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Background Long-term pouch surveillance outcomes for familial adenomatous polyposis (FAP) are unknown. We aimed to quantify surveillance outcomes and to determine which of selected possible predictive factors are associated with pouch dysplasia. Methods Retrospective analysis of collected data on 249 patients was performed, analyzing potential risk factors for the development of adenomas or advanced lesions ( ≥ 10 mm/high grade dysplasia (HGD)/cancer) in the pouch body and cuff using Cox proportional hazards models. Kaplan–Meier analyses included landmark time-point analyses at 10 years after surgery to predict the future risk of advanced lesions. Results Of 249 patients, 76 % developed at least one pouch body adenoma, with 16 % developing an advanced pouch body lesion; 18 % developed an advanced cuff lesion. Kaplan–Meier analysis showed a 10-year lag before most advanced lesions developed; cumulative incidence of 2.8 % and 6.4 % at 10 years in the pouch body and cuff, respectively. Landmark analysis suggested the presence of adenomas prior to the 10-year point was associated with subsequent development of advanced lesions in the pouch body (hazard ratio [HR] 4.8, 95 %CI 1.6–14.1; P = 0.004) and cuff (HR 6.8, 95 %CI 2.5–18.3; P Conclusions Pouch adenoma progression is slow and most advanced lesions occur after 10 years. HGD and cancer were rare events. Pouch phenotype in the first decade is associated with the future risk of developing advanced lesions and may guide personalized surveillance beyond 10 years.

INTRODUCTION: Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli gene that results in intestinal adenoma formation and colorectal cancer, necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis. METHODS: Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labeled with anti-IgA/IgG and analyzed by flow cytometry. RESULTS: Proportions of resident memory CD103-expressing CD8 + and γδ T-cell receptor + intraepithelial lymphocytes were dramatically reduced in both the left and right colon of patients with FAP compared with healthy controls. In lamina propria, T cells expressed less CD103, and CD4 + CD103 + cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA + peripheral B cells, and CD4 T-cell memory responses to commensal bacteria were increased in FAP. DISCUSSION: Loss of resident memory T cells and γδ T cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this precancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function, and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

Background Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here, we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC). Results In healthy individuals, Bt OMVs stimulated significant (p < 0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p < 0.001 and p < 0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p < 0.01 and p < 0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). Conclusions Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients.

ObjectivesFamilial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli (APC) gene that results in intestinal adenoma formation and colorectal cancer (CRC), necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis.MethodsColonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labelled with anti-IgA/IgG and analyzed by flow cytometry.ResultsProportions of resident memory CD103-expressing CD8+ and γδ T cell receptor+ intraepithelial lymphocytes were dramatically reduced in both left and right colon of patients with FAP compared to healthy controls. In lamina propria, T-cells expressed less CD103 and CD4+ CD103+ cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA+ peripheral B cells and CD4 T-cell memory responses to commensal bacteria were increased in FAP.ConclusionsLoss of resident memory T-cells and γδ T-cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this pre-cancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli gene that results in intestinal adenoma formation and colorectal cancer, necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis.Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labeled with anti-IgA/IgG and analyzed by flow cytometry.Proportions of resident memory CD103-expressing CD8 + and γδ T-cell receptor + intraepithelial lymphocytes were dramatically reduced in both the left and right colon of patients with FAP compared with healthy controls. In lamina propria, T cells expressed less CD103, and CD4 + CD103 + cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA + peripheral B cells, and CD4 T-cell memory responses to commensal bacteria were increased in FAP.Loss of resident memory T cells and γδ T cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this precancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function, and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

We sought methods of rectifying microbiota:immune dysregulation as a route to prophylaxis and improved immunotherapy of colorectal cancer (CRC). CRC develops in gut epithelium, accompanied by low level inflammatory signaling, intestinal microbial dysbiosis and immune dysfunction. We examined populations of intraepithelial lymphocytes in non-affected colonic mucosa of CRC and healthy donors and circulating immune memory to commensal bacterial species and yeasts. Colonic tissue in CRC was significantly depleted of γδ T cells and resident memory T cells, populations with a regulatory CD39-expressing phenotype. T cell memory responses to a panel of commensals were distinct in CRC, while B cell memory responses to several bacteria/yeast were significantly increased, accompanied by increased proportions of effector memory B cells, transitional B cells and plasmablasts in blood. IgA responses to mucosal microbes were unchanged. Our data describe a novel immune signature with similarities to and differences from that of inflammatory bowel disease. They implicate B cell dysregulation as a potential contributor to parainflammation and identify pathways of weakened barrier function and tumor surveillance in CRC-susceptible individuals.

