Debabrata Majumdar

Keratins in colorectal epithelial function and disease

Keratins are the largest subgroup of intermediate filament proteins, which are an important constituent of the cellular cytoskeleton. The principally expressed keratins (K) of the intestinal epithelium are K8, K18 and K19. The specific keratin profile of a particular epithelium provides it with strength and integrity. In the colon, keratins have been shown to regulate electrolyte transport, likely by targeting ion transporters to their correct location in the colonocytes.

Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated cancer.

Background Keratins are intermediate filament (IF) proteins, which form part of the epithelial cytoskeleton and which have been implicated pathology of inflammatory bowel diseases (IBD). Methods In this study biopsies were obtained from IBD patients grouped by disease duration and subtype into eight categories based on cancer risk and inflammatory status: quiescent recent onset (<5 years) UC (ROUC); UC with primary sclerosing cholangitis; quiescent long-standing pancolitis (20–40 years) (LSPC); active colitis and non-inflamed proximal colonic mucosa; pancolitis with dysplasia-both dysplastic lesions (DT) and distal rectal mucosa (DR); control group without pathology. Alterations in IF protein composition across the groups were determined by quantitative proteomics. Key protein changes were validated by western immunoblotting and immunohistochemical analysis. Result Acute inflammation resulted in reduced K8, K18, K19 and VIM (all p<0.05) compared to controls and non inflamed mucosa; reduced levels of if– associated proteins were also seen in DT and DR. Increased levels of keratins in LSPC was noted relative to controls or ROUC (K8, K18, K19 and VIM, p<0.05). Multiple K8 forms were noted on immunoblotting, with K8 phosphorylation reduced in progressive disease along with an increase in VIM:K8 ratio. K8 levels and phosphorylation are reduced in acute inflammation but appear restored or elevated in subjects with clinical and endoscopic remission (LSPC) but not apparent in subjects with elevated risk of cancer. Conclusions These data suggest that keratin regulation in remission may influence subsequent cancer risk.

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography “shotgun” proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel‐free LC‐MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF‐7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.

PillCam® Colon Capsule is an Effective Tool in Management of Patients With Suspected or Known Inflammatory Bowel Disease: Preliminary Results From a UK Tertiary Care Centre

Introduction PillCam Colon capsule endoscopy (CCE) is a relatively new technique for imaging the colon. Its role in patients with inflammatory bowel disease (IBD) has not been fully explored. A recent European multicentre trial has shown that PillCam reactivated in the small bowel (SB) in about 98% of patients. Ileocolonic mucosal visualisation thus achieved by CCE may be a potential strategy in assessment of patients with known or suspected IBD. Methods Consecutive patients with proven or suspected IBD were included in the study. The CCE was performed as per standard protocol (with PEG and sodium phosphate as laxative agents). Patient demographics, clinical presentation and indication for CCE as well as nature and location of the findings, colonic transit time, complications and clinical implications of the findings, were analysed. Bowel cleanliness was described using a 4 point validated scale (1 – excellent, 2 – good, 3 – fair, 4 – poor). Data was expressed as median and IQR. Results 29 patients (86.2% females) with proven (n=10) or suspected IBD (n=19) underwent CCE between October 2007 and 2010. 41.4% of all patients had refused colonoscopy; 31% had a failed attempt while the rest were offered CCE as the initial imaging test. Median age was 44 (IQR 29–55.5) years. PillCam colon 1 and 2 were respectively used in 26 (89.7%) and 3 (11.3%) patients. CCE failed in 2 (6.9%) patients, all in the known IBD patients. Complete colonic mucosal visualisation was noted in 62.1% of all patients. 8 of patients with IBD had Crohn9s disease (CD) (n=4 Crohn9s colitis, n=3 both small (SB) and large bowel disease, n=1, only SB disease) while 2 had ulcerative colitis. 3 patients with CD had previously undergone surgery. The predominant indication was assessment of disease activity. In patients who had a successful test, mucosal visualisation enabled adequate assessment of disease activity in all (100%), thus guiding effective therapy. In suspected IBD patients, majority had diarrhoea (100%) and abdominal pain (63.2%). Pathology was detected in 63.2% of patients. SB Crohn9s was diagnosed in 4 (21.1%) patients while proctitis was noted in 1 (5.3%). Other significant findings included non-steroidal anti-inflammatory medication related injury (5.3%), colonic polyps (10.5%), angioectasia (21.1%) and diverticular disease (26.3%). Capsule transit times in the SB and colon were 71 (IQR 47–136) and 80 (IQR 48.5–216.5) min respectively; bowel cleanliness was 2 (IQR 2–3)). Conclusion In patients with proven or suspected IBD, CCE is an effective investigation modality. Its strengths lie in its minimally invasive nature coupled with its ability to assess disease activity thus guiding treatment strategies in most cases.

