Faculty Bibliography

BACKGROUND & AIMS/OBJECTIVE:Progression of liver fibrosis still occurs in some patients with chronic hepatitis B virus (HBV) infection despite antiviral therapy. We aimed to identify risk factors for fibrosis progression in patients who received antiviral therapy. METHODS:We conducted a longitudinal study of patients with chronic HBV infection and liver biopsies collected before and after 78 weeks of anti-HBV therapy. Fibrosis progression was defined as Ishak stage increase ≥ 1 or as predominantly progressive classified by P-I-R system (Beijing Classification). Levels of HBV DNA and HBV RNA in blood samples were measured by real-time quantitative PCR. HBV RNA in liver tissue was detected by in situ hybridization. RESULTS:A total of 239 patients with chronic HBV infection with paired liver biopsies were included. Among the 163 patients with significant fibrosis at baseline (Ishak ≥ stage 3), fibrosis progressed in 22 patients (13%), was indeterminate in 24 patients (15%), and regressed in 117 patients (72%). Univariate and multivariate analyses revealed that independent risk factors for fibrosis progression were higher rate of detected HBV DNA at week 78 (odds ratio, 4.84; 95% CI, 1.30-17.98; P=.019) and alcohol intake (odds ratio, 23.84; 95% CI, 2.68-212.50; P=.004). HBV DNA was detected in blood samples from a significantly higher proportion of patients with fibrosis progression (50%) at week 78 than patients with fibrosis regression (19%) or indeterminate fibrosis (26%) (P=.015), despite low viremia (20-200 IU/mL) in all groups. The decrease of serum HBV RNA from baseline in the fibrosis regression group was larger than that in the fibrosis progression group. CONCLUSIONS:In a longitudinal study of patients with chronic HBV infection, we associated liver fibrosis progression at week 78 of treatment with higher rates of detected HBV DNA. We propose that a low level of residual HBV may still promote fibrosis progression, and that patients' levels of HBV DNA should be carefully monitored.

The autonomic nervous system plays a role in a variety of liver regenerative and metabolic functions, including modulating bile secretion and cholangiocyte and hepatobiliary progenitors of the canals of Hering. However, the nature and location of nerves which link to the proximal biliary tree have remained uncertain. We investigate the anatomic relationship of nerves to the proximal biliary tree including the putative stem/progenitor cell niche of the canal of Hering. Using double immunostaining (fluorescence, histochemistry) to highlight markers of cholangiocytes (biliary-type keratins), nerves (S100, neurofilament protein, PGP9.5, tyrosine hydroxylase), and stellate cells (CRBP-1), we examined sections from normal adult livers from autopsy or surgical resections. There is extensive contact between nerves and interlobular bile ducts, bile ductules, and canals of Hering (CoH). In multiple serial sections from 4 normal livers, biliary-nerve contacts were seen in all of these structures and were more common in the interlobular bile ducts (78/137; 57%) than in the ductules and CoH (95/294; 33%) (p < 0.001). Contacts appear to consist of nerves in juxtaposition to the biliary basement membrane, though crossing through basement membrane to interface directly with cholangiocytes is also present. These nerves are positive for tyrosine hydroxylase and are, thus, predominately adrenergic. Electron microscopy confirms nerves closely approximating ductules. Nerve fiber-hepatic stellate cell juxtaposition is observed but without stellate cell approximation to cholangiocytes. We present novel findings of biliary innervation, perhaps mediated in part, by direct cholangiocyte-nerve interactions. The implications of these findings are protean for studies of neuromodulation of biliary physiology and hepatic stem/progenitor cells.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death in the world. Although most cases occur in stiff, cirrhotic livers, and stiffness is a significant risk factor, HCC can also arise in non-cirrhotic livers in the setting of non-alcoholic fatty liver disease (NAFLD). We hypothesized that lipid droplets in NAFLD might apply mechanical forces to the nucleus, functioning as mechanical stressors akin to stiffness. We investigated the effect of lipid droplets on cellular mechanosensing and found that primary human hepatocytes loaded with the fatty acids oleate and linoleate exhibited decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers. The presence of large lipid droplets in hepatocytes resulted in increased nuclear localization of the mechano-sensor Yes-associated protein (YAP). In cirrhotic livers from patients with NAFLD, hepatocytes filled with large lipid droplets showed significantly higher nuclear localization of YAP as compared to cells with small lipid droplets. This work suggests that lipid droplets induce a mechanical signal that disrupts the ability of the hepatocyte to sense its underlying matrix stiffness, and that the presence of lipid droplets can induce intracellular mechanical stresses.

