IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors on the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing identified IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells.

Hepatic inflammation is a key pathologic feature of nonalcoholic steatohepatitis (NASH). Natural killer T (NKT) cells and clusters of differentiation (CD)8+ T‐cells are known to play an important role in obesity‐related adipose tissue inflammation. We hypothesized that these same inflammatory phenotypes would be present in progressive NASH. We used a previously established high‐fat high‐carbohydrate (HFHC) murine obesogenic diet model of progressive NASH to investigate the role of NKT cells and CD8+ T‐cells in C57Bl6/J mice. To better understand the impact of these cell populations, CD1d‐deficient and CD8+ T‐cell‐depleted mice were subjected to an HFHC diet for 16 weeks. C57Bl6/J mice fed an HFHC diet had increased body weight, liver triglyceride content, serum alanine aminotransferase levels, and increased NKT‐cell and CD8+ T‐cell infiltration in the liver. In addition, human liver sections from patients with NASH showed increased CD8+ T‐cells. In comparison, CD1d‐deficient and CD8 T‐cell‐depleted mice fed an HFHC diet had a lower hepatic triglyceride content, lower alanine aminotransferase levels, lower activated resident macrophages and infiltrating macrophages, improved nonalcoholic fatty liver disease activity scores, and reduced α‐smooth muscle actin, collagen type 1 alpha 1, and collagen type 1 alpha 2 messenger RNA expression. Further, while CD1d‐deficient mice were protected against weight gain on the HFHC diet, CD8 T‐cell‐depleted mice gained weight on the HFHC diet. Conclusion: We found that NASH has an immunological signature that includes hepatic infiltrating NKT and CD8+ T‐cells. Depletion of these cells resulted in reduced NASH progression and thus presents novel therapeutic avenues for the treatment of NASH. (Hepatology Communications 2017;1:299–310)

Biliary atresia (BA) is a progressive obliterative extrahepatic cholangiopathy of infants. Despite successful surgical drainage of bile, most children with BA experience progression of cirrhosis and liver failure. There is evidence that trans-differentiation of epithelial cells into collagen-producing mesenchymal cells during injury occurs secondary to Fibroblast Growth Factor (FGF) and Transforming Growth Factor-β (TGFβ) signaling activation. Therefore, we hypothesized that TGFβ-mediated epithelial-mesenchymal trans-differentiation (EMT) of hepatic progenitor cells contributes to periportal fibrosis associated with BA.

High fat diet induced models of simple hepatic steatosis have been shown to result in a decreased hepatic natural killerT (NKT) cell population. Interestingly, NKT cells are known to correlate positively with the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity. Recent data implicates refined carbohydrates (Fructose) in the progression of hepatic steatosis to advanced fibrotic NASH in humans. This has also been replicated in a novel model of obesity and NASH with fibrosis in mice. We therefore hypothesized that NKT cells populate the livers in a murine model of advanced NASH with fibrosis. Methods: Adult male C57BL6 mice were randomly assigned to control (C) or high fat high carbohydrate diet (HFHC). The C group received regular chow and water while the HFHC group received diet with Medium Chain Triglycerides (MCT; 58% kcal) and water supplemented with 55% fructose & 45% sucrose. Mice were sacrificed at 16 wks of age and underwent hepatic mononuclear cell phenotyping by flow cytometry. Results: Mice receiving HFHC diet gained significantly more weight than C fed mice starting at 4 wk (p<0.01), with a Mean ± SE weight of 45.84±1.4g at 16 wk compared to 31.33±1.0g for C fed mice (p<0.0001). Fasting glucose was higher in HFHC mice at 16 wks (176.3±9.2 mg/dl; p=0.001). Liver weight at 16 weeks was higher in the HFHC group (2.44±0.2g) compared to the C fed mice (1.47±0.03g; p<0.01). Steatosis as measured by liver triacylglycerol (TG) content was also higher in HFHC mice at 16 wks (2354±487.8 mg/dl/100 mg wet liver) compared to the C fed mice (p= 0.001). Inflammation as measured by serum alanine aminotransferase (ALT) levels was higher in HFHC mice (135.8±24.28 IU/L) at 16 wks compared to the C fed mice (p=0.019). Flow cytometric analysis identified increased NK-T cell (CD3+NK1.1+) signature in mice receiving HFHC (3.7±2.3x104 cells) compared to C fed mice (1.1±0.6x104 cells; p=0.02). While hepatic CD1d-restricted invariant NK-T CD4+ and CD8+ cell populations remained unchanged, total CD3+CD8+ T-cells significantly increased in the HFHC group (1.93±0.79x105) vs. C fed (1.0±0.7x105; p=0.04). A corresponding increase in CD8+ T-cells expressing the activation marker CD69 was also seen [(2.02±1.01x104 (C) vs 6.85±2.6x104 (HFHC); p<0.01]. Liver fibrosis was visible at sacrifice in majority of the HFHC fed mice (Stage 1c and 2) while none was seen in C diet fed mice. Conclusion: HFHC diet produces a relatively rapid and progressive liver injury resulting in NASH with fibrosis in a background of diet-induced obesity. In this system, which closely resembles the human phenotype of advanced NASH with fibrosis, we observed a significant increase in hepatic NK-T and CD8+ T-cells. These cell types may potentially typify the hepatic inflammasome in the progression of human disease to NASH with fibrosis.