PTU-070 The fate of epithelial keratins in recent-onset and long-standing colitis in remission

Introduction Intermediate filaments (IF), which mainly consist of keratins (K), are one of the main components of the human cell cytoskeleton. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. We have previously shown increased expression of epithelial keratins in the IF fraction from long-standing UC (>20 years) in remission (LSPC) relative to recent –onset ulcerative colitis (≤5 years) in remission (ROUC) using proteomic iTRAQ-based analysis.1Whether this reduced expression is due to change in solubilisation or reduced total expression is unknown. We aimed to clarify the fate of epithelial keratins during UC disease evolution in the two cohorts. Method Soluble proteomes were extracted from individual biopsies in patients with LSPC in clinical, endoscopic and histological remission (n = 10) and ROUC in remission (n = 8). Median interval from last relapse to the index endoscopy in LSPC and ROUC was 31 and 1 month, respectively. Median histological activity index in both groups were 0 (range 0–1). An iTRAQ-compatible extraction protocol for soluble proteins was applied. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Validation of iTRAQ results was carried out with immunoblotting on pooled and individual samples using monoclonal antibody against K8. These outcomes were then compared with our previous insoluble proteome analyses.1 Results Comparative iTRAQ analysis of the soluble fraction showed no statistically significant log fold changes in K8, K18 and K19 levels in LSPC relative to ROUC and normal controls. Median relative K8 concentration in IF/soluble proteome fraction in individual LSPC and ROUC samples were 1.54/10.34 and 0.03/10.93, respectively. Relative K8 concentration was significantly higher in LSPC relative to ROUC in the IF fraction (p = 0.001), whereas the same comparison did not reach statistical significance for the soluble fraction (p = 0.82). Conclusion This study suggests relative total increase in expression of epithelial keratins in colonic epithelial cells during LSPC compared to the levels in ROUC. Comparing K8 IF and soluble bands in ROUC showes change in solubilisation relative to control, which in turn suggest effect of recent inflammation on increased keratin solubility in this group. Whether restoration of K8 is a marker of deep remission beyond histological and endoscopic remission needs to be further investigated. Disclosure of interest None Declared. Reference Corfe BM, Majumdar D, Assadsangabi A, Marsh AMR, Cross SS, Connolly JB, Evans CA, Lobo AJ. Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated-cancer. BMJ Open Gastroenterol., In press

