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The article presents a study on a stem/progenitor cell population within liver regenerative potency which is harbored in human duodenal sub mucosal glands. Topics include information on common hepato-bilio-pancreatic progenitors; early stage of development in the primitive duodenum; and immunohistochemistry and immunofluorescence of duodenum.

The article offers information about the hepatic stem/progenitor cell activation and ductularcanalicular junctions in primary biliary cholangitis It mentions that hepatic stem/progenitor cells (HpSCs) are facultative bipotential stem cells, located in the canals of Hering and surrounded by a specialized niche.

Background: Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes. Results: Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCβ-I phosphorylation but decreased PKCa phosphorylation. Administration of an antiprolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCβ-I and dephosphorylation of PKCα. Conclusion: We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCβ-I and dephosphorylation of PKCα; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients. [ABSTRACT FROM AUTHOR]

• In Italy, recent regulation has updated the post-mortem body disposition. • Body donation is admitted for study, training and scientific research purposes. • Unsolved issues concern the grantees of teaching and training activities. • The Italian College of Anatomists has made constructive criticisms to the law. • Suggestions are provided to manage the donation process and use of the body. In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations. [ABSTRACT FROM AUTHOR]

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis. [ABSTRACT FROM AUTHOR]

Cholangiocarcinomas (CCAs) are heterogeneous biliary tract malignancies with dismal prognosis, mainly due to tumor aggressiveness, late diagnosis, and poor response to current therapeutic options. High-throughput technologies have been used as a fundamental tool in unveiling CCA molecular landscape, and several molecular classifications have been proposed, leading to various targeted therapy trials. In this review, we aim to analyze the critical issues concerning the status of precision medicine in CCA, discussing molecular signatures and clusters, related to both anatomical classification and different etiopathogenesis, and the latest therapeutic strategies. Furthermore, we propose an integrated approach comprising the CCA molecular mechanism, pathobiology, clinical and histological findings, and treatment perspectives for the ultimate purpose of improving the methods of patient allocations in clinical trials and the response to personalized therapies. [ABSTRACT FROM AUTHOR]

