen. Our results suggest that therapeutic strategies aimed at developing antagonist peptides might be a useful treatment (immunotherapy) for allergic asthma.

Background & Aim Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. Design Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. Results We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p

Dietz, Niki M., John R. Halliwill, John M. Spielmann, Lori A. Lawler, Bettina G. Papouchado, Tamara J. Eickhoff, and Michael J. Joyner. Sympathetic withdrawal and forearm vasodilation during vasovagal syncope in humans. J. Appl. Physiol. 82(6): 1785–1793, 1997.—Our aim was to determine whether sympathetic withdrawal alone can account for the profound forearm vasodilation that occurs during syncope in humans. We also determined whether either vasodilating β2-adrenergic receptors or nitric oxide (NO) contributes to this dilation. Forearm blood flow was measured bilaterally in healthy volunteers ( n = 10) by using plethysmography during two bouts of graded lower body negative pressure (LBNP) to syncope. In one forearm, drugs were infused via a brachial artery catheter while the other forearm served as a control. In the control arm, forearm vascular resistance (FVR) increased from 77 ± 7 units at baseline to 191 ± 36 units with −40 mmHg of LBNP ( P < 0.05). Mean arterial pressure fell from 94 ± 2 to 47 ± 4 mmHg just before syncope, and all subjects demonstrated sudden bradycardia at the time of syncope. At the onset of syncope, there was sudden vasodilation and FVR fell to 26 ± 6 units ( P < 0.05 vs. baseline). When the experimental forearm was treated with bretylium, phentolamine, and propranolol, baseline FVR fell to 26 ± 2 units, the vasoconstriction during LBNP was absent, and FVR fell further to 16 ± 1 units at syncope ( P < 0.05 vs. baseline). During the second trial of LBNP, mean arterial pressure again fell to 47 ± 4 mmHg and bradycardia was again observed. Treatment of the experimental forearm with the NO synthase inhibitor NG-monomethyl-l-arginine in addition to bretylium, phentolamine, and propranolol significantly increased baseline FVR to 65 ± 5 units but did not prevent the marked forearm vasodilation during syncope (FVR = 24 ± 4 vs. 29 ± 8 units in the control forearm). These data suggest that the profound vasodilation observed in the human forearm during syncope is not mediated solely by sympathetic withdrawal and also suggest that neither β2-adrenergic-receptor-mediated vasodilation nor NO is essential to observe this response.

Non-alcoholic steatohepatitis (NASH) is typically associated with pro-apoptotic caspase activation. A potential role for pro-inflammatory caspases remains incompletely understood. Our aims were to examine a potential role of caspase-1 in the development of liver damage and fibrosis in NASH. C57BL/6 wild type (WT) developed marked steatohepatitis, activation, fibrosis and increased hepatic caspase-1 and interleukin-1β expression when placed on the methionine- and choline-deficient (MCD) diet. Marked caspase-1 activation was detected in the liver of MCD-fed mice. Hepatocyte and non-parenchymal fractionation of the livers further demonstrated that caspase-1 activation after MCD feeding was mainly localized to non-parenchymal cells. Caspase-1-knockout (Casp1(-/-)) mice on the MCD diet showed marked reduction in mRNA expression of genes involved in inflammation and fibrogenesis (tumor necrosis factor-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; F4/80 was 1.5-fold greater in WT vs Casp1(-/-) MCD-fed mice; α-smooth muscle actin was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice; collagen 1-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; transforming growth factor-β was 2.4-fold greater in WT vs Casp1(-/-) MCD-fed mice; cysteine- and glycine-rich protein 2 was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp1(-/-) MCD-fed mice compared with WT MCD-fed animals. However, serum alanine aminotransferase levels, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells were similar in Casp1(-/-) and WT mice on the MCD diet. Selective Kupffer cell depletion by clodronate injection markedly suppressed MCD-induced caspase-1 activation and protected mice from fibrogenesis and fibrosis associated with this diet. The conclusion of this study is that it uncovers a novel role for caspase-1 in inflammation and fibrosis during NASH development.