PTU-069 The fate of epithelial keratins in active ulcerative colitis

Introduction Intermediate filaments (IF), which mainly consist of keratins (K), are one of the main components of the human cell cytoskeleton. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. Keratin alterations may play a role in the pathophysiology of ulcerative colitis (UC). We have previously shown reduced expression of Keratins in IF fraction from distal active UC relative to proximal inactive mucosa as well as controls using proteomic iTRAQ-based analysis.1Whether this reduced expression is due to change in solubilisation, reduced total expression and/or degradation, is unknown. We aimed to clarify the fate of epithelial keratins during UC disease activity. Method Soluble proteome fractions were extracted from rectal biopsies in patients (n = 10) with active distal colitis (ACT) as well as endoscopically and histologically uninflamed proximal colonic mucosa (INACT) in the same patients. Median histological activity index in ACT and INACT were 2 (range 1–3) and 0 (range 0). Control colonic biopsies (n = 7) from normal individuals were fractionated similarly. An iTRAQ-compatible extraction protocol for soluble proteins was developed and applied. Labelled peptides from pooled patients in each group were analysed by SCX-LC-MS/MS and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Validation of iTRAQ results was carried out with immunoblotting on pooled and individual samples using monoclonal antibody against K8. These outcomes were then compared with our previous insoluble proteome analyses.1 Results Comparative iTRAQ analysis of the soluble fraction showed significantly lower log fold changes in K8, K18 and K19 levels in ACT relative to INACT (0.42, 0.53, 0.42) and normal controls (0.61, 0.76, 0.41), respectively. The results were similar to our recent iTRAQ analysis of the IF fraction.1Immunoblotting further confirmed these findings: median relative K8 concentration in IF/soluble proteome fractions in individual ACT and INACT samples was 0.18/1 (p = 0.02) and 1.21/1.02 (p = 0.02), respectively. Conclusion This study suggests relative total reduction in expression of epithelial keratins in actively inflamed colonic mucosa of UC patients compared to un-inflamed proximal mucosa and normal controls. Keratins may have utility as a tissue biomarker of UC disease activity. Disclosure of interest None Declared. Reference Corfe BM, Majumdar D, Assadsangabi A, Marsh AMR, Cross SS, Connolly JB, Evans CA, Lobo AJ. Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated-cancer. BMJ Open Gastroenterol., In press

PWE-037 Panenteric Capsule Endoscopy: An Alternative Non-invasive Tool To Screen For Idiopathic Inflammatory Bowel Disease (ibd)

Introduction Compared to conventional endoscopy, capsule endoscopy (CE) is potentially safer, non-invasive, performed in out-patients and may be an alternative first line investigation in patients with suspected inflammatory bowel disease (IBD). In colon CE (CCE), a dormant mode (to save battery) is followed by device activation when small bowel mucosa is recognised. In this pilot study patients with suspected small and/or large bowel disease underwent a pan-enteric assessment using combined small bowel (SBCE) and CCE. Methods Patients underwent combined SCE and CCE using a novel protocol. Patients had new GI symptoms (group A: symptoms alone or those with additional abnormal results - GI symptoms plus) or underwent assessment of known IBD (group B). Main outcome measures: diagnostic yield (relevant findings only), complications, CE completion rates and colon cleanliness (scored 1–4: excellent to poor). Results Patients (group A, n = 56; group B, n = 26; mean age 41) had refused (50%), had incomplete (21%) prior colonoscopy or chose to have CCE (29%). Group A patients had diarrhoea (62%) and abdominal pain (54%); 17 had GI symptoms plus anaemia (13), acute phase response (9), hypoalbuminaemia (4), radiological abnormalities (3). Mean SBCE and CCE SB examination times: 255 and 92 mins respectively. Mean C examination time: 167mins; median cleanliness score 2. SBCE was complete in 73 (89%) and CCE in 58 patients (71%). In group B, pathology was identified in 62%, 16/26 (all active Crohn’s) which was significantly higher than in Group A (20%: 11/56, p = 0.0003). New diagnoses in Group A: Crohn’s disease (n = 5) and one each of NSAID colitis, proctitis, leiomyoma, angioectasia, diverticulae and idiopathic ulcerated small bowel stricture. 9/11 were in the symptoms plus group. 95% of pathology identified on SBCE was also identified on CCE. No complications were reported. Conclusion 62% of patients known to have IBD had active disease, but diagnostic yield was as high as 20% in those with new symptoms. IBD was the commonest and no complications occurred. Studies of the respective roles of faecal biomarkers, CE and histology in the diagnosis of IBD are needed. Almost all small bowel pathology was recognised by CCE suggesting its use as a remote panenteric endoscopic tool only awaits further battery development. Disclosure of Interest None Declared.