R1679 --> Introduction: PBC is a cholestatic disease characterized by bile duct loss, ductular reaction and liver fibrosis. The canals of Hering (CoH) are the site of ductulo‐canalicular junction (DCJ) that maintain proper bile drainage from the liver, and contain the hepatic progenitor cell (HPC) population. CoH loss is noted in PBC and correlates with scoring. Secretin binds to its receptor (SR, expressed by large cholangiocytes) to promote biliary proliferation during cholestasis; however, during large duct damage, small cholangiocytes acquire large cholangiocyte phenotypes including SR expression. Secretin/SR expression is downregulated in late‐stage PBC; therefore, we aimed to understand the role of the secretin/SR axis during PBC. We hypothesized that secretin treatment restores DCJ through HPC activation and ameliorates liver phenotypes associated with late‐stage PBC. Methods: Female 34 wk old wild‐type (WT) and dominant‐negative transforming growth factor‐β receptor II (dnTGFβRII, model of late‐stage PBC) mice were treated with control or secretin (2.5 nmol/kg BW) for 8 wk by IP implanted minipumps. Cholangitis activity, hepatitis activity and bile duct loss were determined by the Nakanuma scoring system. Ductular reaction was measured by semi‐quantitative CK‐19 immunostaining. Liver fibrosis was evaluated by Sirius Red staining, collagen‐1a immunostaining and qPCR for α‐SMA. Hepatic stellate cell (HSC) presence was determined by desmin immunostaining. Cholestasis was indicated by hepatic total bile acid (TBA) levels using EIA. DCJ presence was evaluated by co‐immunostaining for ABC transporter bile salt export pump (BSEP)/CK‐19 and semi‐quantification. HPC activation was assessed by Sox9 staining and semi‐quantification. Results: dnTGFβRII mice had an increase in cholangitis and hepatitis activity; however, 8 wks of secretin treatment significantly reduces cholangitis, but not hepatitis, activity. dnTGFβRII mice present with ductular reaction and significant bile duct loss, which was reversed with 8 wks secretin treatment. Hepatic TBA levels, liver fibrosis and HSC presence are increased in dnTGFβRII mice, but significantly reduced following 8 wks of secretin treatment. In line with human studies, dnTGFβRII mice have significant loss of DCJ and reduced HPC number; however, 8 wks of secretin treatment restores these parameters. Conclusion: Long‐term secretin treatment (i) reduces ductular reaction, (ii) reverses bile duct loss, (iii) decreases liver fibrosis, (iv) promotes HPC activity and (v) restores DCJ presence in a model of late‐stage PBC. Secretin maintenance of the DCJ may be due to (i) the restoration of bile flow and release of toxic bile acids or (ii) secretin binding to SR acquired on small cholangiocytes/HPCs. Treatment with secretin may be a therapeutic option for late‐stage PBC patients. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease (1). Numerous evidence suggested that gut microbiota may be implicated in the pathogenesis of NAFLD (2). small intestinal bacterial overgrowth contributing to increased serum endotoxemia has been described in NAFLD, with Escherichia coli being the most abundant bacterium. The aim of this study is to investigate the mechanisms by which lipopolysaccharides (LPS) from gut microbiota favors the progression of NAFLD and its effects on liver histology and progenitor cell niche. We studied Escherichia Coli LPS in patients with biopsy-proven NAFLD and matched serum samples: 25 simple steatosis (nonalcoholic fatty liver) and 25 nonalcoholic steatohepatitis (NASH). Biopsies were examined by routine histology and immunohistochemistry. In vitro, hepatocytes and macrophages were cultured in media with LPS addition and Tolllike receptor 4 (TLR4) pathway was analyzed by western blot. In mice, NASH was induced by methionine-choline deficient (MCD) and high fat diets; TAK-242, a TLR4 inhibitor was administrated to MCD mice by daily intraperitoneal injection. NASH patients had higher serum LPS and hepatocytes LPS localization than controls, which was correlated with serum zonulin and phosphorylated Nuclear Factor-κB expression. In vitro, LPS can activate TLR4 pathway in hepatocyte and liver macrophages, triggering pro-inflammatory phenotypes. TLR4+ macrophages were higher in NASH than simple steatosis or controls and correlated with serum LPS. TLR4+ portal macrophages are spatially associated with hepatic progenitor cells and correlated with ductular reaction (hallmark of progenitor cell activation). In mice with NASH, LPS serum levels and LPS hepatocyte localization were increased compared with control mice and associated with Nuclear Factor-κB activation. Treatment with TLR4 inhibitor resulted in lower liver inflammation in mice with NASH. In conclusion, In NAFLD, Escherichia Coli LPS may increase liver damage by inducing macrophage polarization, liver inflammation and progenitor cell niche activation through TLR4 pathway. [ABSTRACT FROM AUTHOR]

