69 results on '"Duckworth CA"'
Search Results
2. OC-043 Nfkb1 deficiency alters susceptibility to helicobacter spp. induced il-1Β secretion in bone marrow derived dendritic cells
- Author
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Tang, JM, Pritchard, DM, Duckworth, CA, Caamano, J, and Burkitt, MD
- Published
- 2015
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3. The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer
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Evans, JP, Winiarski, BK, Sutton, PA, Jones, RP, Ressel, L, Duckworth, CA, Pritchard, DM, Lin, ZX, Vicky, FL, Tweedle, EM, Costello, E, Goldring, CE, Copple, IM, Park, BK, Palmer, DH, and Kitteringham, NR
- Subjects
respiratory system ,neoplasms ,digestive system ,environment and public health ,digestive system diseases - Abstract
© Evans et al. Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines in vitro and in an orthotopic syngeneic mouse model utilising bioluminescent imaging. Using a tissue microarray, Nrf2 was found to be overexpressed (p < 0.01) in primary CRC and metastatic tissue relative to normal colon, with a positive correlation between Nrf2 expression in matched primary and metastatic samples. In vitro experiments in CRC cell lines revealed that Nrf2 siRNA and brusatol, which is known to inhibit Nrf2, decreased viability and sensitised cells to irinotecan toxicity. Furthermore, brusatol effectively abrogated CRC tumour growth in subcutaneously and orthotopicallyallografted mice, resulting in an average 8-fold reduction in luminescence at the study end-point (p=0.02). Our results highlight Nrf2 as a promising drug target in the treatment of CRC.
- Published
- 2018
4. PTU-047 Anti-CD3 Antibody Induces T-Cell Mediated Apoptosis and Shedding of Murine Small Intestinal Epithelial Cells
- Author
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Elramli, AH, primary, Duckworth, CA, additional, Campbell, BJ, additional, and Pritchard, DM, additional
- Published
- 2016
- Full Text
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5. The Effect of Carrier Temperature on Pesticide Emulsification or Dispersion Characteristics
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Duckworth, CA, primary and Cearnal, KS, additional
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6. PWE-109 A Metabolomic Profiling Study Of A Chemically-induced Mouse Model Of Intestinal Inflammation
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Reade, S, primary, Duckworth, CA, additional, Khalid, T, additional, Mayor, A, additional, Aggio, R, additional, Pritchard, DM, additional, and Probert, CS, additional
- Published
- 2014
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7. Sequence of Contact X-ray Brachytherapy (CXB) and External Beam Radiation (EBRT) in organ-preserving treatment for small rectal cancer.
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Than NW, Pritchard DM, Hughes DM, Duckworth CA, Wong H, Haq MU, Sripadam R, and Myint AS
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- Humans, Male, Female, Retrospective Studies, Aged, Middle Aged, Adenocarcinoma radiotherapy, Adenocarcinoma pathology, Adenocarcinoma mortality, Propensity Score, Aged, 80 and over, Rectal Neoplasms radiotherapy, Rectal Neoplasms pathology, Rectal Neoplasms mortality, Brachytherapy methods, Brachytherapy adverse effects, Organ Sparing Treatments methods
- Abstract
Background and Purpose: External Beam Radiotherapy (EBRT) followed by Contact X-ray Brachytherapy (CXB) and vice versa are viable alternatives to surgery for selected rectal cancer patients who have small tumours (≤3 cm). However, the optimal sequence of treatment needs to be established. We compared two approaches using Propensity Score (PS) matching and inverse probability treatment weighting (IPTW) analyses to investigate whether the sequence of treatment affected patient outcomes., Materials and Methods: This retrospective analysis (2008-2019) included patients with rectal adenocarcinoma (cT1-3,N0-1,M0, grade 1-2, size ≤ 3 cm) who received both EBRT and CXB, irrespective of treatment sequence. PS matching and IPTW were conducted to balance covariate standardised mean differences between groups. Oncological outcomes and rate of post-treatment rectal bleeding were assessed., Results: Following PS matching and IPTW analyses from 251 eligible patients; 103 starting with EBRT (median follow-up: 37 [IQR:18-56] months) and 148 with CXB (median follow-up: 32 [IQR:16-54] months, a significant improvement in 3-year overall survival (77% vs 85%, p = 0.02, [HR:0.58 (95% CI:0.37-0.91)]) and a higher risk of post-treatment rectal bleeding (grade 1 (26%) and grade 2 (6%)) were found in patients who started with CXB (p = 0.08). No significant differences were observed in local regrowth (18% vs 12%, p = 0.47), distant relapse (10% vs 6%, p = 0.53), 3-year organ preservation rates (70% vs 75%, p = 0.20, [HR:0.66 (95% CI: 0.35-1.26)]), or disease-free survival (78% vs 82%, p = 0.17, [HR: 0.47 (95% CI: 0.16-1.38)]) CONCLUSION: In patients with rectal cancer (≤3 cm), commencing with CXB rather than EBRT, was associated with improved overall survival, but had a higher risk of G1/2 rectal bleeding. No statistically significant differences were observed in other oncological outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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8. Contact X-ray brachytherapy (CXB) as a salvage treatment for rectal cancer patients who developed local tumor re-growth after watch-and-wait approach.
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Than NW, Pritchard DM, Duckworth CA, Hughes DM, Wong H, Sripadam R, and Myint AS
- Abstract
Purpose: A watch-and-wait approach is an alternative to surgery for rectal cancer patients who have achieved a clinical complete response (cCR) following neoadjuvant (chemo)radiotherapy. However, approximately 25-38% of patients experience subsequent local tumor re-growth that requires salvage surgery. We evaluated the effectiveness of contact X-ray brachytherapy (CXB) as an alternative method of salvage therapy for those patients who were either unfit for or refused surgery. Oncological outcomes, tolerability, and feasibility of subsequent surgery for local treatment failure following CXB were reported., Material and Methods: From 2009-2021, all patients treated with CXB as salvage therapy for local rectal cancer re-growth after watch-and-wait approach at our center were analyzed., Results: Contact X-ray brachytherapy as a salvage treatment (range, 90-110 Gy) was offered to 56 patients who experienced tumor re-growth following (chemo)radiation and watch-and-wait protocol. Median age was 76 (IQR = 66-83) years. Most patients (82%) had early-stage re-growth (ycT1/ycT2, ycN0), and 18% had more advanced stages (ycT3/ycT4, ycN0). After a median of 37-month follow-up (IQR = 19-53), 48% of patients who had early-stage re-growth achieved a sustained complete remission after CXB compared with 20% of those who had more advanced tumor stages. Disease-free and overall survivals for the whole cohort were 69% and 100% at 1-year, 51% and 82% at 3-year, and 51% and 65% at 5-years. CXB effectively controlled local re-growth-related symptoms. Mild post-CXB side effects occurred in 18% of cases. All (100%) eight patients who developed further local relapse, and 29% of those who had residual disease post-CXB salvage were successfully managed with subsequent surgery., Conclusions: Contact X-ray brachytherapy offers a new treatment option for patients in this situation whose other therapy options are not suitable for or refused initial surgery. Early local tumor re-growth responded best with minimal treatment-related toxicity and excellent symptom control. Disease-free and overall survival rates were acceptable, and delaying surgical salvage for local re-growth did not compromise patients' eventual long-term outcomes., Competing Interests: Approval of the Bioethics Committee was not required. The authors report no conflict of interest. Supplementary material is available on journal’s website., (Copyright © 2024 Termedia.)
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- 2024
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9. A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.
- Author
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Johnston LJ, Barningham L, Campbell EL, Cerovic V, Duckworth CA, Luu L, Wastling J, Derricott H, and Coombes JL
- Abstract
Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all- trans -retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b
+ SIRPα+ CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)- Published
- 2023
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10. A dynamic model of the intestinal epithelium integrates multiple sources of preclinical data and enables clinical translation of drug-induced toxicity.
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Gall L, Jardi F, Lammens L, Piñero J, Souza TM, Rodrigues D, Jennen DGJ, de Kok TM, Coyle L, Chung SW, Ferreira S, Jo H, Beattie KA, Kelly C, Duckworth CA, Pritchard DM, and Pin C
- Subjects
- Mice, Humans, Animals, Fluorouracil toxicity, Fluorouracil metabolism, Intestinal Mucosa metabolism, Diarrhea chemically induced, Doxorubicin toxicity, Citrulline, Drug-Related Side Effects and Adverse Reactions
- Abstract
We have built a quantitative systems toxicology modeling framework focused on the early prediction of oncotherapeutic-induced clinical intestinal adverse effects. The model describes stem and progenitor cell dynamics in the small intestinal epithelium and integrates heterogeneous epithelial-related processes, such as transcriptional profiles, citrulline kinetics, and probability of diarrhea. We fitted a mouse-specific version of the model to quantify doxorubicin and 5-fluorouracil (5-FU)-induced toxicity, which included pharmacokinetics and 5-FU metabolism and assumed that both drugs led to cell cycle arrest and apoptosis in stem cells and proliferative progenitors. The model successfully recapitulated observations in mice regarding dose-dependent disruption of proliferation which could lead to villus shortening, decrease of circulating citrulline, increased diarrhea risk, and transcriptional induction of the p53 pathway. Using a human-specific epithelial model, we translated the cytotoxic activity of doxorubicin and 5-FU quantified in mice into human intestinal injury and predicted with accuracy clinical diarrhea incidence. However, for gefitinib, a specific-molecularly targeted therapy, the mice failed to reproduce epithelial toxicity at exposures much higher than those associated with clinical diarrhea. This indicates that, regardless of the translational modeling approach, preclinical experimental settings have to be suitable to quantify drug-induced clinical toxicity with precision at the structural scale of the model. Our work demonstrates the usefulness of translational models at early stages of the drug development pipeline to predict clinical toxicity and highlights the importance of understanding cross-settings differences in toxicity when building these approaches., (© 2023 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)
- Published
- 2023
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11. C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression.