The origin and initiating features of PBC remain obscure despite decades of study. However, recent papers have demonstrated loss of canals of Hering (CoH) to be the earliest histologic change in liver biopsy specimens from patients with primary biliary cholangitis (PBC). We posit that CoH loss prior to significant inflammation or evidence of bile duct injury might be a very early, perhaps even an initiating lesion of PBC. As a potential target of inflammatory or toxic injury, CoH loss may initiate rather than follow the cascade of events leading to duct injury and loss and their sequelae. Toxins may be exogenous in origin, such as environmental toxins or drug exposures, or endogenous, resulting from genetic or epigenetic alterations in canalicular bile transporters upstream from the CoH. In turn, this hypothesis suggests that loss of CoH would lead to altered bile flow and composition injurious to downstream bile ducts, because bile composition has not been modulated by normal CoH physiologic functions or because, in the absence of CoH, canalicular fluid flow into the biliary tree is disrupted interfering with soluble trophic factors important for bile duct integrity. Regardless of the pathogenic mechanism causing CoH loss, only following such loss would the characteristic diagnostic findings of PBC become evident: damage to downstream interlobular and sub-lobular bile ducts. To the extent that the causal mechanisms for CoH loss can be identified, clinical identification (as through early identification of CoH loss) and intervention (depending on the inciting cause) may offer promise for treatment of this enigmatic disease.

Background: As hepatocellular carcinoma (HCC) develops from premalignant lesions (low and high grade dysplastic nodules; LGDN and HGDN), there is a corresponding accumulation of molecular alterations, some of which have been well described. However, the molecular features of lesions comprising the hepatocellular dysplasia-carcinoma sequence as they relate to different etiologies have not yet been explored. Herein, we characterize the molecular landscape of such lesions in cirrhotic explants with alcohol-related liver disease (ALD) and chronic hepatitis C (CHC).
Design(s): Tissue was assessed from 27 liver explants (14 CHC, 13 ALD) including 10 LGDNs (5 CHC, 5 ALD), 8 HGDNs (3 CHC, 5 ALD), 10 HCCs arising from HGDNs (5 CHC, 5 ALD), and 10 small HCCs defined as HCC < 2 cm (5 CHC, 5 ALD), as well as non-lesional cirrhotic parenchyma and matched normal non-liver tissue (e.g. porta hepatis structures). DNA was extracted from FFPE tissue for next generation sequencing (NGS) using a customized NGS580 panel targeting all exonic and select intronic areas in 580 cancer related genes. Data was analyzed using customized bioinformatics pipelines with an R package.
Result(s): TERT promoter HS C228T mutations were identified in 6 of 10 (60%) CHC related HCCs and only 1 of 9 (11%) alcohol related HCC (Figure 1). There was a significant association between TERT promoter HS C228T mutations and CHC related HCCs (p<0.02). In contrast, ALD related lesions showed deleterious events affecting tumor suppressor genes (NF1, BRCA1; CDKN2C) and histone methylation/chromatin remodeling genes (KMT2A; KMT2C; ASXL1; RAD21), which were found in 6 of 13 ALD related lesions (46.2% [3 of 5 small HCCs, 2 of 4 HGDNs, and 1 of 4 HCCs arising in HGDNs]). These events only occurred in 3 of 14 (21.4%) CHC related lesions (Figures 1 and 2). Within the CHC group, 1 HCC arising in HGDN showed copy number gains (CNG) in MARK4, ERCC2, FGFR4 and FLT4 and two HGDNs showed CNGs in NOTCH1 and TERT, respectively. No differences in the tumor mutational burden (TMB) were noted between CHC related and ALD related lesions, nor across the DN-HCC sequence. Non-lesional liver and LGDNs did not show recurrent mutations pertaining to a specific pathway. (Figure presented)
Conclusion(s): Our findings suggest that the pathways of hepatocarcinogenesis are distinct in ALD and CHC. While upregulation of telomerase activity (TA) and cancer cell immortalization play a pivotal role in CHC related HCC, defective chromatin remodeling appears to contribute to tumorigenesis in ALD related HCC