Advancement in cancer research has shed light on the importance of the tumor microenvironment, which is made of extracellular matrix (ECM) and various cell types that interact with cancer cells autocrinally and paracrinally. Nerves take part in the tumor microenvironment and increasing evidence show that they have a key role in tumor growth, invasion, metastasis and survival [1]. In particular, Schwann cells (SC) are now being investigated as main players of the nerve boost for the tumor progression, since their well-known regenerative potential can be exploited by cancer cells to thrive [2]. While some data about these phenomena are available for pancreatic, lung, and some head and neck cancers, data about cholangiocarcinoma are lacking. The aims of the project are to investigate whether SC are involved in cholangiocarcinoma (CCA) progression and to describe the putative underlying cellular and molecular mechanisms. To do so, we set up a 3D SC migration assay where we placed nerve explants from a mouse pup into an ECM scaffold, and seeded some CCA cells next to it. We observed the system by phase contrast for 7 days, then fixed it and stained it by immunofluorescence for activated SC marker GFAP. Our preliminary data show migration of activated SC from the nerve towards CCA cells (carcinotropism) starting at day 4-5. We also performed neuritogenesis assays using PC12, a well characterized cell line widely utilized in literature to study neuritogenesis. PC12 differentiate in a neurite expressing phenotype if placed in culture with conditioned media containing neuritogenic factors (i.e. neurotrophins). We tested the conditioned media from HuCCT-1 (human CCA) and found some neuritogenic activity in the media compared to the positive control (NGF). We then performed Western Blot analysis on untreated HuCCT-1 cells investigating autophagy (TFEB, c-FLIP, LC3B, p62), cell death (cleaved Caspase-3) and proliferation (PCNA) markers to compare them to those of HuCCT-1 cells treated with conditioned media from unactivated and activated human primary SC in order to study the modulation of cancer cell behavior by activated SC. Our data show that CCA cells do have neuritogenic activity suggesting they can lure new nerve fibers towards them to establish a contact; also, activated SC from nerve explants leave the nerve and reach CCA cells in our 3D ECM system, indicating a "priming" of SC by HuCCT-1 cells which may benefit from factors released by activated GFAP+ SC. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the histopathologic modifications in liver and visceral adipose tissue (VAT), and to correlate these changes with clinical measures, adipokine production, and proinflammatory cytokines in a population of adolescents with obesity with nonalcoholic fatty liver disease (NAFLD) who underwent laparoscopic sleeve gastrectomy (LSG).Study Design: Twenty adolescents with obesity who underwent LSG and with biopsy-proven NAFLD were included. Patients underwent clinical evaluation and blood tests at baseline and 1 year after the surgical procedure. Liver and VAT specimens were processed for routine histology, immunohistochemistry, and immunofluorescence.Results: In adolescents with obesity and NAFLD, hepatic histologic alterations were uncorrelated with VAT inflammation. LSG induced in both liver and VAT tissue histopathology amelioration and macrophage profile modification that were correlated with body mass index and improvement in insulin resistance. The adipokine profile in liver and VAT was associated with weight loss and histologic improvement after LSG. Serum proinflammatory cytokines were correlated with liver and VAT histopathology and IL-1β and IL-6 levels were independently predicted by liver necroinflammatory grade.Conclusions: This study suggests a unique adipose tissue/fatty liver crosstalk in pediatric patients. LSG induces a similar pattern of histologic improvement in the liver and in VAT. Besides VAT, our results strengthen the role of the liver in adipocytokine production and its contribution to systemic inflammation in pediatric patients with NAFLD. [ABSTRACT FROM AUTHOR]

Background: Intrahepatic cholangiocarcinoma (iCCA) is a malignancy that arises from the intrahepatic biliary tree, showing high mortality rates due to its late clinical presentation and limited treatment options. iCCA is characterized by a dense, reactive desmoplastic stroma marked by a dramatic accumulation of extracellular matrix (ECM). Although recent results strongly suggest a relationship between increasing desmoplastic stroma and the enhanced malignant behaviour of iCCA, the importance of ECM proteins in the pathogenesis of iCCA still have to be addressed. Methods: iCCA ECM fibrillar structural organization was characterized by histological analysis. ECM proteome profiles from decellularized iCCA and surrounding noncancerous tissues were analysed by nLC coupled to MALDI-TOF/TOF analysis. Results: iCCA tissues displayed high levels of collagen fibers and low abundance of reticular and elastic fibers, suggesting stiffness and loss of polarity. The ECM proteome profiles of iCCA samples, when compared to those obtained from the surrounding noncancerous tissues showed a dismantling of the basement membrane, a reduced angiogenesis and a downregulation of oncosuppressive activity. In particular, we focused on the effects of the overexpression of collagen type III alpha 1 chain (COL3A1) in iCCA, thus providing evidences that COL3A1 promotes iCCA cells migration and is a component of tumor-associated aligned collagen. Conclusions: Overall, this study contributes to the understanding of molecular basis underlying desmoplasia in iCCA and indicates the type III collagen as a promising therapeutic target. [ABSTRACT FROM AUTHOR]

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial–mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFβ receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFβ signaling in regulating this process. [ABSTRACT FROM AUTHOR]