- Author
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Wan Y, Adair K, Herrmann A, Shan X, Xia L, Duckworth CA, and Yu LG
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- Chick Embryo, Humans, Animals, Mice, Galactose, Glycosylation, Macrophages, Tumor Microenvironment, Galectin 3 genetics, Colonic Neoplasms genetics
- Abstract
Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. β1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1
-/- /Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression., (© 2023. The Author(s).)- Published
- 2023
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12. Mouse organoids as an in vitro tool to study the in vivo intestinal response to cytotoxicants.
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Jardi F, Kelly C, Teague C, Fowler-Williams H, Sevin DC, Rodrigues D, Jo H, Ferreira S, Herpers B, Van Heerden M, de Kok T, Pin C, Lynch A, Duckworth CA, De Jonghe S, Lammens L, and Pritchard DM
- Subjects
- Humans, Animals, Mice, Caspase 3 metabolism, Diarrhea chemically induced, Organoids, Intestinal Mucosa, Fluorouracil toxicity, Apoptosis
- Abstract
Cross-species comparison of drug responses at the organoid level could help to determine the human relevance of findings from animal studies. To this end, we first need to evaluate the in vitro to in vivo translatability of preclinical organoids. Here, we used 5-fluorouracil (5-FU) as an exemplar drug to test whether the in vivo gut response to this cytotoxicant was preserved in murine intestinal organoids. Mice treated with 5-FU at 20 or 50 mg/kg IV (low and high dose, respectively) displayed diarrhea at clinically relevant exposures. 5-FU also induced intestinal lesions, increased epithelial apoptosis, and decreased proliferation in a dose-dependent manner. To enable comparison between the in vitro and in vivo response, top nominal in vitro drug concentrations that caused significant cytotoxicity were chosen (dose range 1-1000 µM). The inferred intracellular concentration in organoids at 1000 µM was within the tissue exposure range related to intestinal toxicity in vivo. 5-FU at ≥ 100 µM decreased ATP levels and increased Caspase-3 activity in intestinal organoids. In keeping with the in vivo findings, 5-FU increased the percentage of Caspase-3-positive cells and reduced Ki67 staining. At the transcriptome level, there was an overlap in the activity of pathways related to 5-FU's mode of action, lipid and cholesterol metabolism and integrin signaling across in vivo gut and organoids. The predicted activity state of upstream regulators was generally well preserved between setups. Collectively, our results suggest that despite their inherent limitations, organoids represent an adequate tool to explore the intestinal response to cytotoxicants., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2023
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13. Galectin-2 in Health and Diseases.
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Negedu MN, Duckworth CA, and Yu LG
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- Pregnancy, Female, Humans, Galectin 2 genetics, Galectins, Placenta metabolism, Neoplasms metabolism, Arthritis, Rheumatoid
- Abstract
Galectin-2 is a prototype member of the galactoside-binding galectin family. It is predominately expressed in the gastrointestinal tract but is also detected in several other tissues such as the placenta and in the cardiovascular system. Galectin-2 expression and secretion by epithelial cells has been reported to contribute to the strength of the mucus layer, protect the integrity of epithelia. A number of studies have also suggested the involvement of galectin-2 in tissue inflammation, immune response and cell apoptosis. Alteration of galectin-2 expression occurs in inflammatory bowel disease, coronary artery diseases, rheumatoid arthritis, cancer, and pregnancy disorders and has been shown to be involved in disease pathogenesis. This review discusses our current understanding of the role and actions of galectin-2 in regulation of these pathophysiological conditions.
- Published
- 2022
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14. Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa.
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Papoutsopoulou S, Tang J, Elramli AH, Williams JM, Gupta N, Ikuomola FI, Sheibani-Tezerji R, Alam MT, Hernández-Fernaud JR, Caamaño JH, Probert CS, Muller W, Duckworth CA, and Pritchard DM
- Subjects
- Animals, Immunoglobulin A metabolism, Immunoglobulins metabolism, Intestinal Mucosa metabolism, Lipopolysaccharides pharmacology, Mice, NF-kappa B metabolism, Proteomics, NF-kappa B p52 Subunit genetics, NF-kappa B p52 Subunit metabolism, Plasma Cells metabolism
- Abstract
The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb 2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2
-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut. NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.- Published
- 2022
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15. Interleukin-10 Deficiency Impacts on TNF-Induced NFκB Regulated Responses In Vivo.
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Papoutsopoulou S, Pollock L, Williams JM, Abdul-Mahdi MMLF, Dobbash R, Duckworth CA, and Campbell BJ
- Abstract
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has a major protective role against intestinal inflammation. We recently revealed that intestinal epithelial cells in vitro regulate NFκB-driven transcriptional responses to TNF via an autocrine mechanism dependent on IL-10 secretion. Here in this study, we investigated the impact of IL-10 deficiency on the NFκB pathway and its downstream targets in the small intestinal mucosa in vivo. We observed dysregulation of TNF, IκBα, and A20 gene and protein expression in the small intestine of steady-state or TNF-injected Il10
-/- mice, compared to wild-type C57BL6/J counterparts. Upon TNF injection, tissue from the small intestine showed upregulation of NFκB p65[RelA] activity, which was totally diminished in Il10-/- mice and correlated with reduced levels of TNF, IκBα, and A20 expression. In serum, whilst IgA levels were noted to be markedly downregulated in IL-10-deficient- mice, normal levels of mucosal IgA were seen in intestine mucosa. Importantly, dysregulated cytokine/chemokine levels were observed in both serum and intestinal tissue lysates from naïve, as well as TNF-injected Il10-/- mice. These data further support the importance of the IL-10-canonical NFκB signaling pathway axis in regulating intestinal mucosa homeostasis and response to inflammatory triggers in vivo.- Published
- 2022
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16. Soluble Non-Starch Polysaccharides From Plantain ( Musa x paradisiaca L.) Diminish Epithelial Impact of Clostridioides difficile .
- Author
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Simpson HL, Roberts CL, Thompson LM, Leiper CR, Gittens N, Trotter E, Duckworth CA, Papoutsopoulou S, Miyajima F, Roberts P, O'Kennedy N, Rhodes JM, and Campbell BJ
- Abstract
Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea. Adhesion of this Gram-positive pathogen to the intestinal epithelium is a crucial step in CDI, with recurrence and relapse of disease dependent on epithelial interaction of its endospores. Close proximity, or adhesion of, hypervirulent strains to the intestinal mucosa are also likely to be necessary for the release of C. difficile toxins, which when internalized, result in intestinal epithelial cell rounding, damage, inflammation, loss of barrier function and diarrhoea. Interrupting these C. difficile -epithelium interactions could therefore represent a promising therapeutic strategy to prevent and treat CDI. Intake of dietary fibre is widely recognised as being beneficial for intestinal health, and we have previously shown that soluble non-starch polysaccharides (NSP) from plantain banana ( Musa spp.), can block epithelial adhesion and invasion of a number of gut pathogens, such as E. coli and Salmonellae. Here, we assessed the action of plantain NSP, and a range of alternative soluble plant fibres, for inhibitory action on epithelial interactions of C. difficile clinical isolates, purified endospore preparations and toxins. We found that plantain NSP possessed ability to disrupt epithelial adhesion of C. difficile vegetative cells and spores, with inhibitory activity against C. difficile found within the acidic (pectin-rich) polysaccharide component, through interaction with the intestinal epithelium. Similar activity was found with NSP purified from broccoli and leek, although seen to be less potent than NSP from plantain. Whilst plantain NSP could not block the interaction and intracellular action of purified C. difficile toxins, it significantly diminished the epithelial impact of C. difficile , reducing both bacteria and toxin induced inflammation, activation of caspase 3/7 and cytotoxicity in human intestinal cell-line and murine intestinal organoid cultures. Dietary supplementation with soluble NSP from plantain may therefore confer a protective effect in CDI patients by preventing adhesion of C. difficile to the mucosa, i.e. a "contrabiotic" effect, and diminishing its epithelial impact. This suggests that plantain soluble dietary fibre may be a therapeutically effective nutritional product for use in the prevention or treatment of CDI and antibiotic-associated diarrhoea., Competing Interests: NO’K is a present, and CR a past, employee of Provexis Plc. BC, HS and JR received additional funding support from the biotech partner Provexis Plc as part of the BBSRC Industrial CASE studentship (BB/I016783/1). JR is listed as inventor on patents held by Provexis Plc, in a licence agreement with the University of Liverpool, for use of complex oligosaccharides in the prevention or treatment of inflammatory bowel disease (US8945639B2), and for use of soluble fibres in antibiotic-associated diarrhoea (US20120165289A1). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Simpson, Roberts, Thompson, Leiper, Gittens, Trotter, Duckworth, Papoutsopoulou, Miyajima, Roberts, O’Kennedy, Rhodes and Campbell.)