Society for Pediatric Pathology Interim Meeting, Columbus, Ohio, October 5–8, 2006 Abstracts are listed in presentation order, beginning with thes are listed in presentation order, beginning with the Platform Presentations Platform Presentations: 1. Microarray Analysis of Ventilated Preterm Lungs Identifies Differential Expression of Angiogenesis-Related Genes. ME De Paepe, D Greco, F Gundogan, S Gundavarapu, Q Mao, Women and Infants Hospital and Brown Medical School, Providence, RI. Background: Preterm infants exposed to mechanical ventilation and high concentrations of oxygen are at risk for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disorder characterized by arrested alveolar development. Recent studies have described disruption of microvascular development in BPD, resembling the primitive angioarchitectural patterns seen in canalicular/saccular stages of lung development. The molecular regulation of this BPD-associated dysangiogenesis remains undetermined. The aim of this study was to investigate the expression of angiogenesisrelated genes in ventilated preterm human lungs. Design: RNA was extracted from lung tissues obtained at post-mortem examination (2001–2006). Infants in the Ventilated group (N 1⁄4 7) were between 23 to 29 weeks postmenstrual age at the time of death and had received ventilatory support for at least 5 days. Age-matched Control infants (N1⁄4 7) had lived for less than six hours. Angiogenic gene expression was studied by ribonuclease protection assay (RPA) and pathway-focused microarray, allowing simultaneous interrogation of 115 angiogenesis-related genes. MannWhitney U test was used for statistical analysis. Results: By RPA, ventilated lungs showed significantly downregulated expression of angiogenic regulators traditionally implicated in lung development, such as vascular endothelial growth factor (VEGF), angiopoietin and their respective receptors. In contrast, expression of endoglin, a TGF-â co-receptor, was significantly upregulated. Microarray analysis confirmed the RPA results and, in addition, identified significant (. 1.5 fold) upregulation of 15, and downregulation of 14 angiogenesis-related genes. The differentially expressed genes included growth factors, adhesion molecules, cytokines/chemokines, transcription factors and matrix pro-

BACKGROUND & AIMS Discovery of the pathogenic mechanisms of biliary atresia has been limited by the inability to study extrahepatic biliary tissues from patients at early phases of disease. Here, we used a rotavirus-induced model of biliary atresia to investigate the entire biliary transcriptome for molecular networks activated at the onset and different phases of progression to duct obstruction. METHODS We injected Balb/c mice with saline or rotavirus intraperitoneally within 24 hours of birth, microdissected the gallbladder and extrahepatic bile ducts en bloc 3, 7, and 14 days later, generated biotinylated RNA pools, and hybridized them against microarrays containing 45,101 gene products. RESULTS Data filtering, cluster analysis, and functional assignment of the gene expression platform revealed 2 unique patterns of expression. The first was an overarching expression of genes regulating immunity, enzymes, and structural proteins at all phases of atresia. Within this pattern, the sequential expression of the interferon inducers Irf7 and Irf9 at the onset of injury, and interferon-gamma and interferon-gamma-activated genes (Stat1, Igtp, Cxcl9, Cxcl10) at the time of duct obstruction, pointed to a prominent proinflammatory circuit. The second was the time-restricted expression of genes regulating biological networks previously unrecognized in biliary atresia, such as the complement components C3ar-1 and C1q-alpha/beta. CONCLUSIONS The coordinate expression of functionally related genes in the biliary transcriptome underscores a predominant proinflammatory footprint and provides a basis for identification of gene groups that may play regulatory roles in the pathogenesis of duct injury and obstruction in experimental biliary atresia.