Additional file 1: Supplementary Figure 1. Bt OMVs induce diverse cytokines from whole colon tissue and marked IL-10 response from colonic LP mDC. Supplementary Figure 2. Plasmacytoid DC cytokine response to Bt and Bt OMVs in healthy controls. Supplementary Figure 3. Loss of CD103+ DC in UC and CD colonic LP. Supplementary Figure 4. Bt OMVs do not induce IL-10-expressing mDC in colonic LP in ulcerative colitis.

Background and Aims The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD]. Methods Resident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed. Results Systemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses. Conclusions A previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology.

Introduction There is limited experience of endoscopic submucosal dissection (ESD) for resection of colorectal lesions in the West and outcome data tends to be worse than that reported from Japanese centres. We report the outcomes of ESD in a single, tertiary UK referral centre. Methods A prospective database was analysed including 165 consecutive patients (mean age: 64.6±12.6 years, 62.4% males) with 173 colorectal neoplasms resected by ESD between 3/2012 and 12/2017. Two experienced colonoscopists performed all procedures. Results The median (IQR) lesion size was 3.5 cm (2–5), and 140 (80.9%) were located in the rectum. Overall, 49.7% were granular-type laterally spreading tumours (LST), 19.7% were non-granular LST, and 30.6% were polypoid lesions. In 29 (16.7%) cases a flexible endosurgical platform was used to assist ESD of complex rectal polyps [median(IQR) size: 6 cm (5–8)] by dynamic trans-anal retraction (Trans-Anal Submucosal Endoscopic Resection; TASER). Histology showed low-grade adenoma/dysplasia in 83 (47.9%), high-grade adenoma/dysplasia in 52 (30%), T1 cancer ( Conclusions The current study demonstrates favourable clinical outcomes of selected colorectal ESD in a Western endoscopy setting. Further studies addressing the cost-effectiveness of ESD and comparing its long-term outcome with endoscopic mucosal resection in the West are needed.

Objective Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. Design Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. Results A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1β) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4 + FoxP3 + IL-10 + (regulatory) T cells . There were enhanced proportions of CD103 + Sirpα − DC in the colon, with increased proportions of CD103 + Sirpα + DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103 + Sirpα + DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103 + DC, in particular CD103 + Sirpα + DC. However, expression of ILT3 was associated with CD103 − DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. Conclusions The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

This is an open access article under the terms of the Creative Commons Attribution License.-- et al., [Scope]: The human/microbiota cross-talk is partially mediated by bacteria-derived peptides like Serine-Threonine peptide (STp), which is resistant to gut proteolysis, is found in the human healthy colon and induces regulatory properties on gut dendritic cells (DCs); here we characterized human gut DC in ulcerative colitis (UC) patients and studied the effect of STp on their properties. [Methods and results]: Human colonic DC from healthy controls and UC patients were isolated, conditioned for 24 h +/- STp and characterized by flow cytometry, immunohistochemistry, and electron microscopy. Expression of immature DC markers DC-SIGN and ILT3, and Toll-like receptors were increased on gut UC-DC. Langerin (involved in phagocytosis), lymph node homing marker CCR7, and activation markers CD40/CD80/CD86 were decreased in UC. Gut DC had restricted stimulatory capacity for T-cells in UC. Conditioning of DC with STp in vitro reduced Toll-like receptor expression, increased CD40 and CD80 expression, and restored their stimulatory capacity. [Conclusion]: Colonic DCs display an abnormal immature phenotype in UC, which was partially restored following STp treatment. Bacteria-derived metabolites, like STp, seem to have a role in gut homeostasis that is missing in UC so they might lead a new era of probiotic products setting the basis for nondrug dietary therapy in inflammatory bowel disease. © 2013 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim., The author(s) gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC), this research was funded by the BBSRC Institute Strategic Programme for Gut Health and Food Safety BB/J004529/1. The authors also thank the St Mark’s Hospital Foundation, The Association for International Cancer Research (AICR), Scotland, and the Junta de Castilla y León (GRS175/B/07).

