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Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)–dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation., This work was supported by the UK Medical Research MRC (MR/K017047/1) (to S.J.F.), the Computational and Chemical Biology of Stem Cell Niche (MR/L012766/1) (to S.J.F.), the UK Regenerative Medicine Platform (MR/K026666/1) (to S.J.F.), and the Wellcome Trust Institutional Translational Partnership Award (WT iTPA) (to S.F.-G.). J.M.B. was supported by the Spanish Carlos III Health Institute (ISCIII) (PI15/01132, PI18/01075, and Miguel Servet Program CON14/00129 and CPII19/00008) cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER); “Instituto de Salud Carlos III” (CIBERehd), Spain; “Euskadi RIS3” (2019222054 and 2020333010); and the Department of Industry of the Basque Country (Elkartek: KK-2020/00008). This research was funded in whole or in part by The Wellcome Trust (grant number 209710/Z/17/Z), a cOAlition S organization.

Background & Aim Following acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI). Methods Several phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice. Results BMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings. Conclusion We identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury. Lay summary After an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury., Graphical abstract, Highlights • Primary BMDMs localised to liver and spleen within hours following intravenous injection in mice. • AAMs were highly phagocytic and i.v. transfer elicited reductions in necrotic area, HMGB1 translocation, and hepatic neutrophil infiltration. • AAM injection reduced inflammatory mediators and stimulated hepatocyte/endothelium proliferation in injured liver. • Injection of clinical-grade human AAMs could partially recapitulate the efficacy of murine AAMs in immunocompetent mice.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

Cellular senescence is an irreversible cell cycle arrest implemented by the cell as a result of stressful insults. Characterized by phenotypic alterations, including secretome changes and genomic instability, senescence is capable of exerting both detrimental and beneficial processes. Accumulating evidence has shown that cellular senescence plays a relevant role in the occurrence and development of liver disease, as a mechanism to contain damage and promote regeneration, but also characterizing the onset and correlating with the extent of damage. The evidence of senescent mechanisms acting on the cell populations of the liver will be described including the role of markers to detect cellular senescence. Overall, this review intends to summarize the role of senescence in liver homeostasis, injury, disease, and regeneration.

Following injury, the liver's epithelial cells regenerate efficiently with rapid proliferation of hepatocytes and biliary cells. However, when proliferation of resident epithelial cells is impaired, alternative regeneration mechanisms can occur. Intricate lineage-tracing strategies and experimental models of regenerative stress have revealed a degree of plasticity between hepatocytes and biliary cells. New technologies such as single-cell omics, in combination with functional studies, will be instrumental to uncover the remaining unknowns in the field. In this review, we evaluate the experimental and clinical evidence for epithelial plasticity in the liver and how this influences the development of therapeutic strategies for chronic liver disease.

Liver disease cases are rapidly expanding across the globe and the only effective cure for end-stage disease is a transplant. Transplant procedures are costly and current supply of donor livers does not satisfy demand. Potential drug treatments and regenerative therapies that are being developed to tackle these pressing issues require effective in-vitro culture platforms. Electrospun scaffolds provide bio-mimetic structures upon which cells are cultured to regulate function in-vitro. This study aims to shed light on the effects of electrospun PCL morphology on the culture of an immortalised hepatic cell line and mouse primary hepatocytes. Each cell type was cultured on large 4–5 µm fibres and small 1–2 µm fibres with random, aligned and highly porous cryogenically spun configurations. Cell attachment, proliferation, morphology and functional protein and gene expression was analysed. Results show that fibre morphology has a measurable influence on cellular morphology and function, with the alteration of key functional markers such as CYP1A2 expression.