- Published
- 2021
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17. Identifying key regulators of the intestinal stem cell niche.
- Author
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Duckworth CA
- Subjects
- Animals, Signal Transduction, Intestines cytology, Stem Cell Niche
- Abstract
The intestinal tract is lined by a single layer of epithelium that is one of the fastest regenerating tissues in the body and which therefore requires a very active and exquisitely controlled stem cell population. Rapid renewal of the epithelium is necessary to provide a continuous physical barrier from the intestinal luminal microenvironment that contains abundant microorganisms, whilst also ensuring an efficient surface for the absorption of dietary components. Specialised epithelial cell populations are important for the maintenance of intestinal homeostasis and are derived from adult intestinal stem cells (ISCs). Actively cycling ISCs divide by a neutral drift mechanism yielding either ISCs or transit-amplifying epithelial cells, the latter of which differentiate to become either absorptive lineages or to produce secretory factors that contribute further to intestinal barrier maintenance or signal to other cellular compartments. The mechanisms controlling ISC abundance, longevity and activity are regulated by several different cell populations and signalling pathways in the intestinal lamina propria which together form the ISC niche. However, the complexity of the ISC niche and communication mechanisms between its different components are only now starting to be unravelled with the assistance of intestinal organoid/enteroid/colonoid and single-cell imaging and sequencing technologies. This review explores the interaction between well-established and emerging ISC niche components, their impact on the intestinal epithelium in health and in the context of intestinal injury and highlights future directions and implications for this rapidly developing field., (© 2021 The Author(s).)
- Published
- 2021
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18. Appearance of peanut agglutinin in the blood circulation after peanut ingestion promotes endothelial secretion of metastasis-promoting cytokines.
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Wang W, Sindrewicz-Goral P, Chen C, Duckworth CA, Pritchard DM, Rhodes JM, and Yu LG
- Subjects
- Animals, Cell Adhesion Molecules metabolism, Cells, Cultured, Endothelium, Vascular drug effects, Endothelium, Vascular metabolism, Female, Humans, Inflammation Mediators metabolism, Mice, Mice, Inbred BALB C, Mucin-1 metabolism, Peanut Agglutinin pharmacology, Signal Transduction, Arachis, Cytokines metabolism, Neoplasm Metastasis pathology, Peanut Agglutinin blood
- Abstract
Peanut agglutinin (PNA) is a carbohydrate-binding protein in peanuts that accounts for ~0.15% peanut weight. PNA is highly resistant to cooking and digestion and is rapidly detectable in the blood after peanut consumption. Our previous studies have shown that circulating PNA mimics the actions of endogenous galactoside-binding protein galectin-3 by interaction with tumour cell-associated MUC1 and promotes circulating tumour cell metastatic spreading. The present study shows that circulating PNA interacts with micro- as well as macro-vascular endothelial cells and induces endothelial secretion of cytokines MCP-1 (CCL2) and IL-6 in vitro and in vivo. The increased secretion of these cytokines autocrinely/paracrinely enhances the expression of endothelial cell surface adhesion molecules including integrins, VCAM and selectin, leading to increased tumour cell-endothelial adhesion and endothelial tubule formation. Binding of PNA to endothelial surface MCAM (CD146), via N-linked glycans, and subsequent activation of PI3K-AKT-PREAS40 signalling is here shown responsible for PNA-induced secretion of MCP-1 and IL-6 by vascular endothelium. Thus, in addition to its influence on promoting tumour cell spreading by interaction with tumour cell-associated MUC1, circulating PNA might also influence metastasis by enhancing the secretion of metastasis-promoting MCP-1 and IL-6 from the vascular endothelium., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2021
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19. Retraction notice to " IP1867B suppresses the Insulin-like Growth Factor 1 Receptor (IGF1R) ablating epidermal growth factor receptor inhibitor resistance in adult high grade gliomas" [Canc. Lett., 458 (2019) pages 29-38].
- Author
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Mihajluk K, Simms C, Reay M, Madureira PA, Howarth A, Murray P, Nasser S, Duckworth CA, Pritchard DM, Pilkington GJ, and Hill R
- Published
- 2021
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20. Dilodendron bipinnatum Radlk. extract alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration, TNF-α and IL-1β concentrations, IL-17 and COX-2 expressions, supporting mucus production and promotes an antioxidant effect.
- Author
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Oliveira RG, Damazo AS, Antonielli LF, Miyajima F, Pavan E, Duckworth CA, Lima JCDS, Arunachalam K, and Martins DTO
- Subjects
- Animals, Antioxidants chemistry, Antioxidants therapeutic use, Brazil, Caco-2 Cells, Cell Survival drug effects, Colitis, Ulcerative chemically induced, Colitis, Ulcerative pathology, Cyclooxygenase 2 metabolism, Disease Models, Animal, Edema chemically induced, Edema prevention & control, Glutathione metabolism, Humans, Interleukin-17 metabolism, Interleukin-1beta metabolism, Interleukin-8 metabolism, Lipopolysaccharides toxicity, Mast Cells drug effects, Peroxidase metabolism, Plant Extracts chemistry, Plant Extracts therapeutic use, Rats, Wistar, Trinitrobenzenesulfonic Acid toxicity, Tumor Necrosis Factor-alpha metabolism, Rats, Antioxidants pharmacology, Colitis, Ulcerative prevention & control, Mucus metabolism, Plant Bark chemistry, Plant Extracts pharmacology, Sapindaceae chemistry
- Abstract
Ethnopharmacological Relevance: Dilodendron bipinnatum (Sapindaceae) stem bark decoction and macerate were used to treat uterine inflammation, pain in general, dermatitis and bone fractures. These homemade preparations also have diuretic, stimulant, expectorants and sedative effects and are effective in treating worm infections in the Brazilian Pantanal population. Our previous research confirmed the anti-inflammatory activity of the hydroethanolic extract of inner stem bark of D. bipinnatum (HEDb)., Aim: This work aimed to investigate the efficacy of HEDb in ameliorating experimental colitis in rats and to elucidate the possible mechanisms involved in the anti-ulcerative colitis properties of HEDb in rats and Caco-2 cell line., Materials and Methods: The effects on cell viability, IL-8 and TNF-α in human colon adenocarcinoma (Caco-2) were determined by flow cytometer and ELISA. Wistar rats (n = 6-7) were orally gavaged with, vehicle (0.9% saline), HEDb at doses of 20, 100 or 500 mg/kg, or mesalazine at a dose of 500 mg/kg, at 48, 24 and 1 h prior to the administration of trinitrobenzene sulfonic acid via rectal administration to induce colitis. The anti-inflammatory effects of HEDb were assessed macroscopically, by myeloperoxidase (MPO) activity and for glutathione (GSH) concentration in the colon. Additionally, colonic histopathological analyses of UC severity were conducted by different staining methods (H&E, PAS and toluidine blue). Pro-inflammatory cytokines TNF-α and IL-1β were quantified in colonic tissue by ELISA and colonic expressions of COX-2 and IL-17 were analyzed by western blotting., Results: HEDb was shown to be non-cytotoxic with mean viability of 80% in Caco-2 cells. HEDb pre-treatments of 1, 5 or 20 μg/mL significantly reduced TNF-α production in Caco-2 cells by 21.8% (p < 0.05), 60.5 and 82.1% (p < 0.001) respectively following LPS treatment compared to LPS alone. However, no change in IL-8 production was observed. HEDb pre-treatment of rats subjected to TNBS significantly (p < 0.001) reduced colonic lesion score. Higher doses (100 and 500 mg/kg) caused a sharp downregulation of haemorrhagic damage, leukocyte infiltration, edema and restoration of mucus production. Moreover, mast cell degranulation was inhibited. Colonic MPO activity was reduced following all doses of HEDb, reaching 51.1% ± 1.51 (p < 0.05) with the highest dose. GSH concentration was restored by 58% and 70% following 100 and 500 mg/kg of HEDb, respectively. The oral treatment of HEDb at doses 20, 100 and 500 mg/kg decreased the concentrations of TNF-α and IL-1β at all doses in comparison to vehicle treated control. In addition, HEDb inhibited the COX-2 and IL-17 expressions with maximal effect at 500 mg/kg (60.3% and 65% respectively; p < 0.001). In all trials, the effect of HEDb at all doses being 20, 100 and 500 mg/kg was statistically comparable to mesalazine (500 mg/kg)., Conclusions: HEDb reduces colonic damage in the TNBS colitis model and relieves oxidative and inflammatory events, at least in part, by increasing mucus production, reducing leukocyte migration and reducing TNF-α (in vivo and in vitro), IL-1β, IL-17 and COX-2 expression. Therefore, HEDb requires further investigation as a candidate for treating IBD., (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2021
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21. Canthin-6-one ameliorates TNBS-induced colitis in rats by modulating inflammation and oxidative stress. An in vivo and in silico approach.