Summary Human monocyte-derived dendritic cells (DC) (MoDC) are utilized for immunotherapy. However, in-vitro immunological effects are often not mirrored in vivo. We studied the tissue-homing potential of MoDC. Circulating monocytes and DC expressed different tissue-homing markers and, during in-vitro development of MoDC, homing marker expression was lost resulting in a ‘homeless’ phenotype. Retinoic acid (RA) induced gut-homing markers (β7 and CCR9) and a regulatory phenotype and function [decreased human leucocyte antigen D-related (HLA-DR) and increased ILT3 and fluorescein isothiocyanate (FITC-dextran uptake) in MoDC]. RA-MoDC were less stimulatory and primed conditioned T cells with a gut-homing profile (β7+CLA−). Unlike the normal intestinal microenvironment, that from inflamed colon of ulcerative colitis (UC) patients did not induce regulatory properties in MoDC. However, RA-MoDC maintained their regulatory gut-specific properties even in the presence of UC microenvironment. Therefore, MoDC may be ineffectual for immunotherapy because they lack tissue-homing and tissue-imprinting specificity. However, MoDC rehabilitation with gut-homing potential by RA could be useful in promoting immunotherapy in pathologies such as UC.

Open Access funded by Biotechnology and Biological Sciences Research Council. Under a Creative Commons license.-- et al., [Background & Aims]: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103 DC specialize in generating immune tolerance with the functionality of CD11b subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. [Methods]: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. [Results]: Colonic DC identified were myeloid (mDC, CD11cCD123) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103SIRPα DC were the major population and with CD103SIRPα DC were CD1cILT3CCR2 (although CCR2 was not expressed on all CD103SIRPα DC). CD103SIRPα DC constituted a minor subset that were CD141ILT3CCR2. Proximal colon samples had higher total DC counts and fewer CD103SIRPα cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4CD45RA T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c, but not CD141 mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. [Conclusions]: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization., This study was funded by the Biotechnology and Biological Sciences Research Council, BBSRC Institute Strategic Programme for Gut Health and Food Safety, grant BB/J004529/1; 16S rRNA gene sequencing was provided by the Wellcome Trust (grant number WT 098051) (to P.S., J.P., A.W.W.); core funding support from the Scottish Government Rural and Environmental Science and Analysis Service (to A.W.W. and the Rowett Institute of Nutrition and Health, University of Aberdeen); the Association for International Cancer Research (AICR), Scotland, grant number 120234 (to H.O.A.); and the Junta de Castilla y León, Spain (SAN196/VA16/07 and SAN196/VA17/07).

month 12.: 944.03±260.50; NS). There was strong correlation between ghrelin levels and hemoglobin-A1c levels (r = 0.74). Ghrelin concentration does not correlated neither to clinical activity indexes (partial Mayo score: r = 0.391; Crohn’s disease activity index: r = 0.120457), nor inflammatory parameters (Creactive protein: r = 0.11). Conclusions: Anti-TNF-alpha therapy improves insulin resistance in IBD patients. Improving metabolic parameters of patients treated with anti-TNF agents is an additional benefit of this kind of medications in IBD.