Background Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation. Methods iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses. Results The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA+ fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocyte-like cells, or cells presenting both morphologies. Conclusion The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

The interplay between the inflammatory infiltrate and tissue resident cell populations invokes fibrogenesis. However, the temporal and mechanistic contributions of these cells to fibrosis are obscure. To address this issue, liver inflammation, ductular reaction (DR), and fibrosis were induced in C57BL/6 mice by thioacetamide administration for up to 12 weeks. Thioacetamide treatment induced two phases of liver fibrosis. A rapid pericentral inflammatory infiltrate enriched in F4/80(+) monocytes co-localized with SMA(+) myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1 to 6 weeks). An expansion of bone marrow-derived macrophages preceded a second phase, characterized by accelerated progression of fibrosis (>6 weeks) after DR migration from the portal tracts to the centrilobular site of injury, in association with an increase in DR/macrophage interactions. Although chemokine (C-C motif) ligand 2 (CCL2) mRNA was induced rapidly in response to thioacetamide, CCL2 deficiency only partially abrogated fibrosis. In contrast, colony-stimulating factor 1 receptor blockade diminished C-C chemokine receptor type 2 [CCR2(neg) (Ly6C(lo))] monocytes, attenuated the DR, and significantly reduced fibrosis, illustrating the critical role of colony-stimulating factor 1-dependent monocyte/macrophage differentiation and linking the two phases of injury. In response to liver injury, colony-stimulating factor 1 drives early monocyte-mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation, and their association with the advancing DR, the formation of fibrotic septa, and the progression of liver fibrosis to cirrhosis.

Although nonalcoholic fatty liver disease (NAFLD) is conventionally assessed histologically for lobular features of inflammation, development of portal fibrosis appears to be associated with disease progression. We investigated the composition of the portal inflammatory infiltrate and its relationship to the ductular reaction (DR), a second portal phenomenon implicated in fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis as well as influence the fate of the DR hepatic progenitor cells (HPCs), regulating the balance between liver repair and fibrosis. The presence of portal inflammation in NAFLD was strongly correlated with disease severity (fibrosis stage) and the DR. The portal infiltrate was characterized by immunostaining NAFLD liver biopsy sections (n = 33) for broad leukocyte subset markers (CD68, CD3, CD8, CD4, CD20, and neutrophil elastase) and selected inflammatory markers (matrix metalloproteinase 9 and interleukin [IL]-17). Cells expressing all markers examined were identified throughout the liver lobules and in portal tracts, although portal tracts were more densely populated (P

Non-alcoholic fatty liver disease (NAFLD) is a rapidly growing global health problem. It can be separated histologically into two broad groups: steatosis, which usually follows a benign clinical course and non-alcoholic steatohepatitis (NASH) that typically has hepatocyte ballooning, necroinflammatory activity and can progress to fibrosis and cirrhosis. More recently the histological spectrum has expanded with the recognition of a paediatric pattern of NASH that has portal-based inflammation and fibrosis without ballooning. An overlap pattern is also described. There is increasing interest in the portal changes of NASH as these correlate with the progression of fibrosis. Disease-associated hepatocyte senescence appears to trigger an alternative regenerative pathway and the development of a periportal ductular reaction (DR), which in turn may have a role in progressive fibrogenesis. Portal inflammation, particularly in association with the DR, is an area of recent study.

The development of portal fibrosis following the iron loading of hepatocytes is the first stage of fibrogenesis in hereditary hemochromatosis. In other chronic liver diseases it has been shown that a ductular reaction (DR) appears early, correlates with fibrosis progression, and is a consequence of activation of an alternative pathway of hepatocyte replication. This study was designed to investigate the presence of the DR in hemochromatosis and describe its associations. Liver biopsies from 63 C282Y homozygous patients were assessed for hepatic iron concentration (HIC) and graded for iron loading, fibrosis stage, steatosis, and inflammation. Immunostaining allowed quantification of the DR, hepatocyte senescence and proliferation, and analysis incorporated clinical data. Hepatocyte senescence was positively correlated with HIC, serum ferritin, and oxidative stress. A DR was demonstrated and occurred prior to histological fibrosis. HIC, age, hepatocyte senescence and proliferation, portal inflammation, and excessive alcohol consumption all had significant associations with the extent of the DR. In multivariate analysis, iron loading, hepatocyte replicative arrest, and portal inflammation remained independently and significantly associated with the DR. Of factors associated with fibrosis progression, the DR (odds ratio [OR] 10.86 P

