TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. METHODS: The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. RESULTS: Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206+ macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. CONCLUSION: These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. LAY SUMMARY: Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation.

Evaluating the taxa that provide shared pollination services across multiple crops and regions.

Many pollinator species visit multiple crops in multiple regions, yet we know little about their pollination service provisioning at local and regional scales. We investigated the floral visitors (n = 13,200), their effectiveness (n = 1718 single visits) and response to landscape composition across three crops avocado, mango and macadamia within a single growing region (1 year), a single crop (3 years) and across different growing regions in multiple years. In total, eight wild visitor groups were shared across all three crops. The network was dominated by three pollinators, two bees (Apis mellifera and Tetragonula spp.) and a fly, Stomorhina discolor. The visitation network for the three crops was relatively generalised but with the addition of pollen deposition data, specialisation increased. Sixteen managed and wild taxa were consistently present across three years in avocado, yet their contribution to annual network structure varied. Node specialisation (d') analyses indicated many individual orchard sites across each of the networks were significantly more specialised compared to that predicted by null models, suggesting the presence of site-specific factors driving these patterns. Identifying the taxa shared across multiple crops, regions and years will facilitate the development of specific pollinator management strategies to optimize crop pollination services in horticultural systems.

Toward an integrated approach to crop production and pollination ecology through the application of remote sensing.

Insect pollinators provide an essential ecosystem service by transferring pollen to crops and native vegetation. The extent to which pollinator communities vary both spatially and temporally has important implications for ecology, conservation and agricultural production. However, understanding the complex interactions that determine pollination service provisioning and production measures over space and time has remained a major challenge. Remote sensing technologies (RST), including satellite, airborne and ground based sensors, are effective tools for measuring the spatial and temporal variability of vegetation health, diversity and productivity within natural and modified systems. Yet while there are synergies between remote sensing science, pollination ecology and agricultural production, research communities have only recently begun to actively connect these research areas. Here, we review the utility of RST in advancing crop pollination research and highlight knowledge gaps and future research priorities. We found that RST are currently used across many different research fields to assess changes in plant health and production (agricultural production) and to monitor and evaluate changes in biodiversity across multiple landscape types (ecology and conservation). In crop pollination research, the use of RST are limited and largely restricted to quantifying remnant habitat use by pollinators by ascertaining the proportion of, and/or isolation from, a given land use type or local variable. Synchronization between research fields is essential to better understand the spatial and temporal variability in pollinator dependent crop production. RST enable these applications to be scaled across much larger areas than is possible with field-based methods and will facilitate large scale ecological changes to be detected and monitored. We advocate greater use of RST to better understand interactions between pollination, plant health and yield spatial variation in pollinator dependent crops. This more holistic approach is necessary for decision-makers to improve strategies toward managing multiple land use types and ecosystem services.

Notch3 drives development and progression of cholangiocarcinoma.

The prognosis of cholangiocarcinoma (CC) is dismal. Notch has been identified as a potential driver; forced exogenous overexpression of Notch1 in hepatocytes results in the formation of biliary tumors. In human disease, however, it is unknown which components of the endogenously signaling pathway are required for tumorigenesis, how these orchestrate cancer, and how they can be targeted for therapy. Here we characterize Notch in human-resected CC, a toxin-driven model in rats, and a transgenic mouse model in which p53 deletion is targeted to biliary epithelia and CC induced using the hepatocarcinogen thioacetamide. We find that across species, the atypical receptor NOTCH3 is differentially overexpressed; it is progressively up-regulated with disease development and promotes tumor cell survival via activation of PI3k-Akt. We use genetic KO studies to show that tumor growth significantly attenuates after Notch3 deletion and demonstrate signaling occurs via a noncanonical pathway independent of the mediator of classical Notch, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). These data present an opportunity in this aggressive cancer to selectively target Notch, bypassing toxicities known to be RBPJ dependent.

WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited.

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

Distance learning improves attainment of professional milestones in the early years of surgical training.