In inflammatory bowel disease (IBD) models, the body's innate and adaptive immune systems influence differentiation of multipotent gut stem cells. These stem cells differentiate into mucous producing goblet cells, enterocytes, enteroendocrine cells or Paneth cells. Differentiation related to inflammatory stress has been well studied in the lung and liver but gut stem cell differentiation in IBD has only a small body of literature. Hypothesis: Changing environments in the TNBS mouse model of chronic colitis modulate the lineage of colonic mucosal stem cells. Methods: BALB/c mice were given weekly colonic exposure to TNBS, to induce colitis, or saline as a control. Colon, spleen and mesenteric lymph nodes were collected from groups of mice (n=4-5) every other week from 5-17 weeks(W). Sections of tissue were prepared for microscopic, protein and real-time PCR (RT-PCR) analysis. Colonic muscle segments were isolated to assess smooth muscle contractility. BrdU was administered to each mouse 2 hours prior to harvest. Results: Mircoscopic analysis of TNBS-treated colon demonstrates increased submucosal cellularity and collagen deposition at 5W. This cellularity decreases but the collagen deposition is unchanged and is increasingly organized (strands) in the subsequent study intervals. During this time there was a 25% increase in thickness of the colonic mucosa and corresponding increase in the number of BdrU positive cells that peak at 15W. There are prominent goblet cells and significant mucous production associated with elevated levels of IL-13 and MUC2 and low levels of IL-2, IL-17, TGF β1 and TNFα. From 13-17W there was marked disruption of normal mucosal architecture. BrdU positive crypt cells drop precipitously at 17W coincident with a decrease in goblet cells and an increase in lymphocytic and stromal cells in the lamina propria. At this time there is a marked upregulation in IL-17, TNFα and IFNγ. Conclusions: The development of chronic inflammation is a transition phasing response to a changing microenvironment in the chronic mouse model. First, an acute inflammatory response is apparent at 5W. This is followed by a second mucosal proliferative phase characterized by proliferation of goblet cells due to elevated levels of IL-13. There is suppression of fibrosis during this period. The 3rd phase, evolving between 13-17W, is marked by architectural disruption, an increase in proinflammatory cytokines, a dramatic decrease in crypt goblet cell replication and a dramatic increase in stromal cellularity and fibrosis.

There is a gap in the formal assessment of technical skills in polypectomy that is now considered an integral part of colonoscopy. Polypectomy has been shown to reduce the incidence of colorectal cancer but does have associated complications. Polypectomy competency assessment should arguably be a part of the certification process for all endoscopists. A polypectomy competency assessment tool (Direct Observation of Polypectomy Skills [DOPyS]) has been developed and its reliability examined. This study examined the ability of the DOPyS to reliably distinguish between endoscopists with different levels of experience, ie, its construct validity.To determine the construct validity of the DOPyS.Videos of 32 polypectomies (endoscopic view only) were collected from 2 expert (1000 colonoscopies) endoscopists (17 polyps) and 6 intermediate-level (100-500 colonoscopies) endoscopists (15 polyps). The videos were edited to include only the entire polypectomy procedure, arranged in random order, and assessed blindly by 4 experienced endoscopists, only 2 of whom were familiar with polypectomy assessment by using the DOPyS before scoring. The differences in overall competency scores (range 1-4; competency, scores ≥ 3) for the expert and intermediate groups were compared by using the Fisher exact test.Single center.The analysis suggested that both trained assessors familiar with the DOPyS could reliably distinguish between the expert and intermediate endoscopists (P = .049 and P.001), with the expert group scoring higher than the intermediate one. For the assessors with no previous experience of the DOPyS, no such difference could be seen (P = .71 and P = .15).Small sample and polyp size.The results of the analysis suggested that the DOPyS could reliably differentiate between polypectomies performed by endoscopists of different levels of experience, but only if the assessors were trained in the use of the assessment tool. Training is therefore required to use this tool reliably.

Introduction There is a void in the formal assessment of technical skills in polypectomy which is now considered an integral part of colonoscopy. It has been shown to reduce the incidence of colorectal cancer but does have associated complications. Polypectomy competency assessment should arguably be a part of the certification process for all endoscopists. A polypectomy competency assessment tool (Direct Observation of Polypectomy Skills-DOPyS) has been developed and its construct validity, examined. Methods To determine construct validity of the DOPyS. Thirty-two polypectomy videos (incorporating the endoscopic view only) were collected from real patient procedures performed by two experienced (>1000 colonoscopies) endoscopists (17 videos) and six intermediate level (100–500 colonoscopies) endoscopists (15 videos). The videos were edited to include only the entire polypectomy procedure, arranged in random order, and assessed blindly by two experienced endoscopy assessors. Both assessors underwent a polypectomy assessment training session using the DOPyS prior to scoring. The DOPyS incorporates a 33-point procedure-specific checklist and an overall competency score. The differences in overall competency scores (range 1–4; scores ≥3=competency) for the expert and intermediate groups were compared, and considered significant (p value Results Conclusion The results of the analysis suggested that both blinded assessors were able to reliably differentiate between endoscopists of varying experience using the DOPyS (the expert group had higher scores than the intermediate group). Large-scale validation studies are required to further strengthen evidence for the use of this tool for routine polypectomy competency assessment. Competing interests None.