Background and Aims: Liver and systemic inflammatory factors influence monocyte phenotype and function, which has implications for hepatic recruitment and subsequent inflammatory and fibrogenic responses, as well as host defence. Methods: Peripheral blood monocyte surface marker (CD14, CD16, CD163, CSF1R, CCR2, CCR4, CCR5, CXCR3, CXCR4, CX3CR1, HLA-DR, CD62L, SIGLEC-1) expression and capacity for phagocytosis, oxidative burst and LPS-stimulated TNF production were assessed in patients with hepatitis C (HCV) (n = 39) or non-alcoholic fatty liver disease (NAFLD) (n = 34) (classified as non-advanced disease, compensated cirrhosis and decompensated cirrhosis) and healthy controls (n = 11) by flow cytometry. Results: The selected markers exhibited similar monocyte-subset-specific expression patterns between patients and controls. Monocyte phenotypic signatures differed between NAFLD and HCV patients, with an increased proportion of CD16+ non-classical monocytes in NAFLD, but increased expression of CXCR3 and CXCR4 in HCV. In both cohorts, monocyte CCR2 expression was reduced and CCR4 elevated over controls. CD62L expression was specifically elevated in patients with decompensated cirrhosis and positively correlated with the model-for-end-stage-liver-disease score. Functionally, monocytes from patients with decompensated cirrhosis had equal phagocytic capacity, but displayed features of dysfunction, characterised by lower HLA-DR expression and blunted oxidative responses. Lower monocyte TNF production in response to LPS stimulation correlated with time to death in 7 (46%) of the decompensated patients who died within 8 months of recruitment. Conclusions: Chronic HCV and NAFLD differentially affect circulating monocyte phenotype, suggesting specific injury-induced signals may contribute to hepatic monocyte recruitment and systemic activation state. Monocyte function, however, was similarly impaired in patients with both HCV and NAFLD, particularly in advanced disease, which likely contributes to the increased susceptibility to infection in these patients.

Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIghi macrophages differed between patients and in the same patient over time, and a high proportion of CRIghi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIglo macrophages, CRIghi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIghi cells, human macrophages, and mouse F4/80hi resident peritoneal macrophages and among CRIglo macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIghi and CRIglo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.

Background Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro- and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice). Results Fibrosis and HPC activation were exacerbated in LysM-Wls mice compared to littermate controls, in the absence of an apparent increase in myofibroblast activation or interstitial collagen mRNA expression, in both the TAA and CDE models of chronic liver disease. Increased Epcam mRNA levels paralleled the increased HPC activation and more mature ductular reactions, in LysM-Wls mice. Increased Epcam expression in LysM-Wls HPC was also observed, consistent with a more cholangiocytic phenotype. No differences in the mRNA expression levels of key pro-inflammatory and pro-fibrotic cytokines or the macrophage-derived HPC mitogen, Tweak, were observed. LysM-Wls mice exhibited increased expression of Timp1, encoding the key Mmp inhibitor Timp1 that blocks interstitial collagen degradation, and, in the TAA model, reduced expression of the anti-fibrotic matrix metalloproteinases, Mmp12 and Mmp13, suggesting a role for macrophage-derived Wnt proteins in restraining fibrogenesis during ongoing liver injury. Conclusion In summary, these data suggest that macrophage-derived Wnt proteins possess anti-fibrogenic potential in chronic liver disease, which may be able to be manipulated for therapeutic benefit. Electronic supplementary material The online version of this article (doi:10.1186/s13069-015-0036-7) contains supplementary material, which is available to authorized users.

Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.