Peribiliary glands (PBGs) represent the niche of biliary tree stem/progenitor cells (BTSCs) [1]. BTSCs are multipotent stem cells able to differentiate into hepatocytes, cholangiocytes, and pancreatic islets. Primary sclerosing cholangitis (PSC) is a chronic inflammation involving extra-hepatic biliary tree, and is complicated by the risk of cholangiocarcinoma (CCA) development [2]. We aimed to evaluate the involvement of PBGs and BTSCs in PSC and their role in CCA insurgence [2]. Specimens from normal liver (N=5), PSC (N=20), and CCA arising in PSC patients (N=20) were included and processed for histology, immunohistochemistry and immunofluorescence. In vitro experiments were performed on human BTSCs and primary CCA cell cultures. PSC-affected ducts were characterized by the activation of BTSCs and by PBG hyperplasia. In PSC, ducts showed higher microvascular density around PBGs compared to normal ducts. In CCA arising in PSC patients, PBGs showed dysplastic and neoplastic aspects. Compared to non-cancerous ducts, neoplastic ducts showed higher inflammation, wall thickness, and PBG activation. CCAs were characterized by higher expression of epithelial-to-mesenchymal transition (EMT) traits in PBGs and by the absence of primary cilia in BTSCs compared to control ducts. In vitro study confirmed that human BTSCs, under inflammatory milieu, increased proliferation rate and expression of EMT traits, and lost primary cilia compared to control conditions. In conclusion, patients affected by PSC are characterized by PBG involvement and activation of BTSC niche; the insurgence of CCA was characterized by involvement of PBG niche, suggesting a key role of this cell compartment in progressive tumorigenesis. [ABSTRACT FROM AUTHOR]

Hepatic stem/progenitor cells (HpSCs) are facultative bipotential stem cells [1], located in Canals of Hering and surrounded by a specialized niche [2]. We aimed to investigate the modulation of HpSC niche and the modification of adipocytokine expression induced by laparoscopic sleeve gastrectomy (LSG) in adolescents with nonalcoholic fatty liver disease (NAFLD). Twenty obese adolescents who underwent LSG and with biopsy-proven NAFLD were included. At baseline (T0) and 1 year after treatment (T1), patients underwent clinical evaluation, blood tests, and liver biopsy. HpSCs, hepatic stellate cells (HSCs), macrophages, and adipocytokines were evaluated by immunohistochemistry and immunofluorescence. Liver biopsies after LSG demonstrated a significant improvement of NAFLD Activity Score and fibrosis. Immunohistochemistry indicated a significant reduction of hepatocyte cell cycle arrest, HpSC activation, activated HSC, and macrophage number after LSG compared with T0. Hepatocyte expression of adiponectin was significant higher after LSG than at T0. Moreover, LSG caused decreased resistin expression in Sox9+ HpSCs compared to T0. The number of S100A9+ macrophages was also reduced by LSG correlating with resistin expression in HpSC. Finally, serum levels of proinflammatory cytokines significantly correlated with macrophages and activated HSC numbers. The histologic improvement induced by LSG is associated with the reduced activation of local cellular cross-talks, thus, strengthening the role of stem cell niche and hepatic adipocytokine production in the pathogenesis of NAFLD. [ABSTRACT FROM AUTHOR]

Objectives: To investigate whether the modulation of local cellular cross-talks and the modification of hepatic adipocytokine expression could mechanistically explain the improvement of liver histopathology after laparoscopic sleeve gastrectomy (LSG) in adolescents with nonalcoholic fatty liver disease (NAFLD).Study Design: Twenty obese (body mass index of ≥35 kg/m2) adolescents who underwent LSG and with biopsy-proven NAFLD were included. At baseline (T0) and 1 year after treatment, patients underwent clinical evaluation, blood tests, and liver biopsy. Hepatic progenitor cells, hepatic stellate cells (HSCs), macrophages, and adipocytokines were evaluated by immunohistochemistry and immunofluorescence.Results: Liver biopsy samples after LSG demonstrated a significant improvement of NAFLD Activity Score and fibrosis. Immunohistochemistry indicated a significant reduction of hepatocyte cell cycle arrest, ductular reaction, activated HSC, and macrophage number after LSG compared with T0. The activation state of HSC was accompanied by modification in the expression of the autophagy marker LC3. Hepatocyte expression of adiponectin was significant higher after LSG than into T0. Moreover, LSG caused decreased resistin expression in Sox9+ hepatic progenitor cells compared with T0. The number of S100A9+ macrophages was also reduced by LSG correlating with resistin expression. Finally, serum levels of proinflammatory cytokines significantly correlated with macrophages and activated HSC numbers.Conclusions: The histologic improvement induced by LSG is associated with the reduced activation of local cellular compartments (hepatic progenitor cells, HSCs, and macrophages), thus, strengthening the role of cellular interactions and hepatic adipocytokine production in the pathogenesis of NAFLD. [ABSTRACT FROM AUTHOR]