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Arunachalam K, Damazo AS, Macho A, Matchado MS, Pavan E, Figueiredo FF, Oliveira DM, Duckworth CA, Thangaraj P, Leonti M, and Martins DTO
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- Animals, Anti-Inflammatory Agents chemistry, Anti-Inflammatory Agents pharmacology, Anti-Inflammatory Agents therapeutic use, Carbolines chemistry, Carbolines pharmacology, Colitis chemically induced, Colitis drug therapy, Fungicides, Industrial chemistry, Fungicides, Industrial pharmacology, Fungicides, Industrial therapeutic use, Indole Alkaloids chemistry, Indole Alkaloids pharmacology, Inflammation chemically induced, Inflammation drug therapy, Inflammation metabolism, Inflammation Mediators antagonists & inhibitors, Male, Oxidative Stress physiology, Protein Structure, Secondary, Rats, Rats, Wistar, Carbolines therapeutic use, Colitis metabolism, Computer Simulation, Indole Alkaloids therapeutic use, Inflammation Mediators metabolism, Oxidative Stress drug effects, Trinitrobenzenesulfonic Acid toxicity
- Abstract
Canthin-6-one (Cant) is an indole alkaloid found in several botanical drugs used as medicines, reported to be gastroprotective, anti-inflammatory, anti-microbial, anti-diarrheal and anti-proliferative. We aimed to explore Cant in the management of colitis using a trinitrobenzenesulfonic acid (TNBS)-induced rat model. Cant (1, 5 and 25 mg/kg) was administered by oral gavage to Wistar rats followed by induction of colitis with TNBS. Macroscopic and histopathological scores, myeloperoxidase (MPO), malondialdehyde (MDA) and reduced glutathione (GSH) were assessed in colon tissues. Pro- (TNF-α, IL-1β and IL-12p70) and anti-inflammatory (IL-10) cytokines, and vascular endothelial growth factor (VEGF) were also quantified. Mitogen-activated protein kinase 14 (MAPK14) and Toll-like receptor-8 (TLR8), as putative targets, were considered through in silico analysis. Cant (5 and 25 mg/kg) reduced macroscopic and histological colon damage scores in TNBS-treated rats. MPO and MDA were reduced by up to 61.69% and 92.45%, respectively, compared to TNBS-treated rats alone. Glutathione concentration was reduced in rats administered with TNBS alone (50.00% of sham group) but restored to 72.73% (of sham group) with Cant treatment. TNF-α, IL-1β, IL-12p70 and VEGF were reduced, and anti-inflammatory IL-10 was increased following Cant administration compared to rats administered TNBS alone. Docking ligation results for MAPK14 (p38α) and TLR8 with Cant, confirmed that these proteins are feasible putative targets. Cant has an anti-inflammatory effect in the intestine by down-regulating molecular immune mediators and decreasing oxidative stress. Therefore, Cant could have therapeutic potential for the treatment of inflammatory bowel disease and related syndromes., (Copyright © 2021 Elsevier Inc. All rights reserved.)
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- 2021
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22. Long-Term Iron Deficiency and Dietary Iron Excess Exacerbate Acute Dextran Sodium Sulphate-Induced Colitis and Are Associated with Significant Dysbiosis.
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Mahalhal A, Burkitt MD, Duckworth CA, Hold GL, Campbell BJ, Pritchard DM, and Probert CS
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- Anemia, Iron-Deficiency, Animals, Bacteria genetics, Colitis physiopathology, Colon pathology, Dextran Sulfate pharmacology, Diet, Dietary Supplements adverse effects, Disease Models, Animal, Dysbiosis etiology, Female, Gastrointestinal Microbiome drug effects, Gastrointestinal Microbiome genetics, Inflammation, Inflammatory Bowel Diseases pathology, Iron, Dietary adverse effects, Mice, Mice, Inbred C57BL, Microbiota, RNA, Ribosomal, 16S genetics, Colitis metabolism, Iron metabolism, Iron Overload physiopathology
- Abstract
Background: Oral iron supplementation causes gastrointestinal side effects. Short-term alterations in dietary iron exacerbate inflammation and alter the gut microbiota, in murine models of colitis. Patients typically take supplements for months. We investigated the impact of long-term changes in dietary iron on colitis and the microbiome in mice., Methods: We fed mice chow containing differing levels of iron, reflecting deficient (100 ppm), normal (200 ppm), and supplemented (400 ppm) intake for up to 9 weeks, both in absence and presence of dextran sodium sulphate (DSS)-induced chronic colitis. We also induced acute colitis in mice taking these diets for 8 weeks. Impact was assessed (i) clinically and histologically, and (ii) by sequencing the V4 region of 16S rRNA., Results: In mice with long-term changes, the iron-deficient diet was associated with greater weight loss and histological inflammation in the acute colitis model. Chronic colitis was not influenced by altering dietary iron however there was a change in the microbiome in DSS-treated mice consuming 100 ppm and 400 ppm iron diets, and control mice consuming the 400 ppm iron diet. Proteobacteria levels increased significantly, and Bacteroidetes levels decreased, in the 400 ppm iron DSS group at day-63 compared to baseline., Conclusions: Long-term dietary iron alterations affect gut microbiota signatures but do not exacerbate chronic colitis, however acute colitis is exacerbated by such dietary changes. More work is needed to understand the impact of iron supplementation on IBD. The change in the microbiome, in patients with colitis, may arise from the increased luminal iron and not simply from colitis.
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- 2021
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23. A "Watch and Wait" Strategy Involving Regular Endoscopic Surveillance Is Safe for Many Patients with Small, Sporadic, Grade 1, Non-Ampullary, Non-Functioning Duodenal Neuroendocrine Tumours.
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Exarchou K, Moore AR, Smart HL, Duckworth CA, Howes N, and Pritchard DM
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- Aged, Female, Follow-Up Studies, Humans, Male, Middle Aged, Retrospective Studies, Duodenal Neoplasms diagnosis, Neuroendocrine Tumors diagnosis, Outcome and Process Assessment, Health Care, Watchful Waiting
- Abstract
Introduction: Duodenal neuroendocrine tumours (d-NETs) are rare but are increasing in incidence. Current ENETS guidelines advocate resection of all localized d-NETs. However, "watch and wait" may be appropriate for some localized, small, grade 1, non-functioning, non-ampullary d-NETs. We evaluated whether patients with such d-NETs who chose "watch and wait" involving regular endoscopic surveillance had equivalent disease-related outcomes to patients undergoing endoscopic or surgical resection., Methods: Retrospective review of patients with histologically confirmed d-NETs at Liverpool ENETS Centre of Excellence 2007-2020., Results: Sixty-nine patients were diagnosed with d-NET of which 50 were sporadic, non-functioning, non-ampullary tumours. Patient treatment groups were similar in terms of age, gender, and tumour location and grade, but unsurprisingly, larger tumours (median diameter 17 mm [p < 0.0001]) were found in the surgically treated group. Five patients underwent surgical resection with no evidence of tumour recurrence or disease-related death. Twelve patients underwent endoscopic resection (ER), with 1 local recurrence detected during follow-up. Thirty patients (28 with d-NETs ≤10 mm) underwent "watch and wait" with resection only if tumours increased in size. The d-NETs in 28/30 patients remained stable or decreased in size over a median 27 months (IQR: 15-48, R: 3-98). In 7 patients, the d-NET was completely removed by avulsion during diagnostic biopsy and was not seen at subsequent endoscopies. Only 2 patients showed increased d-NET size during surveillance, of whom only one was fit for ER. No NET-related deaths were documented during follow-up., Conclusions: All of the localized, ≤10 mm, grade 1, non-functioning, non-ampullary d-NETs in this cohort behaved indolently with very low risks of progression and no tumour-related deaths. "Watch and wait," therefore, appears to be a safe alternative management strategy for selected d-NETs., (© 2020 S. Karger AG, Basel.)
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- 2021
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24. Using systems medicine to identify a therapeutic agent with potential for repurposing in inflammatory bowel disease.
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Lloyd K, Papoutsopoulou S, Smith E, Stegmaier P, Bergey F, Morris L, Kittner M, England H, Spiller D, White MHR, Duckworth CA, Campbell BJ, Poroikov V, Martins Dos Santos VAP, Kel A, Muller W, Pritchard DM, Probert C, and Burkitt MD
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- Animals, Cells, Cultured, Clarithromycin pharmacology, Clarithromycin therapeutic use, Colitis chemically induced, Colitis metabolism, Colitis pathology, DNA metabolism, Dextran Sulfate, Gene Regulatory Networks, Humans, Inflammatory Bowel Diseases metabolism, Lipopolysaccharides, Luciferases metabolism, Mice, Inbred C57BL, NF-kappa B metabolism, Organoids drug effects, Organoids metabolism, Protein Binding drug effects, Signal Transduction, Transcription Factors metabolism, Tumor Necrosis Factor-alpha metabolism, Drug Repositioning, Inflammatory Bowel Diseases drug therapy, Inflammatory Bowel Diseases pathology, Systems Analysis
- Abstract
Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper., Competing Interests: Competing interestsV.A.P.M.d.S. is a shareholder and director of GeneXplain GmbH. A.K. is a shareholder and director of LifeGlimmer GmbH., (© 2020. Published by The Company of Biologists Ltd.)
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- 2020
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25. Corrigendum to "IP1867B suppresses the insulin-like growth factor 1 receptor (IGF1R) ablating epidermal growth factor receptor inhibitor resistance in adult high grade gliomas." [Cancer Lett. 458C (2019) 29-38].
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Mihajluk K, Simms C, Reay M, Madureira PA, Howarth A, Murray P, Nasser S, Duckworth CA, Pritchard DM, Pilkington GJ, and Hill R
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- 2020
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26. Netazepide Inhibits Expression of Pappalysin 2 in Type 1 Gastric Neuroendocrine Tumors.