Ethanol metabolism by liver generates short lived reactive oxygen species that damage liver but also affects distal organs through unknown mechanisms. We hypothesized that dissemination of liver oxidative stress proceeds through release of biologically active oxidized lipids to the circulation. We searched for these by tandem mass spectrometry in plasma of rats fed a Lieber-DeCarli ethanol diet or in patients with established alcoholic liver inflammation, steatohepatitis. We found a severalfold increase in plasma peroxidized phosphatidylcholines, inflammatory and pro-apoptotic oxidatively truncated phospholipids, and platelet-activating factor, a remarkably potent and pleiotropic inflammatory mediator, in rats chronically ingesting ethanol. Circulating peroxidized phospholipids also increased in humans with established steatohepatitis. However, reactive oxygen species generated by liver ethanol catabolism were not directly responsible for circulating oxidized phospholipids because the delayed appearance of these lipids did not correlate with ethanol exposure, hepatic oxidative insult, nor plasma alanine transaminase marking hepatocyte damage. Rather, circulating oxidized lipids correlated with steatohepatitis and tumor necrosis factor-alpha deposition in liver. The organic osmolyte 2-aminoethylsulfonic acid (taurine), which reduces liver endoplasmic reticulum stress and inflammation, even though it is not an antioxidant, abolished liver damage and the increase in circulating oxidized phospholipids. Thus, circulating oxidized phospholipids are markers of developing steatohepatitis temporally distinct from oxidant stress associated with hepatic ethanol catabolism. Previously, circulating markers of the critical transition to pathologic steatohepatitis were unknown. Circulating oxidatively truncated phospholipids are pro-inflammatory and pro-apoptotic mediators with the potential to systemically distribute the effect of chronic ethanol exposure. Suppressing hepatic inflammation, not ethanol catabolism, reduces circulating inflammatory and apoptotic agonists.

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are growing public health problems, and are strongly associated. The link between the two conditions remains poorly understood. Hepatic interleukin-6 (IL-6), a major proinflammatory cytokine, expression is increased in animal models of NAFLD, while in mice, selective sustained upregulation of IL-6 in the liver results in systemic insulin resistance. The extent and clinical significance of hepatic IL-6 expression in human NAFLD, as well as potential mechanisms by which steatosis may increase IL-6 production in the liver, have not been examined.To ascertain the occurrence and significance of IL-6 expression in the liver in human NAFLD.Plasma was obtained at time of liver biopsy from 50 consecutive patients with suspected NAFLD. Histology was assessed blindly. Hepatic IL-6 expression was assessed by immunohistochemistry, while plasma IL-6 levels were determined by an enzyme-linked immunosorbent assay.IL-6 expression was markedly increased in the livers of patients with nonalcoholic steatohepatitis (NASH) as compared to patients with simple steatosis (P0.005) or normal biopsies (P0.010), confirming the presence of hepatic IL-6 expression in human NASH. A positive correlation was observed between hepatocyte IL-6 expression and degree of inflammation and stage of fibrosis. Furthermore, liver IL-6 expression positively correlated with plasma IL-6 levels and degree of systemic insulin resistance. Culture of liver cells with saturated, but not mono- or polyunsaturated, FFA resulted in a significant increase in IL-6 messenger RNA (mRNA) and protein expression.Collectively, these data suggest that increased hepatic IL-6 production may play an important role in NASH development, as well as in systemic insulin resistance and diabetes.

Many patients with nonalcoholic fatty liver disease (NAFLD) have a benign clinical course, but a subgroup of patients progress to advanced fibrosis and cirrhosis. However, there are no available clinical tools to predict fibrosis progression in this population. Activated hepatic stellate cells (HSCs) are the source of collagen deposition in the liver. We aimed at determining whether an HSC activation score predicts fibrosis progression in NAFLD patients.The cohort consisted of 39 untreated patients with NAFLD with paired liver biopsies performed 5-59 months apart (mean, 22 months). Patients were divided into 2 groups on the basis of whether fibrosis progression was noted on their second liver biopsy. Liver tissue was immunostained for alpha-smooth muscle actin, and the HSC score was determined independently by 2 pathologists in the NAFLD population and in control subjects without liver disease.The HSC activation score was significantly increased in patients with fibrosis progression versus patients in whom no fibrosis progression was observed (4.8 +/- 0.5 vs 1.8 +/- 0.6, respectively; P.001). The HSC score was accurate in predicting fibrosis progression, with a positive predictive value of 90%, specificity of 94%, and an area under the receiver operating characteristic curve of 0.82. However, the negative predictive value and sensitivity were 56% and 41%, respectively. The inter-pathologist agreement for the HSC score was excellent (kappa coefficient, 0.95).These findings suggest that the HSC activation score is a suitable clinical tool to determine the risk of fibrosis progression in patients with NAFLD.