OC-091 Are There Changes in Keratin Expression Profile in Active Colitis and in Colitis Phenotypes Associated with Increased Cancer Risk?

Introduction Colitis in homozygous mK8-/- mice as a result of a primary epithelial defect and heterogeneous missense mutations in keratin (K) 8 and K18 in IBD suggest a possible association between simple epithelial keratins and IBD. Our previous work using mass spectrometry (MS) and western immunoblotting suggests alterations in the insoluble forms of K8, K18 and K19 in intestinal epithelial cells in colitis phenotypes compared to controls. We have previously shown an increase in insoluble keratin in epithelium from patients with longstanding pancolitis (LSPC) and decreased levels in epithelium from patients with dysplasia (in both the lesion and rectal mucosa), recent onset colitis (ROUC) and PSC associated colitis. There is also a reduction in insoluble keratins in active areas of inflammation compared to inactive areas. Methods Paired biopsies were taken from patients with active colitis (one from the actively inflamed area and another from a proximal inactive area, n = 10) and patients with dysplasia (biopsies taken from the dysplastic area and an area of rectal mucosa, n = 3). Single rectal biopsies were taken from patients with ROUC (n = 8), LSPC (n = 10) and PSC colitis (n = 7). These tissue sections underwent IHC staining for K8, K18 and K19. Semi-quantitative assessment of keratin expression was performed using a pixel counting algorithm and a manual scoring system, which scored staining intensity at the surface and base of the crypts and extent of crypt staining. Results There was no difference in K8 expression measured by manual scoring between the groups. K18 showed a significant difference in pixel count scores between the active and inactive inflammation (0.078 p = 0.022) reflecting a shift towards more extensive, moderate and weak staining in the active group. There was also a significant change in the manual scores for the ratio of surface intensity to crypt intensity (p = 0.028) representing a greater surface compared to crypt K18 expression. All other groups were similar in K18 expression and pattern. There was no difference in K19 expression measured by manual and pixel count scoring between the groups. Conclusion The results suggest a difference between MS and IHC approaches. If the IHC data hold true they may suggest that the changes in keratin expression profile that occur with duration of disease, inflammation and dysplasia may be a result of changes in keratin solubility rather than a change in the total expression of keratins. However, possible explanations include too small sample size for IHC and lack of sensitivity of IHC by comparison with MS. Disclosure of Interest None Declared

PWE-219 First reported experience of colon capsule endoscopy (CCE) in routine clinical practice

Introduction The advantages of colon capsule endoscopy (CCE) over other imaging modalities include the absence of intubation, sedation or irradiation. Recent multicentre trials suggest a sensitivity of approaching 90% in detecting significant polyps1 2 but there are no data regarding use in routine clinical practice. Methods Alternative modalities of colonic investigation were discussed with all patients requiring investigation. Data were collected prospectively on those undergoing colon capsule endoscopy following a standard bowel preparation. Small bowel patency was confirmed in patients with Crohns disease using the Agile patency system. Results 86 patients (67F; median age 42 (range 18–95)) underwent CCE (CC1, n=34; CC2, n=52). 81.4% had refused (n=43) or had had incomplete (n=27) colonoscopy. Indications: symptoms without alarm features (n=31), symptoms with alarm features (weight loss, bleeding, condition associated with malignancy; n=14), Crohns disease (n=17), symptoms with abnormal blood test results; n=15), anaemia (n=6), miscellaneous (n=3). CCE was complete in 79.5% (n=66), incomplete in 19.8% (n=17), 3.5% failed (one patient did not swallow the capsule; two provided no images). Median (range) time in the small and large bowel were 63.5 (0–424) and 121 (0–1020) min respectively and bowel cleanliness score 2 (1–4: excellent-poor). Findings were normal (31.4%), inflammatory bowel disease (IBD 25.6%: Crohns disease, n=13; ulcerative proctitis, n=1; NSAID colopathy, n=1; inflammation of uncertain significance, n=7), polyps (22.1%), diverticulosis (12.8%), angioectasia (5.8%), miscellaneous (3.5%), no images (3.5%). These were considered relevant to the indications in 25.6% (n=22, 15 of which were IBD). Outcomes included discharge (47.7%) and management change based on the findings (37.2%, including commencing (16.3%) or cessation (2.3%) of IBD therapy, further investigation (14.0%), advice regarding polyp surveillance (3.5%) and other treatment (2.3%). Half of the 20 patients with incomplete or failed studies were offered further investigations, six studies were considered sufficient to exclude organic disease, three showed active Crohns disease and one patient was too ill for further investigation. There were no complications. Conclusion CCE is an alternative for patients who refuse or have incomplete colonoscopy and which provides both small and large bowel visualisation. Although one in five studies were incomplete, sufficient information was provided to enable discharge in almost half the patients with functional bowel disorders and the identification of IBD in one quarter. Competing interests None declared. References 1. Eliakim R, Yassin K, Niv Y, et al. Prospective multicenter performance evaluation of the second-generation colon capsule compared with colonoscopy. Endoscopy 2009;41:1026–31. 2. Spada C, Hassan C, Munoz-Navas M, et al. Second-generation colon capsule endoscopy compared with colonoscopy. Gastrointest Endosc 2011;74:581–9.