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Lloyd KA, Parsons BN, Burkitt MD, Moore AR, Papoutsopoulou S, Boyce M, Duckworth CA, Exarchou K, Howes N, Rainbow L, Fang Y, Oxvig C, Dodd S, Varro A, Hall N, and Pritchard DM
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- Animals, Benzodiazepines pharmacology, Benzodiazepinones therapeutic use, Cell Line, Tumor, Disease Models, Animal, Gastric Mucosa cytology, Gastric Mucosa pathology, Gastrins antagonists & inhibitors, Gastrins blood, Gastrins metabolism, Gene Knockdown Techniques, Humans, Mice, Mice, Transgenic, Neuroendocrine Tumors blood, Neuroendocrine Tumors pathology, Organoids, Phenylurea Compounds therapeutic use, Pregnancy-Associated Plasma Protein-A analysis, Pregnancy-Associated Plasma Protein-A antagonists & inhibitors, Pregnancy-Associated Plasma Protein-A genetics, Primary Cell Culture, Receptor, Cholecystokinin B antagonists & inhibitors, Receptor, Cholecystokinin B metabolism, Stomach Neoplasms blood, Stomach Neoplasms pathology, Treatment Outcome, Benzodiazepinones pharmacology, Neuroendocrine Tumors drug therapy, Phenylurea Compounds pharmacology, Pregnancy-Associated Plasma Protein-A metabolism, Stomach Neoplasms drug therapy
- Abstract
Background & Aims: In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling., Methods: We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGS
GR gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples., Results: Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGSGR cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGSGR cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5., Conclusions: In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
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27. Development of an orthotopic syngeneic murine model of colorectal cancer for use in translational research.
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Evans JP, Winiarski BK, Sutton PA, Ressel L, Duckworth CA, Pritchard DM, Palmer DH, Goldring CE, and Kitteringham NR
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- Animals, Cell Line, Tumor, Liver Neoplasms, Experimental secondary, Male, Mice, Mice, Inbred BALB C, Pilot Projects, Translational Research, Biomedical, Colorectal Neoplasms pathology, Disease Models, Animal
- Abstract
Improving outcomes in colorectal cancer requires more accurate in vivo modelling of the disease in humans, allowing more reliable pre-clinical assessment of potential therapies. Novel imaging techniques are necessary to improve the longitudinal assessment of disease burden in these models, reducing the number of animals required for translational studies. This report describes the development of an immune-competent syngeneic orthotopic murine model of colorectal cancer, utilising caecal implantation of CT26 cells stably transfected with the luciferase gene into immune-competent BALB/c mice, allowing serial bioluminescent imaging of cancer progression. Luminescence in the stably transfected CT26 cell line, after pre-conditioning in the flank of a BALB/c mouse, accurately reflected cell viability and resulted in primary caecal tumours in five of eight (63%) mice in the initial pilot study following caecal injection. Luminescent signal continued to increase throughout the study period with one mouse (20%) developing a liver metastasis. Histopathological assessment confirmed tumours to be consistent with a poorly differentiated adenocarcinoma. We have now performed this technique in 68 immune-competent BALB/c mice. There have been no complications from the procedure or peri-operative deaths, with primary tumours developing in 44 (65%) mice and liver metastases in nine (20%) of these. This technique provides an accurate model of colorectal cancer with tumours developing in the correct microenvironment and metastasising to the liver with a similar frequency to that seen in patients presenting with colorectal cancer, with serial bioluminescent reducing the murine numbers required in studies by removing the need for cull for assessment of disease burden.
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- 2019
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28. NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells.
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Jones LG, Vaida A, Thompson LM, Ikuomola FI, Caamaño JH, Burkitt MD, Miyajima F, Williams JM, Campbell BJ, Pritchard DM, and Duckworth CA
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- Animals, Apoptosis drug effects, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Cell Degranulation drug effects, Cell Proliferation drug effects, Culture Media, Conditioned pharmacology, Dendritic Cells drug effects, Enterocytes cytology, Enterocytes drug effects, Epithelial Cells drug effects, Epithelial Cells metabolism, Interferon-gamma pharmacology, Intestine, Small metabolism, Lipopolysaccharides pharmacology, Mice, Inbred C57BL, Paneth Cells drug effects, Paneth Cells metabolism, Reproducibility of Results, Tumor Necrosis Factor-alpha pharmacology, Bone Marrow Cells cytology, Dendritic Cells metabolism, Enterocytes metabolism, NF-kappa B p52 Subunit metabolism, Organoids metabolism, Signal Transduction
- Abstract
Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2
-/- mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1-/- mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.- Published
- 2019
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29. RETRACTED: IP1867B suppresses the insulin-like growth factor 1 receptor (IGF1R) ablating epidermal growth factor receptor inhibitor resistance in adult high grade gliomas.
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Mihajluk K, Simms C, Reay M, Madureira PA, Howarth A, Murray P, Nasser S, Duckworth CA, Pritchard DM, Pilkington GJ, and Hill R
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- Animals, Aspirin pharmacology, Brain Neoplasms genetics, Brain Neoplasms metabolism, Brain Neoplasms pathology, Cell Hypoxia physiology, Drug Resistance, Neoplasm, Drug Synergism, ErbB Receptors antagonists & inhibitors, ErbB Receptors biosynthesis, ErbB Receptors genetics, Excipients administration & dosage, Female, Glioma genetics, Glioma metabolism, Glioma pathology, Humans, Insulin-Like Growth Factor I antagonists & inhibitors, Insulin-Like Growth Factor I biosynthesis, Insulin-Like Growth Factor I genetics, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, Mice, SCID, NF-kappa B antagonists & inhibitors, NF-kappa B metabolism, Neoplasm Grading, Receptor, IGF Type 1 biosynthesis, Receptor, IGF Type 1 genetics, Temozolomide administration & dosage, Temozolomide pharmacology, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols pharmacology, Aspirin administration & dosage, Brain Neoplasms drug therapy, Glioma drug therapy, Receptor, IGF Type 1 antagonists & inhibitors
- Abstract
This article has been retracted at the request of the Editor-in-Chief due to concerns regarding the legitimacy of images and data presented in the paper. Though a corrigendum (Can. Lett. Vol. 469, 2020, pages 524-535) was previously published to address some of these concerns, this corrigendum has also been found to contain errors and therefore cannot stand. Specific concerns are listed below. The Editor and Publisher received a letter from the University of Portsmouth alerting us to an investigation into alleged research misconduct. The University concluded their investigation with external experts and determined that misconduct did take place in relation to the research involved in this paper. Upon our separate investigation, it has been determined that the paper headline relies on showing that there was considerable reduction of IGF1R, IL6R and EGFR post treatment in all cell lines. During review, it was determined that this cannot be concluded from the presented data. For example, in SEBTA-003 the EGFR levels go up and there is no difference in IGFR1. It is apparent from Fig 4d that in the SEBTA-003 cell line the EGFR level does not go down, which is stated in the Results section on page 32, it is rather going up. The data for IGFR1 are inconclusive and there are concerns regarding the blot. The general implications would be that the effects of the drug IP1867B does not seem to be the same for all tested cell lines, and this should have been discussed in detail by the authors. Additionally, in subsequent experiments (Fig. 4g and h) the SEBTA-003 cell line (no reduction of EGFR, rather increased expression) and the other 3 cell lines (reduction of EGFR) show similar responses. This is particularly evident in Fig. 4g: Two cell lines are compared, SEBTA-003 (increased EGFR expression) and UP-029 (decreased EGFR expression), both behave similarly after exposure to drugs. The corrigendum (https://doi.org/10.1016/j.canlet.2019.10.002) issue is with respect to the Supplemental Figure 6i EGFR, particularly panel IP1867B. The Corrigendum states that the left part is a cut out of the very right part. If so, the bands for IP1867B should show the same staining pattern - but they do not. Also, in the Corrigendum, there are incorrect mentions between day 14 in the Figure and day 19 in the Figure legend. All authors were informed of the retraction in advance. Drs. Pritchard and Duckworth agreed to the retraction. The corresponding author, Dr Hill, did not agree to the retraction. No response had been received from Drs. Mihajluk, Simms, Reay, Madureira, Howarth, Murray, Nasser and Pilkinton at the time of the retraction being published., (Copyright © 2019 Elsevier B.V. All rights reserved.)
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- 2019
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30. An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.
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Luu L, Johnston LJ, Derricott H, Armstrong SD, Randle N, Hartley CS, Duckworth CA, Campbell BJ, Wastling JM, and Coombes JL
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- Animals, Cell Culture Techniques, Cholesterol, Collagen, Disease Models, Animal, Epithelial Cells parasitology, Epithelial Cells pathology, Humans, Intestinal Mucosa diagnostic imaging, Intestinal Mucosa pathology, Mice, Mice, Inbred C57BL, Host-Parasite Interactions physiology, Intestinal Mucosa metabolism, Intestinal Mucosa parasitology, Toxoplasma pathogenicity, Toxoplasma physiology
- Abstract
When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii . Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.
- Published
- 2019
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31. DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.
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Frau A, Kenny JG, Lenzi L, Campbell BJ, Ijaz UZ, Duckworth CA, Burkitt MD, Hall N, Anson J, Darby AC, and Probert CSJ
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- DNA Primers genetics, DNA, Fungal genetics, Feces microbiology, Humans, RNA, Ribosomal, 18S genetics, DNA, Fungal isolation & purification, Gastrointestinal Microbiome genetics
- Abstract
Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.