Summary: Background: Porto‐sinusoidal vascular disorder (PSVD) is characterised by lesions involving portal veins and sinusoids in absence of cirrhosis with an unclear pathophysiology. However, its association with immunodeficiency, bowel disorders and abdominal bacterial infections supports the role of altered intestinal permeability and gut‐derived endotoxins. The study aimed at assessing the association between serological markers of increased intestinal permeability, pro‐aggregating/procoagulant state and liver injury in PSVD and portal hypertension. Methods: Thirty‐three patients with biopsy‐proven PSVD and portal hypertension and 33 healthy subjects were submitted to a venous blood sampling for the measurement of zonulin and lipopolysaccharides (LPS) as markers of intestinal permeability, of s‐Glycoprotein VI, sP‐selectin, ADAMTS13 and von Willebrand factor (vWF), as markers of platelet aggregation and microvascular inflammation, factor VIII and F1 + 2 as markers of hypercoagulability. In 17 PSVD patients, histomorphological and immunohistochemical study on liver biopsies was performed. Results: Compared with controls, PSVD patients had higher levels of LPS, zonulin, vWF, factor VIII and sP‐selectin, F1 + 2. ADAMTS13 was reduced. Serum LPS correlated with zonulin, sP‐selectin, FVIII and vWF. At histological analysis, PSVD specimens had increased LPS localisation, toll‐like receptor‐4(TLR4)‐positive macrophages and platelet number compared with samples from healthy liver donors. TLR4+ macrophage number correlated with portal inflammation and fibrosis. Sinusoid dilation and capillarisation were observed. PSVD biopsies showed signs of biliary damage and reduced ductular reaction without alteration in Sox9+ cell population. Conclusions: PSVD patients display an altered intestinal permeability and endotoxemia correlated to a pro‐aggregating/procoagulant state; histologically, PSVD was associated with increased TLR4+ cell involvement and platelet clumps within sinusoids. Our study suggests that LPS‐TLR4 pathway could contribute to the pathophysiological basis of PSVD with portal hypertension. [ABSTRACT FROM AUTHOR]

The activation of hepatic stem/progenitor cells (HPCs) is characterized by the appearance of ductular reaction (DR) in the liver parenchyma [1]. The aims of the present study were to evaluate the activation of HPCs in human cholangiopathies. Human liver tissue was obtained from liver donors (N=5), Primary Sclerosing Cholangitis (PSC; N=20), and Primary Biliary Cholangitis (PBC; N=20) patients. Ductular reaction extension was evaluated by Keratin(K) 7 immunoreactivity. HPC phenotype and signalling pathways were investigated by immunohistochemistry and immunofluorescence [2]. Ductular reaction in PBC is more extensive, appears earlier, and has a higher proliferation index compared to PSC. In PBC the extension of DR strongly correlates with clinical prognostic scores. A higher percentage of Sox9+ and K19+ cells characterized DR in PBC versus PSC. In cirrhotic-PSC, the HPC compartment showed signs of hepatocyte commitment. The study of the HPC niche indicated lower levels of laminin and NOTCH1 but higher expression of WNT pathway components in PSC compared to PBC. In conclusion, PSC and PBC are characterized by different patterns of HPC niche activation, reflecting the involvement of different portions of the biliary tree as primary target of damage. These aspects could have implications in the pathogenesis of cholangiopathies and could add prognostic value. [ABSTRACT FROM AUTHOR]