The development of portal fibrosis following the iron loading of hepatocytes is the first stage of fibrogenesis in hereditary hemochromatosis. In other chronic liver diseases it has been shown that a ductular reaction (DR) appears early, correlates with fibrosis progression, and is a consequence of activation of an alternative pathway of hepatocyte replication. This study was designed to investigate the presence of the DR in hemochromatosis and describe its associations. Liver biopsies from 63 C282Y homozygous patients were assessed for hepatic iron concentration (HIC) and graded for iron loading, fibrosis stage, steatosis, and inflammation. Immunostaining allowed quantification of the DR, hepatocyte senescence and proliferation, and analysis incorporated clinical data. Hepatocyte senescence was positively correlated with HIC, serum ferritin, and oxidative stress. A DR was demonstrated and occurred prior to histological fibrosis. HIC, age, hepatocyte senescence and proliferation, portal inflammation, and excessive alcohol consumption all had significant associations with the extent of the DR. In multivariate analysis, iron loading, hepatocyte replicative arrest, and portal inflammation remained independently and significantly associated with the DR. Of factors associated with fibrosis progression, the DR (odds ratio [OR] 10.86 P0.0001) and the presence of portal inflammation (OR 4.31, P=0.028) remained significant after adjustment for cofactors. The extent of the DR regressed following therapeutic venesection.Iron loading of hepatocytes leads to impaired replication, stimulating the development of the DR in hemochromatosis and this correlates strongly with hepatic fibrosis. Portal inflammation occurs in hemochromatosis and is independently associated with the DR and fibrosis, and thus its role in this disease should be evaluated further.

Background Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. Aims To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. Methods Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n=85). Results Portal tracts were more densely populated with macrophages (10.5 +/- 0.36 macrophages/HPF) than lobules (7.2 +/- 0.16 macrophages/HPF, P

Aim: To investigate the influence of macrophages on hepatocyte phenotype and function. Methods: Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophage-conditioned medium on HepG2 mRNA and protein expression determined. The in vivo relevance of these findings was confirmed using liver biopsies from 147 patients with hepatitis C virus (HCV) infection. Results: Conditioned media from macrophages, but not monocytes, induced a transient morphological change in hepatocytes associated with upregulation of vimentin (7.8 +/- 2.5-fold, P = 0.045) and transforming growth factor (TGF)-beta 1 (2.6 +/- 0.2-fold, P < 0.001) and downregulation of epithelial cadherin (1.7 +/- 0.02-fold, P = 0.017) mRNA expression. Microarray analysis revealed significant upregulation of lipocalin-2 (17-fold, P < 0.001) and pathways associated with inflammation, and substantial downregulation of pathways related to hepatocyte function. In patients with chronic HCV, real-time polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA (F0 1.0 +/- 0.3, F1 2.2 +/- 0.2, F2 3.0 +/- 9.3, F3/4 4.0 +/- 0.8, P = 0.003) and protein expression (F1 1.0 +/- 0.5, F2 1.3 +/- 0.4, F3/4 3.6 +/- 0.4, P = 0.014) with increasing liver injury. High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase (MMP)-9 in macrophage-conditioned medium, and a chemical inhibitor of MMP-9 attenuated the change in morphology and mRNA expression of TGF-beta 1 (2.9 +/- 0.2 vs 1.04 +/- 0.1, P < 0.001) in macrophage-conditioned media treated HepG2 cells. In patients with chronic HCV infection, hepatic mRNA expression of CD163 (F0 1.0 +/- 0.2, F1/2 2.8 +/- 0.3, F3/4 5.3 +/- 1.0, P = 0.001) and MMP-9 (F0 1.0 +/- 0.4, F1/2 2.8 +/- 0.3, F3/4 4.1 +/- 0.8, P = 0.011) was significantly associated with increasing stage of fibrosis. Conclusion: Secreted macrophage products alter the phenotype and function of hepatocytes, with increased expression of inflammatory mediators, suggesting that hepatocytes actively participate in liver injury Refereed/Peer-reviewed