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- 2019
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32. Replication of Crohn's Disease Mucosal E. coli Isolates inside Macrophages Correlates with Resistance to Superoxide and Is Dependent on Macrophage NF-kappa B Activation.
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Tawfik A, Knight P, Duckworth CA, Pritchard DM, Rhodes JM, and Campbell BJ
- Abstract
Mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). CD isolates replicate within macrophages but the specificity of this effect for CD and its mechanism are unclear. Gentamicin exclusion assay was used to assess E. coli replication within J774.A1 murine macrophages. E. coli growth was assessed following acid, low-nutrient, nitrosative, oxidative and superoxide stress, mimicking the phagolysosome. Twelve of 16 CD E. coli isolates replicated >2-fold within J774.A1 macrophages; likewise for isolates from 6/7 urinary tract infection (UTI), 8/9 from healthy subjects, compared with 2/6 ulcerative colitis, 2/7 colorectal cancer and 0/3 laboratory strains. CD mucosal E. coli were tolerant of acidic, low-nutrient, nitrosative and oxidative stress. Replication within macrophages correlated strongly with tolerance to superoxide stress (rho = 0.44, p = 0.0009). Exemplar CD E. coli HM605 and LF82 were unable to survive within Nfκb1
-/- murine bone marrow-derived macrophages. In keeping with this, pre-incubation of macrophages with hydrocortisone (0.6 µM for 24 h) caused 70.49 ± 12.11% inhibition of intra-macrophage replication. Thus, CD mucosal E. coli commonly replicate inside macrophages, but so do some UTI and healthy subject strains. Replication correlates with resistance to superoxide and is highly dependent on macrophage NF-κB signalling. This may therefore be a good therapeutic target.- Published
- 2019
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33. Author Correction: Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration.
- Author
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Burkitt MD, Williams JM, Townsend T, Hough R, Duckworth CA, and Pritchard DM
- Abstract
Following the publication of this article [1], it was noted that the author list was incomplete and was missing the following author.
- Published
- 2019
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34. Stem cell models as an in vitro model for predictive toxicology.
- Author
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Lynch S, Pridgeon CS, Duckworth CA, Sharma P, Park BK, and Goldring CEP
- Subjects
- Animals, Gastrointestinal Tract drug effects, Heart drug effects, Humans, Liver drug effects, Models, Biological, Toxicological Phenomena, Drug-Related Side Effects and Adverse Reactions, Stem Cells drug effects
- Abstract
Adverse drug reactions (ADRs) are the unintended side effects of drugs. They are categorised as either predictable or unpredictable drug-induced injury and may be exhibited after a single or prolonged exposure to one or multiple compounds. Historically, toxicology studies rely heavily on animal models to understand and characterise the toxicity of novel compounds. However, animal models are imperfect proxies for human toxicity and there have been several high-profile cases of failure of animal models to predict human toxicity e.g. fialuridine, TGN1412 which highlight the need for improved predictive models of human toxicity. As a result, stem cell-derived models are under investigation as potential models for toxicity during early stages of drug development. Stem cells retain the genotype of the individual from which they were derived, offering the opportunity to model the reproducibility of rare phenotypes in vitro Differentiated 2D stem cell cultures have been investigated as models of hepato- and cardiotoxicity. However, insufficient maturity, particularly in the case of hepatocyte-like cells, means that their widespread use is not currently a feasible method to tackle the complex issues of off-target and often unpredictable toxicity of novel compounds. This review discusses the current state of the art for modelling clinically relevant toxicities, e.g. cardio- and hepatotoxicity, alongside the emerging need for modelling gastrointestinal toxicity and seeks to address whether stem cell technologies are a potential solution to increase the accuracy of ADR predictivity in humans., (© 2019 The Author(s).)
- Published
- 2019
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35. Potential role of fecal volatile organic compounds as biomarkers of chemically induced intestinal inflammation in mice.
- Author
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Reade S, Williams JM, Aggio R, Duckworth CA, Mahalhal A, Hough R, Pritchard DM, and Probert CS
- Subjects
- Acute Disease, Aldehydes analysis, Animals, Biomarkers analysis, Chronic Disease, Colitis chemically induced, Colitis pathology, Dextran Sulfate, Diarrhea chemically induced, Diarrhea metabolism, Disease Models, Animal, Female, Humans, Inflammatory Bowel Diseases diagnosis, Inflammatory Bowel Diseases metabolism, Metabolomics, Mice, Mice, Inbred C57BL, Colitis metabolism, Feces chemistry, Volatile Organic Compounds analysis
- Abstract
Metabolomics studies have the potential to discover biomarkers. Fecal volatile organic compounds (VOCs) have been found to differ in patients with inflammatory bowel disease and irritable bowel syndrome. Murine models of colitis offer an alternative to human studies in which diet can be controlled. We aimed to investigate fecal VOCs from mice in which acute and chronic colitis was induced. Groups of adult C57BL/6 mice underwent treatment with oral dextran sulfate sodium to induce colitis. Control mice received no treatment or had acute osmotic diarrhea induced with magnesium sulfate. Colitis was assessed clinically and by histology. Samples of feces and/or colon contents were collected and volatile compounds determined by solid phase microextraction-GC-MS. Statistics were performed using metabolomics tools. Acute colitis was associated with an increase in aldehydes and chronic colitis with one specific ketone. Osmotic diarrhea was associated with a significant reduction in VOCs, especially alcohols. We provide evidence that the identification of disease-associated VOC concentration ranges, combined with specific marker compounds, would potentially increase the likelihood of finding an inflammatory bowel disease-specific fecal VOC marker profile.-Reade, S., Williams, J. M., Aggio, R., Duckworth, C. A., Mahalhal, A., Hough, R., Pritchard, D. M., Probert, C. S., Potential role of fecal volatile organic compounds as biomarkers of chemically induced intestinal inflammation in mice.
- Published
- 2019
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36. Developing a 3D intestinal epithelium model for livestock species.
- Author
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Derricott H, Luu L, Fong WY, Hartley CS, Johnston LJ, Armstrong SD, Randle N, Duckworth CA, Campbell BJ, Wastling JM, and Coombes JL
- Subjects
- Animals, Cattle, Cell Differentiation, Cryopreservation, Livestock, Mice, Inbred C57BL, Organoids metabolism, Phenotype, Salmonella typhimurium physiology, Swine, Tissue Survival, Toxoplasma physiology, Cell Culture Techniques methods, Intestinal Mucosa metabolism
- Abstract
The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells' ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. 'Rounding' of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.
- Published
- 2019
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37. Correction: The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.
- Author
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Evans JP, Winiarski BK, Sutton PA, Jones RP, Ressel L, Duckworth CA, Pritchard DM, Lin ZX, Fretwell VL, Tweedle EM, Costello E, Goldring CE, Copple IM, Park BK, Palmer DH, and Kitteringham NR
- Abstract
[This corrects the article DOI: 10.18632/oncotarget.25497.].
- Published
- 2019
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38. Oral iron exacerbates colitis and influences the intestinal microbiome.
- Author
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Mahalhal A, Williams JM, Johnson S, Ellaby N, Duckworth CA, Burkitt MD, Liu X, Hold GL, Campbell BJ, Pritchard DM, and Probert CS
- Subjects
- Administration, Oral, Anemia microbiology, Anemia pathology, Animals, Colitis chemically induced, Colitis metabolism, Dextran Sulfate toxicity, Disease Models, Animal, Feces microbiology, Gastrointestinal Microbiome drug effects, Humans, Inflammatory Bowel Diseases microbiology, Inflammatory Bowel Diseases pathology, Mice, RNA, Ribosomal, 16S genetics, Anemia drug therapy, Colitis diet therapy, Inflammatory Bowel Diseases drug therapy, Iron metabolism, Iron, Dietary administration & dosage
- Abstract
Inflammatory bowel disease (IBD) is associated with anaemia and oral iron replacement to correct this can be problematic, intensifying inflammation and tissue damage. The intestinal microbiota also plays a key role in the pathogenesis of IBD, and iron supplementation likely influences gut bacterial diversity in patients with IBD. Here, we assessed the impact of dietary iron, using chow diets containing either 100, 200 or 400 ppm, fed ad libitum to adult female C57BL/6 mice in the presence or absence of colitis induced using dextran sulfate sodium (DSS), on (i) clinical and histological severity of acute DSS-induced colitis, and (ii) faecal microbial diversity, as assessed by sequencing the V4 region of 16S rRNA. Increasing or decreasing dietary iron concentration from the standard 200 ppm exacerbated both clinical and histological severity of DSS-induced colitis. DSS-treated mice provided only half the standard levels of iron ad libitum (i.e. chow containing 100 ppm iron) lost more body weight than those receiving double the amount of standard iron (i.e. 400 ppm); p<0.01. Faecal calprotectin levels were significantly increased in the presence of colitis in those consuming 100 ppm iron at day 8 (5.94-fold) versus day-10 group (4.14-fold) (p<0.05), and for the 400 ppm day-8 group (8.17-fold) versus day-10 group (4.44-fold) (p<0.001). In the presence of colitis, dietary iron at 400 ppm resulted in a significant reduction in faecal abundance of Firmicutes and Bacteroidetes, and increase of Proteobacteria, changes which were not observed with lower dietary intake of iron at 100 ppm. Overall, altering dietary iron intake exacerbated DSS-induced colitis; increasing the iron content of the diet also led to changes in intestinal bacteria diversity and composition after colitis was induced with DSS., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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39. The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.