gp120 is a subunit of the Env (viral envelope protein) of HIV-1. The protein consists of inner and outer domains linked by a bridging sheet. Several gp120 residues that bind the neutralizing antibody 17b as well as the cellular co-receptor CCR5 (CC chemokine receptor 5), are located in the bridging sheet. Peptides that mimic the 17b-binding regions of gp120 would be useful potential immunogens for the generation of neutralizing antibodies against HIV-1. Towards this end, a 26-residue, four-stranded beta-sheet peptide was designed on the basis of the structure of the bridging sheet, and its structure was characterized in methanol by NMR. In methanol, amide and alpha-proton resonances were well resolved and dispersed. A number of interstrand NOEs (nuclear Overhauser effects) were observed, providing good evidence for multiple turn beta-hairpin structure. NOEs also provided good evidence for all Xxx-D-Pro bonds in the trans configuration and all three turns formed by a two residue D-Pro-Gly segment to be of type II' turn. The structure conforms well to the designed four-stranded beta-sheet structure. Approx. 20% of the peptide was estimated to adopt a folded conformation in water, as evidenced by CD spectroscopy. This was consistent with smaller, but still significant, downfield shifts of C(alpha)H protons relative to random-coil values. A second peptide was designed with two disulphide bonds to further constrain the peptide backbone. While structured in methanol, this peptide, like the previous one, also exhibits only partial structure formation in water, as evidenced by CD spectroscopy.

The molecular basis for the embryonic and perinatal clinical forms of biliary atresia is largely undefined. In this study, we aimed to: 1) determine if the clinical forms can be differentiated at the transcriptional level, and 2) search for molecular mechanisms underlying phenotypic differences. To this end, we generated biotinylated cRNA probes from livers of age-matched infants with the embryonic (n = 5) and perinatal (n = 6) forms of biliary atresia at the time of diagnosis and hybridized them against the Affymetrix human HG-U133 A and B microarrays containing 44,760 gene products. Data filtering and two-way cluster analysis of the gene expression platform identified 230 genes with an expression profile that is highly distinctive of the clinical phenotypes. Functionally, the profile did not reveal a higher-order function for a specific cell type; instead, it uncovered a coordinated expression of regulatory genes. These regulatory genes were predominantly represented in the embryonic form (45% of genes), with a unique pattern of expression of genes involved in chromatin integrity/function (Smarca-1, Rybp, and Hdac3) and the uniform overexpression of five imprinted genes (Igf2, Peg3, Peg10, Meg3, and IPW), implying a failure to downregulate embryonic gene programs. In conclusion, embryonic and perinatal forms of biliary atresia are distinguished by gene expression profiling. The coordinate expression of regulators of chromatin structure/function and of imprinted genes provides evidence for a transcriptional basis for the pathogenesis of the embryonic form of biliary atresia. Further studies exploring these biological processes are required to determine the significance of these findings.

Epigraphy is the study of ancient inscriptions. These inscriptions are of great importance as they give the picture of ancient civilization. Since the contribution of the inscription in understanding the ancient history is remarkable, the automation of decoding the inscription into an understandable form is found to be vital. To automate the deciphering of epigraphical script s, we need to segment the characters from the text lines present in the script. The complexity involved in segmentation of epigraphical characters is the spacing between two text lines and adjacent characters. Some times the lines are touching and not spaced uniformly. The text lines are also found to be skewed in some scripts. Hence we propose a novel approach based on nearest neighbors clustering method to segment each text line and character. The proposed method works for skewed document also.

The etiology and pathogenesis of bile duct obstruction in children with biliary atresia are largely unknown. We have previously reported that, despite phenotypic heterogeneity, genomic signatures of livers from patients display a proinflammatory phenotype. Here, we address the hypothesis that production of IFN-gamma is a key pathogenic mechanism of disease using a mouse model of rotavirus-induced biliary atresia. We found that rotavirus infection of neonatal mice has a unique tropism to bile duct cells, and it triggers a hepatobiliary inflammation by IFN-gamma-producing CD4(+) and CD8(+) lymphocytes. The inflammation is tissue specific, resulting in progressive jaundice, growth failure, and greater than 90% mortality due to obstruction of extrahepatic bile ducts. In this model, the genetic loss of IFN-gamma did not alter the onset of jaundice, but it remarkably suppressed the tissue-specific targeting of T lymphocytes and completely prevented the inflammatory and fibrosing obstruction of extrahepatic bile ducts. As a consequence, jaundice resolved, and long-term survival improved to greater than 80%. Notably, administration of recombinant IFN-gamma led to recurrence of bile duct obstruction following rotavirus infection of IFN-gamma-deficient mice. Thus, IFN-gamma-driven obstruction of bile ducts is a key pathogenic mechanism of disease and may constitute a therapeutic target to block disease progression in patients with biliary atresia.

A study of the response to horizontal ground shaking of a rigid cylindrical tank containing an inviscid liquid with a continuous vertical variation in density is presented. In addition to the free vibrational sloshing characteristics of the liquid, the responses examined include the vertical displacements at the free surface, and the impulsive and convective components of the hydrodynamic wall pressures and associated tank forces. The equations of motion for the system are formulated for an arbitrary variation in liquid density but the solutions presented are for a density that increases exponentially from top to bottom. Comprehensive numerical data are included which elucidate the underlying response mechanisms and the effects and relative importance of the various parameters involved. The solution for the continuous density variation considered herein is also compared with a previously reported solution in which the liquid was modelled as a multi-layered, discrete system.