Background: Alcohol-related liver disease (ALD) is characterized by ductular reaction (DR), liver inflammation, steatosis, fibrosis, and cirrhosis. The secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes) regulates liver phenotypes in cholestasis. We evaluated the role of Sct signaling on ALD phenotypes. Methods: We used male wild-type and Sct−/− mice fed a control diet (CD) or ethanol (EtOH) for 8 wk. Changes in liver phenotypes were measured in mice, female/male healthy controls, and patients with alcoholic cirrhosis. Since Cyp4a10 and Cyp4a11/22 regulate EtOH liver metabolism, we measured their expression in mouse/human liver. We evaluated: (i) the immunoreactivity of the lipogenesis enzyme elongation of very-long-chain fatty acids 1 (Elovl, mainly expressed by hepatocytes) in mouse/human liver sections by immunostaining; (ii) the expression of miR-125b (that is downregulated in cholestasis by Sct) in mouse liver by qPCR; and (iii) total bile acid (BA) levels in mouse liver by enzymatic assay, and the mRNA expression of genes regulating BA synthesis (cholesterol 7a-hydroxylase, Cyp27a1, 12a-hydroxylase, Cyp8b1, and oxysterol 7a-hydroxylase, Cyp7b11) and transport (bile salt export pump, Bsep, Na+-taurocholate cotransporting polypeptide, NTCP, and the organic solute transporter alpha (OSTa) in mouse liver by qPCR. Results: In EtOH-fed WT mice there was increased biliary and liver damage compared to control mice, but decreased miR-125b expression, phenotypes that were blunted in EtOH-fed Sct−/− mice. The expression of Cyp4a10 increased in cholangiocytes and hepatocytes from EtOH-fed WT compared to control mice but decreased in EtOH-fed Sct−/− mice. There was increased immunoreactivity of Cyp4a11/22 in patients with alcoholic cirrhosis compared to controls. The expression of miR-125b decreased in EtOH-fed WT mice but returned at normal values in EtOH-fed Sct−/− mice. Elovl1 immunoreactivity increased in patients with alcoholic cirrhosis compared to controls. There was no difference in BA levels between WT mice fed CD or EtOH; BA levels decreased in EtOH-fed Sct−/− compared to EtOH-fed WT mice. There was increased expression of Cyp27a1, Cyp8b1, Cyp7b1, Bsep, NTCP and Osta in total liver from EtOH-fed WT compared to control mice, which decreased in EtOH-fed Sct−/− compared to EtOH-fed WT mice. Conclusions: Targeting Sct/SR signaling may be important for modulating ALD phenotypes. [ABSTRACT FROM AUTHOR]

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA- stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC. [ABSTRACT FROM AUTHOR]

Background & aims: Cholangiocytes are the target cells of liver diseases that are characterized by biliary senescence (evidenced by enhanced levels of senescence-associated secretory phenotype, SASP, e.g., TGF-β1), and liver inflammation and fibrosis accompanied by altered bile acid (BA) homeostasis. Taurocholic acid (TC) stimulates biliary hyperplasia by activation of 3′,5′-cyclic cyclic adenosine monophosphate (cAMP) signaling, thereby preventing biliary damage (caused by cholinergic/adrenergic denervation) through enhanced liver angiogenesis. Also: (i) α-calcitonin gene-related peptide (α-CGRP, which activates the calcitonin receptor-like receptor, CRLR), stimulates biliary proliferation/senescence and liver fibrosis by enhanced biliary secretion of SASPs; and (ii) knock-out of α-CGRP reduces these phenotypes by decreased cAMP levels in cholestatic models. We aimed to demonstrate that TC effects on liver phenotypes are dependent on changes in the α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-β1/VEGF axis. Methods: Wild-type and α-CGRP−/− mice were fed with a control (BAC) or TC diet for 1 or 2 wk. We measured: (i) CGRP levels by both ELISA kits in serum and by qPCR in isolated cholangiocytes (CALCA gene for α-CGRP); (ii) CALCRL immunoreactivity by immunohistochemistry (IHC) in liver sections; (iii) liver histology, intrahepatic biliary mass, biliary senescence (by β-GAL staining and double immunofluorescence (IF) for p16/CK19), and liver fibrosis (by Red Sirius staining and double IF for collagen/CK19 in liver sections), as well as by qPCR for senescence markers in isolated cholangiocytes; and (iv) phosphorylation of PKA/ERK1/2, immunoreactivity of TGF-β1/TGF- βRI and angiogenic factors by IHC/immunofluorescence in liver sections and qPCR in isolated cholangiocytes. We measured changes in BA composition in total liver by liquid chromatography/mass spectrometry. Results: TC feeding increased CALCA expression, biliary damage, and liver inflammation and fibrosis, as well as phenotypes that were associated with enhanced immunoreactivity of the PKA/ERK1/2/TGF-β1/TGF-βRI/VEGF axis compared to BAC-fed mice and phenotypes that were reversed in α-CGRP−/− mice fed TC coupled with changes in hepatic BA composition. Conclusion: Modulation of the TC/ α-CGRP/CALCRL/PKA/ERK1/2/TGF-β1/VEGF axis may be important in the management of cholangiopathies characterized by BA accumulation. [ABSTRACT FROM AUTHOR]