- Author
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Evans JP, Winiarski BK, Sutton PA, Jones RP, Ressel L, Duckworth CA, Pritchard DM, Lin ZX, Fretwell VL, Tweedle EM, Costello E, Goldring CE, Copple IM, Park BK, Palmer DH, and Kitteringham NR
- Abstract
Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines in vitro and in an orthotopic syngeneic mouse model utilising bioluminescent imaging. Using a tissue microarray, Nrf2 was found to be overexpressed (p<0.01) in primary CRC and metastatic tissue relative to normal colon, with a positive correlation between Nrf2 expression in matched primary and metastatic samples. In vitro experiments in CRC cell lines revealed that Nrf2 siRNA and brusatol, which is known to inhibit Nrf2, decreased viability and sensitised cells to irinotecan toxicity. Furthermore, brusatol effectively abrogated CRC tumour growth in subcutaneously and orthotopically-allografted mice, resulting in an average 8-fold reduction in luminescence at the study end-point (p=0.02). Our results highlight Nrf2 as a promising drug target in the treatment of CRC., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.
- Published
- 2018
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40. Comparison of the human gastric microbiota in hypochlorhydric states arising as a result of Helicobacter pylori-induced atrophic gastritis, autoimmune atrophic gastritis and proton pump inhibitor use.
- Author
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Parsons BN, Ijaz UZ, D'Amore R, Burkitt MD, Eccles R, Lenzi L, Duckworth CA, Moore AR, Tiszlavicz L, Varro A, Hall N, and Pritchard DM
- Subjects
- Achlorhydria chemically induced, Achlorhydria etiology, Achlorhydria immunology, Adult, Aged, Autoimmune Diseases drug therapy, Autoimmune Diseases immunology, Autoimmune Diseases microbiology, Cluster Analysis, Cohort Studies, England epidemiology, Female, Gastric Mucosa drug effects, Gastric Mucosa immunology, Gastritis, Atrophic drug therapy, Gastritis, Atrophic immunology, Gastritis, Atrophic microbiology, Helicobacter Infections drug therapy, Helicobacter Infections immunology, Helicobacter Infections microbiology, Helicobacter pylori drug effects, Helicobacter pylori growth & development, Helicobacter pylori immunology, Helicobacter pylori isolation & purification, Hospitals, University, Humans, Male, Middle Aged, Proton Pump Inhibitors adverse effects, Proton Pump Inhibitors therapeutic use, Risk, Stomach Neoplasms epidemiology, Achlorhydria microbiology, Gastric Mucosa microbiology, Gastrointestinal Microbiome drug effects
- Abstract
Several conditions associated with reduced gastric acid secretion confer an altered risk of developing a gastric malignancy. Helicobacter pylori-induced atrophic gastritis predisposes to gastric adenocarcinoma, autoimmune atrophic gastritis is a precursor of type I gastric neuroendocrine tumours, whereas proton pump inhibitor (PPI) use does not affect stomach cancer risk. We hypothesised that each of these conditions was associated with specific alterations in the gastric microbiota and that this influenced subsequent tumour risk. 95 patients (in groups representing normal stomach, PPI treated, H. pylori gastritis, H. pylori-induced atrophic gastritis and autoimmune atrophic gastritis) were selected from a cohort of 1400. RNA extracted from gastric corpus biopsies was analysed using 16S rRNA sequencing (MiSeq). Samples from normal stomachs and patients treated with PPIs demonstrated similarly high microbial diversity. Patients with autoimmune atrophic gastritis also exhibited relatively high microbial diversity, but with samples dominated by Streptococcus. H. pylori colonisation was associated with decreased microbial diversity and reduced complexity of co-occurrence networks. H. pylori-induced atrophic gastritis resulted in lower bacterial abundances and diversity, whereas autoimmune atrophic gastritis resulted in greater bacterial abundance and equally high diversity compared to normal stomachs. Pathway analysis suggested that glucose-6-phospahte1-dehydrogenase and D-lactate dehydrogenase were over represented in H. pylori-induced atrophic gastritis versus autoimmune atrophic gastritis, and that both these groups showed increases in fumarate reductase. Autoimmune and H. pylori-induced atrophic gastritis were associated with different gastric microbial profiles. PPI treated patients showed relatively few alterations in the gastric microbiota compared to healthy subjects.
- Published
- 2017
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41. Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration.
- Author
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Burkitt MD, Williams JM, Townsend T, Hough R, Duckworth CA, and Pritchard DM
- Subjects
- Animals, Female, Gastric Mucosa pathology, Helicobacter Infections chemically induced, Helicobacter Infections genetics, Helicobacter Infections pathology, Helicobacter felis metabolism, Mice, Mice, Knockout, NF-kappa B p52 Subunit genetics, NF-kappa B p52 Subunit metabolism, Proto-Oncogene Proteins c-rel genetics, Proto-Oncogene Proteins c-rel metabolism, Signal Transduction drug effects, Signal Transduction genetics, Stomach Diseases chemically induced, Stomach Diseases genetics, Stomach Diseases pathology, Tamoxifen pharmacology, DNA Damage, Gastric Mucosa metabolism, Helicobacter Infections metabolism, NF-kappa B p50 Subunit deficiency, Stomach Diseases metabolism, Tamoxifen adverse effects
- Abstract
Tamoxifen (TAM) has recently been shown to cause acute gastric atrophy and metaplasia in mice. We have previously demonstrated that the outcome of Helicobacter felis infection, which induces similar gastric lesions in mice, is altered by deletion of specific NF-κB subunits. Nfkb1
-/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection. In contrast, Nfkb2-/- mice were protected from this pathology. We therefore hypothesized that gastric lesions induced by TAM may be similarly regulated by signaling via NF-κB subunits. Groups of five female C57BL/6 (WT), Nfkb1-/- , Nfkb2-/- and c-Rel-/- mice were administered 150 mg/kg TAM by IP injection. Seventy-two hours later, gastric corpus tissues were taken for quantitative histological assessment. In addition, groups of six female WT and Nfkb1-/- mice were exposed to 12 Gy γ-irradiation. Gastric epithelial apoptosis was quantified 6 and 48 h after irradiation. TAM induced gastric epithelial lesions in all strains of mice, but this was more severe in Nfkb1-/- mice than in WT mice. Nfkb1-/- mice exhibited more severe parietal cell loss than WT mice, had increased gastric epithelial expression of Ki67 and had an exaggerated gastric epithelial DNA damage response as quantified by γH2AX. To investigate whether the difference in gastric epithelial DNA damage response of Nfkb1-/- mice was unique to TAM-induced DNA damage or a generic consequence of DNA damage, we also assessed gastric epithelial apoptosis following γ-irradiation. Six hours after γ-irradiation, gastric epithelial apoptosis was increased in the gastric corpus and antrum of Nfkb1-/- mice. NF-κB1-mediated signaling regulates the development of gastric mucosal pathology following TAM administration. This is associated with an exaggerated gastric epithelial DNA damage response. This aberrant response appears to reflect a more generic sensitization of the gastric mucosa of Nfkb1-/- mice to DNA damage.- Published
- 2017
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42. Towards better models and mechanistic biomarkers for drug-induced gastrointestinal injury.
- Author
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Carr DF, Ayehunie S, Davies A, Duckworth CA, French S, Hall N, Hussain S, Mellor HR, Norris A, Park BK, Penrose A, Pritchard DM, Probert CS, Ramaiah S, Sadler C, Schmitt M, Shaw A, Sidaway JE, Vries RG, Wagoner M, and Pirmohamed M
- Subjects
- Animals, Biomarkers metabolism, Drug Design, Drug Discovery methods, Drug Evaluation, Preclinical methods, Gastrointestinal Diseases physiopathology, Humans, Toxicity Tests methods, Drug-Related Side Effects and Adverse Reactions diagnosis, Gastrointestinal Diseases chemically induced, Models, Biological
- Abstract
Adverse drug reactions affecting the gastrointestinal (GI) tract are a serious burden on patients, healthcare providers and the pharmaceutical industry. GI toxicity encompasses a range of pathologies in different parts of the GI tract. However, to date no specific mechanistic diagnostic/prognostic biomarkers or translatable pre-clinical models of GI toxicity exist. This review will cover the current knowledge of GI ADRs, existing biomarkers and models with potential application for toxicity screening/monitoring. We focus on the current gaps in our knowledge, the potential opportunities and recommend that a systematic approach is needed to identify mechanism-based GI biomarkers with potential for clinical translation., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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- View/download PDF
43. Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models.
- Author
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Burkitt MD, Duckworth CA, Williams JM, and Pritchard DM
- Subjects
- Animals, Disease Models, Animal, Humans, Stomach microbiology, Stomach pathology, Helicobacter Infections microbiology, Helicobacter Infections pathology, Helicobacter pylori physiology, Stomach Diseases microbiology, Stomach Diseases pathology
- Abstract
Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems., (© 2017. Published by The Company of Biologists Ltd.)
- Published
- 2017
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44. Importance of the alternative NF-κB activation pathway in inflammation-associated gastrointestinal carcinogenesis.