PMO-249 Quantitative proteomic analysis of intermediate filament profile in ulcerative colitis reveals increased levels of keratins 8, 18 and 19 in patients with longstanding pan colitis which are reduced with development of dysplasia: Abstract PMO-249 Figure 1

Introduction Intermediate filaments (IF), principally keratins (K), are key components of epithelial cytoskeleton. K8, 18 and 19 are expressed in intestinal epithelial cells and play a role in cell-death signalling pathways, in particular apoptosis mediated by tumour necrosis factor-α. Reduced K8 and K20 expression is linked to epithelial-to-mesenchymal transition indicative of increased tumour aggressiveness. We investigated the change in levels of insoluble IF proteins in well-characterised groups of patients at differing risk of UC-associated cancer. Methods Rectal biopsies were obtained from patients with inactive UC with: (1) Long-standing (20–40 years) pancolitis (LSPC) (n=10); (2) Recent onset (<5 years) UC (ROUC) (n=8); (3) UC with primary sclerosing cholangitis (PSC) (n=7); (4) pancolitis with dysplasia (n=4) and 10 controls, with additional biopsies from dysplastic/neoplastic lesions and snap frozen. An iTRAQ (isobaric tagging for relative and absolute quantification)-compatible extraction and solubilisation protocol for IF proteins was developed. Labelled peptides from pooled patients were analysed by SCX-LC-MS/MS (strong cation exchange-reverse phase HPLC tandem mass spectrometry) and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Results Tandem mass spectrometry (MS/MS) identified 52 proteins; 32 (61.5%) were matched by two or more peptides. Abstract PMO-249 figure 1A shows the log fold change in IF levels compared to control, with significant increase in levels of K8, K19 and vimentin in those with LSPC, but marked reduction in IF levels in areas of dysplasia (DT) and rectal mucosa distant from this (DR). Marked increase in levels of keratins was noted in patients with LSPC compared to those with ROUC (Abstract PMO-249 figure 1B), suggesting an effect of disease duration on IF levels.Abstract PMO-249 Figure 1 Tandem mass spectrometry results showing significant log fold changes (p<0.05) in IF levels. Conclusion This is the first study using a quantitative proteomic approach with an iTRAQ based proteomic workflow to analyse changes in IF levels in patients with UC with differing colon cancer risk. LSPC is associated with enhanced mucosal levels of keratins, spectrin and xin, which is reduced in dysplasia and in distant rectal mucosa of those with dysplasia—suggesting a field change. These changes need further characterisation including of post-translational modifications, which may help better understanding of the pathogenesis of colitis associated cancer. Competing interests None declared.