OBJECTIVES: To assess the impact of a surgical sciences e-learning programme in supporting the academic development of surgical trainees during their preparation for professional examination. BACKGROUND: In 2007, a 3-year online part-time Master of Surgical Sciences (MSc) degree programme was launched, utilizing an innovative platform with virtual case scenarios based on common surgical conditions addressed by the curriculum relating to the Membership Examination of the Royal Colleges of Surgeons (MRCS). Multiple-choice questions with feedback and discussion boards facilitated by expert clinical tutors provided formative assessment. Summative assessment comprised written examination at the end of each of the first 2 years (equivalent to MRCS level), culminating in submission of a research dissertation in year 3 toward an MSc. METHODS: Students' age, gender, and level at entry to the programme were documented. Anonymized student feedback from 2008 to 2012 was examined using online questionnaires, and performance in the MSc programme was compared to MRCS examination outcomes for students who had consented to release of their results. RESULTS: A total of 517 surgical trainees from 40 countries were recruited over the 6-year period, and 116 MSc students have graduated to date. Of 368 students, 279 (76%) were foundation doctors (interns) and had not commenced formal surgical training on enrolling in the MSc programme. However, level at entry did not influence performance (P > 0.05 across all 3 years). Average pass rates since the programme launched, for those students completing all of the required assessments, were 84% ± 11% in year 1, 85% ± 10% in year 2, and 88% ± 7% in year 3 of the MSc programme. MSc students had significantly higher MRCS pass rates than nonenrolled trainees (67% vs 51%, P < 0.01, n = 352). There was a significant correlation between MRCS examination performance and overall performance in the MSc (R = 58%; P < 0.01, n = 37). Of 248 respondents, 202 (81%) considered that the MSc would improve their chances of gaining a surgical training post, and 224 (90%) would recommend the programme to their peers. CONCLUSIONS: The online MSc programme supports academic development of trainees in the early years of surgical training, is well received by students, and is associated with improved success in their professional examination.

Elastin accumulation is regulated at the level of degradation by macrophage metalloelastase (MMP-12) during experimental liver fibrosis.

UNLABELLED: Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP-12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl(4) ) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP-12 elastin degradation was defined mechanistically using CD11b-DTR and MMP-12 knockout mice. In a CCl(4) model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP-12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP-12 was bound in greater quantities to its inhibitor TIMP-1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP-12. Exposure of CCl(4) in MMP-12(-/-) mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP-12(-/-) mice compared with WT. CONCLUSION: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage-derived MMP-12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies.

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function.

UNLABELLED: Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex-mismatch and green fluorescent protein expression. BM-derived macrophage (BMM) delivery resulted in early chemokine up-regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up- regulation of the liver progenitor cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor-1, insulin-like growth factor-1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM. CONCLUSION: Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response.

BACKGROUND AND METHODS: In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. HPCs are located within a designated niche in close association with myofibroblasts and bone marrow (BM) derived macrophages. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Using the collagen Iα1((r/r)) mouse (r/r), which produces mutated collagen I resistant to matrix metalloproteinase degradation and has an exaggerated fibrotic response to liver injury, we test the relationship between collagen degradation, laminin deposition, and the HPC response. RESULTS: Chronic fibrotic carbon tetrachloride (CCl4) injury can induce a florid HPC response associated with dense laminin deposition. In the recovery phase after chronic CCl4 injury, r/r mice have a markedly attenuated HPC response compared to wild-types, together with persistence of collagen I and failure to deposit ECM laminin. Similar results were found in r/r mice given the choline-deficient ethionine supplemented diet, another model of the HPC response. In cross-over sex-mismatched BM transplantation (BMT) experiments between r/r mice and wild-types, the blunted HPC response of r/r mice was not rescued by wild-type BMT and likewise not conferred on to wild-type recipients by r/r BMT, demonstrating that the attenuated HPC response in r/r mice is a property intrinsic to the liver. CONCLUSION: Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.

The effect of surgical subspecialization on outcomes in peptic ulcer disease complicated by perforation and bleeding.

BACKGROUND: Emergency surgical services in Edinburgh were restructured in July 2002 to deliver subspecialist management of colorectal and upper-gastrointestinal emergencies on separate sites. The effect of emergency subspecialization on outcome from perforated and bleeding peptic ulceration was assessed. METHODS: All patients admitted with complicated peptic ulceration (January 2000-February 2005) were identified from a prospectively compiled database. RESULTS: Perforation: 148 patients were admitted with perforation before the service reorganization (period A - 31 months) of whom 126 (85.1%) underwent surgery; 135 patients were admitted in period B (31 months) of whom 114 (84.4%) were managed operatively. The in-hospital mortality was lower in period B (14/135, 10.4%) than period A (30/148, 20.3%; P = 0.023; relative risk (RR), 0.51; 95% confidence interval (CI), 0.28-0.91). There was a significantly higher rate of gastric resection in the second half of the study (period A 1/126 vs. period B 8/114; P = 0.015; RR, 8.84; 95% CI, 1.48-54.34). Length of hospital stay was similar for both groups. Bleeding: 51 patients underwent operative management of bleeding peptic ulceration in period A and 51 in period B. There were no differences in length of stay or mortality between these two groups. CONCLUSION: Restructuring of surgical services with emergency subspecialization was associated with lower mortality for perforated peptic ulceration. Subspecialist experience, intraoperative decision-making, and improved postoperative care have all contributed to this improvement.