- Author
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Merga YJ, O'Hara A, Burkitt MD, Duckworth CA, Probert CS, Campbell BJ, and Pritchard DM
- Subjects
- Animals, Gastrointestinal Neoplasms complications, Gastrointestinal Neoplasms epidemiology, Humans, Inflammatory Bowel Diseases complications, NF-kappa B genetics, Gastrointestinal Neoplasms metabolism, Inflammatory Bowel Diseases metabolism, NF-kappa B metabolism, Signal Transduction
- Abstract
Chronic inflammation is a common factor in the development of many gastrointestinal malignancies. Examples include inflammatory bowel disease predisposing to colorectal cancer, Barrett's esophagus as a precursor of esophageal adenocarcinoma, and Helicobacter pylori-induced gastric cancer. The classical activation pathway of NF-κB signaling has been identified as regulating several sporadic and inflammation-associated gastrointestinal tract malignancies. Emerging evidence suggests that the alternative NF-κB signaling pathway also exerts a distinct influence on these processes. This review brings together current knowledge of the role of the alternative NF-κB signaling pathway in the gastrointestinal tract, with a particular emphasis on inflammation-associated cancer development., (Copyright © 2016 the American Physiological Society.)
- Published
- 2016
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45. Intestinal Preparation Techniques for Histological Analysis in the Mouse.
- Author
-
Williams JM, Duckworth CA, Vowell K, Burkitt MD, and Pritchard DM
- Subjects
- Animals, Models, Animal, Histological Techniques methods, Intestines, Mice
- Abstract
The murine intestinal tract represents a difficult organ system to study due to its long convoluted tubular structure, narrow diameter, and delicate mucosa which undergoes rapid changes after sampling prior to fixation. These features do not make for easy histological analysis as rapid fixation in situ, or after simple removal without careful dissection, results in poor postfixation tissue handling and limited options for high quality histological sections. Collecting meaningful quantitative data by analysis of this tissue is further complicated by the anatomical changes in structure along its length. This article describes two methods of intestinal sampling at necropsy that allow systematic histological analysis of the entire intestinal tract, either through examination of cross sections (circumferences) by the gut bundling technique or longitudinal sections by the adapted Swiss roll technique, together with basic methods for data collection. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)
- Published
- 2016
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46. Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis.
- Author
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Duckworth CA, Guimond SE, Sindrewicz P, Hughes AJ, French NS, Lian LY, Yates EA, Pritchard DM, Rhodes JM, Turnbull JE, and Yu LG
- Subjects
- Animals, Binding Sites, Blood Proteins, Cell Adhesion drug effects, Cell Line, Tumor, Circular Dichroism, Colonic Neoplasms pathology, Female, Galectin 3 blood, Galectin 3 metabolism, Galectins, Human Umbilical Vein Endothelial Cells, Humans, Lung Neoplasms secondary, Magnetic Resonance Imaging, Melanoma pathology, Mice, Mice, Inbred BALB C, Mice, Nude, Neovascularization, Pathologic drug therapy, Protein Binding, Protein Structure, Tertiary drug effects, Galectin 3 antagonists & inhibitors, Heparin analogs & derivatives, Heparin pharmacology, Lung Neoplasms drug therapy
- Abstract
Concentrations of circulating galectin-3, a metastasis promoter, are greatly increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these modified heparin derivatives and their ultra-low molecular weight sub-fractions had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives (with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections of the derivatives) abolished the circulating galectin-3-mediated increase in lung metastasis of human melanoma and colon cancer cells in nude mice. Structural analysis using nuclear magnetic resonance and synchrotron radiation circular dichroism spectroscopies showed that the modified heparin derivatives bind to the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified, non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding inhibitors with substantial potential as anti-metastasis/cancer drugs.
- Published
- 2015
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47. bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice.
- Author
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Duckworth CA, Abuderman AA, Burkitt MD, Williams JM, O'Reilly LA, and Pritchard DM
- Subjects
- Animals, Atrophy, Cell Differentiation genetics, Cytokines pharmacology, Female, Gastric Mucosa cytology, Gastric Mucosa metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Primary Cell Culture, Cell Proliferation genetics, Epithelium growth & development, Helicobacter Infections pathology, Helicobacter felis, Stomach cytology, Stomach pathology, bcl-2 Homologous Antagonist-Killer Protein genetics
- Abstract
Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1β-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection., (Copyright © 2015 the American Physiological Society.)
- Published
- 2015
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48. NF-κB1, NF-κB2 and c-Rel differentially regulate susceptibility to colitis-associated adenoma development in C57BL/6 mice.
- Author
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Burkitt MD, Hanedi AF, Duckworth CA, Williams JM, Tang JM, O'Reilly LA, Putoczki TL, Gerondakis S, Dimaline R, Caamano JH, and Pritchard DM
- Subjects
- Adenoma chemically induced, Adenoma etiology, Animals, Azoxymethane toxicity, Cell Transformation, Neoplastic metabolism, Colitis chemically induced, Colonic Neoplasms chemically induced, Colonic Neoplasms etiology, Cytokines metabolism, Dextran Sulfate toxicity, Disease Models, Animal, Disease Susceptibility, Epithelial Cells metabolism, Female, Inflammation, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Signal Transduction, Adenoma metabolism, Colitis complications, Colonic Neoplasms metabolism, NF-kappa B p50 Subunit metabolism, NF-kappa B p52 Subunit metabolism, Proto-Oncogene Proteins c-rel metabolism
- Abstract
NF-κB signalling is an important factor in the development of inflammation-associated cancers. Mouse models of Helicobacter-induced gastric cancer and colitis-associated colorectal cancer have demonstrated that classical NF-κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF-κB proteins, including NF-κB1/p50, NF-κB2/p52, and c-Rel, differentially regulate the development of gastric pre-neoplasia. To investigate the effect of NF-κB subunit loss on colitis-associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1(-/-), Nfkb2(-/-), and c-Rel(-/-) mice. Animals lacking the c-Rel subunit were more susceptible to colitis-associated cancer than wild-type mice, developing 3.5 times more colonic polyps per animal than wild-type mice. Nfkb2(-/-) mice were resistant to colitis-associated cancer, developing fewer polyps per colon than wild-type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2(-/-) mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c-Rel(-/-) mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild-type counterparts. These observations demonstrate different functions of specific NF-κB subunits in this model of colitis-associated carcinogenesis. NF-κB2/p52 is necessary for the development of colitis, whilst c-Rel-mediated signalling regulates colonic epithelial cell turnover following DNA damage., (© 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)
- Published
- 2015
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49. Murine Models of Helicobacter (pylori or felis)-associated Gastric Cancer.
- Author
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Duckworth CA, Burkitt MD, Williams JM, Parsons BN, Tang JMF, and Pritchard DM
- Subjects
- Adenocarcinoma drug therapy, Adenocarcinoma immunology, Adenocarcinoma pathology, Animals, Anti-Bacterial Agents therapeutic use, Antineoplastic Agents therapeutic use, Carcinogenesis drug effects, Carcinogenesis immunology, Carcinogenesis metabolism, Carcinogenesis pathology, Gastric Mucosa drug effects, Gastric Mucosa immunology, Gastric Mucosa microbiology, Gastric Mucosa pathology, Helicobacter Infections drug therapy, Helicobacter Infections immunology, Helicobacter Infections pathology, Helicobacter felis drug effects, Helicobacter felis immunology, Helicobacter pylori drug effects, Helicobacter pylori immunology, Humans, Mice, Paraneoplastic Syndromes drug therapy, Paraneoplastic Syndromes immunology, Paraneoplastic Syndromes pathology, Stomach Neoplasms drug therapy, Stomach Neoplasms immunology, Stomach Neoplasms pathology, Adenocarcinoma microbiology, Disease Models, Animal, Helicobacter Infections microbiology, Helicobacter felis pathogenicity, Helicobacter pylori pathogenicity, Paraneoplastic Syndromes microbiology, Stomach Neoplasms microbiology
- Abstract
Gastric adenocarcinoma is the fifth most common cancer and third most common cause of cancer-related death in the world. The majority of these cancers develop in genetically susceptible individuals who are chronically infected with the Gram-negative bacterium Helicobacter pylori. Often these individuals have also been exposed to certain environmental factors that increase susceptibility, such as dietary components. Murine models of Helicobacter-induced gastric cancer are valuable tools for investigating the mechanisms responsible for the stepwise pathological changes of chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric adenocarcinoma. Helicobacter felis colonization greatly accelerates the development of gastric neoplasia in mice, and causes pathologies similar to those observed with Helicobacter pylori-associated gastric carcinogenesis in humans. These mouse models are therefore useful for investigating genetic and environmental factors that may be involved in the pathogenesis and treatment of gastric cancer. Detailed in these protocols are procedures for inducing Helicobacter-associated carcinogenesis in mice as well as the histological analysis and interpretation of gastric pathology in these animals., (Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.)
- Published
- 2015
- Full Text
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50. Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip.
- Author
-
Williams JM, Duckworth CA, Burkitt MD, Watson AJ, Campbell BJ, and Pritchard DM
- Subjects
- Animals, Apoptosis physiology, Humans, Intestinal Mucosa cytology, Intestine, Small pathology, Mice, Microvilli pathology, NF-kappa B physiology, Tumor Necrosis Factor-alpha physiology, Intestinal Mucosa pathology
- Abstract
The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed., (© The Author(s) 2014.)
- Published
- 2015
- Full Text
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