Background: Fibroconnective tissues of the body are traditionally conceived as layers of densely compacted collagen. Recent in vivo microscopy, however, demonstrates that at least some, including visceral submucosae, dermis, fascia, adventitia and perineurium, are actually a reticular network of fluid-filled sinuses supported by a complex scaffold of thick collagen bundles (Benias et al Sci Rep 2018: 8). The interstitial fluid is rich in hyaluronic acid (HA). Whether these large scale interstitial spaces are continuous between tissues/organs or separate is unclear. Continuity was investigated by two methods: 1. movement of non-biological pigment (tattoo pigment, colloidal silver) in colon and skin specimens; 2. localization of HA by IHC.
Design(s): H&E-stained sections of FFPE tissues from resected colons following endoscopic submucosal tattoo for malignant polyps (n=5) and from skin biopsies with either cosmetic tattoos (n=3) or colloidal silver (n=2) were examined. Location of particles was assessed. The slides were then scanned, decolorized, and stained by multiplex chromogenic IHC assay (Discovery Ultra, Ventana) for HA-binding protein (brown), vimentin (magenta) and CD34 (teal) to label interstitial lining cells.
Result(s): Tattoo pigment and colloidal silver within the interstitial spaces was identified in the dermis (Fig. 1) and colonic submucosa and in the dependent mesenteric and subcutaneous fascias. In all colon specimens HA IHC highlighted the spatial continuity of all layers of the colon from lamina propria through muscularis mucosae to submucosa (Fig. 2A), then through the muscularis propria into mesenteric fascia (Fig. 2B, C). Continuity between these spaces and perivascular stroma/adventitia and perineurium in the bowel wall was also evident. Continuity of HA-filled spaces is also demonstrated from papillary to reticular dermis and then to subcutaneous fascia interface. (Figure presented)
Conclusion(s): Interstitial spaces are neither virtual nor a result of processing artifact, but are filled with physiologically relevant fluid rich in HA. Their continuity across tissue compartments is demonstrated by movement of non-biological pigments and by spatial continuity of HA. The implications of such multisystem continuity are protean, but may particularly explain commonly observed modes of discontinuous cancer spread through tissue planes, such as mesenteric tumor deposits in colon cancer and subcutaneous in-transit melanoma metastasis, and spread of infection (e.g. necrotizing fasciitis)

Background: Alpha-1 antitrypsin deficiency (AATD) arises from inherited autosomal co-dominant SERPINA1 mutations that lead to liver and lung disease. Clinical manifestations within the liver are variable and include chronic hepatitis, cirrhosis, and the development of hepatocellular carcinoma. While the risk of liver disease with homozygous AATD is established, the prevalence of clinically significant liver disease in heterozygotes is not well described. Accumulating evidence suggests that heterozygous AATD (HAATD) acts as a co-factor for other liver diseases, but little is known about its effect on pathologic fibrosis. We investigate the stage of liver fibrosis in patients with histologic evidence of HAATD compared to controls with a wide range of chronic liver diseases.
Design(s): Our database was retrospectively searched to obtain 23 liver biopsy and resection specimens with evidence of chronic liver disease and intracytoplasmic alpha1 anti-trypsin (AAT) globules stained by immunohistochemistry. The group with histologic evidence of possible HAATD was compared to control cases of liver biopsies obtained from patients with chronic liver disease without evidence of AAT globules. Clinicopathologic data included age, gender, race, genotype, chronic liver disease type (chronic cholestatic disease, fatty liver disease, Hepatitis B or C, auto-immune hepatitis, hemochromatosis, and unknown cause), liver disease stage, and clinical follow-up. Statistical analyses were performed to assess correlation between clinicopathologic variables and disease groups.
Result(s): Compared to controls, subjects in the HAATD cohort had a higher rate of stage 4 fibrosis (52.17% vs 21.67%, p = 0.017). The control specimens were relatively evenly distributed among stages 1-4 fibrosis (Table). Of the 7 HAATD patients who underwent subsequent genetic testing, 6 patients were found to have heterozygous SERPINA1 mutations (5 PI*MZ, 1 PI*SZ). There were no significant differences in age, race, or gender between control and HAATD groups. (Table presented)
Conclusion(s): Histologic and immunohistochemical evidence of AAT globules was associated with a higher stage of concurrent liver disease. These results add to the growing body of literature which suggests that HAATD may potentiate the progression of concurrent liver diseases. In addition, histologic and immunohistochemical evidence of AAT globules may be useful in the early diagnosis of HAATD

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

BACKGROUND & AIMS/OBJECTIVE:The extrahepatic bile duct is the primary tissue initially affected by the cholangiopathy biliary atresia. Biliary atresia affects neonates exclusively and current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS:We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes and isolated extrahepatic bile ducts, and a collagen reporter mouse. Methods included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS:The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION/CONCLUSIONS:We identified four key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY/UNASSIGNED:Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction with resulting liver injury. We identify four key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.