Faculty Bibliography

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

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)

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.