Paul Durdey

The roles of enteric bacterial sialidase, sialate O-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or moreO-acetyl esters at positions C7–C9 on the sialic acids retarded the rate of hydrolysis. A specific sialateO-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and aKM of approximately 1mm sialateO-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and aKM of approximately 1mm.Faecal extracts from ulcerative colitis (UC) patients had higher sialateO-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged.Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acidO-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.

Colonic mucins in ulcerative colitis: evidence for loss of sulfation.

Colonic tissue obtained at surgery from control individuals and patients with ulcerative colitis was used to isolate mucins and to prepare mucin glycopolypeptides by pronase digestion. These were compared with mucins labelled with [35S] sulfate and [3H]-glucosamine after organ culture tissue samples from the same patients. A significant loss of mucin sulfation was detected in the colitis patients by both metabolic labelling and chemical analysis of the glycopolypeptides. A change in the size distribution of purified mucin oligosaccharides fractionated on BioGel P6 after release by β-elimination was seen in both radiolabelled and non-labelled colitis mucins compared with controls. Amino acid analysis of the glycopolypeptides showed a close similarity to the expected ratio of serine:threonine:proline for MUC2 and did not vary between control and colitis groups. Analysis of the mucins confirmed >90% purity in the labelling experiments, characteristic behaviour on density gradient centrifugation and agarose gel electrophoresis in control and ulcerative colitis groups and differences in sulfation and turnover at various sites in the normal colon.

Differential expression of the chromosome 11 mucin genes in colorectal cancer

The four secretory mucin genes clustered on chromosome 11, MUC2, MUC5AC, MUC5B and MUC6, were screened in 37 patients with cancers in the left hemi‐colon or rectum and 10 normal rectal controls. The mucin genes were detected by in situ hybridization using oligonucleotide probes to the variable number tandem repeat (VNTR) sequences, while the proteins were stained with non‐VNTR (MUC2, MUC5AC and MUC5B) or VNTR (MUC6) antibodies. Low levels of MUC2 mRNA were detected in non‐mucinous adenocarcinomas (5/27) while a higher proportion of mucinous carcinomas (4/9) was positive. All 25 cases of adjacent normal tissue expressed MUC2 mRNA. No transcripts for MUC5AC, MUC5B or MUC 6 were detected in any of these specimens. MUC2 protein product was detected immunohistochemically in 34/36 carcinoma specimens, with no change from normal controls. There was de novo expression of MUC5AC in 23/36 carcinomas. No MUC5B or MUC6 protein was detected. No difference in MUC2 and MUC5AC protein was found between mucinous and non‐mucinous carcinomas. The level of MUC2 was increased in moderately differentiated cancers compared with normal controls and decreased in the poorly differentiated group. Decreased MUC2 was found in poorly differentiated compared with moderately differentiated tumours. More MUC5AC protein was detected in well and moderately differentiated tumours than in poorly differentiated tumours and in all tumours relative to controls. The pattern of MUC2 staining in cancers was different from control tissue, with strong staining in the perinuclear region and none in goblet cell vesicles. MUC5AC staining was mainly detected in the cytoplasm. Poor detection of MUC2 and MUC5AC mRNA and associated strong staining for the total protein suggests altered biosynthesis and processing, leading to the characteristic subcellular distribution. Hence, change in the synthesis of MUC2 and the de novo appearance of MUC5AC in colorectal carcinomas may be significant events in the adenoma‐carcinoma sequence, with possible implications for tumour prognosis. Copyright

The association between referral source and stage of disease in patients with colorectal cancer

Objective  The colorectal fast track (FT) referral system was set up to ensure patients with suspected cases of colorectal cancer (CRC) received prompt access to specialized services. The aim of this study was to ascertain the association between referral source and the time it took to be seen by a colorectal surgeon to establish whether referral source had any association with the stage of disease at presentation in patients with CRC.

Abnormal subcellular distribution of mature MUC2 and de novo MUC5AC mucins in adenomas of the rectum: Immunohistochemical detection using non-VNTR antibodies to MUC2 and MUC5AC peptide

Anti-mucin variable number tandem repeat (VNTR) antibodies have been used previously to demonstrate the de novo presence of MUC5AC and MUC6 mucin in colorectal adenomas and increased synthesis of MUC2, the major secreted mucin in normal colorectal mucosa. Here we examined secreted mucins in tubular, tubulovillous and villous adenomas of the rectum using non-VNTR antibodies designed to assess mature mucin. Mucin gene messenger RNAs were detected by in situ hybridization. The anti-MUC2 non-VNTR antibody in the goblet cells of adenomas revealed a staining pattern of increased cytoplasmic, Golgi and membrane staining with no change in goblet vesicle reactivity compared with normal controls. In addition, blank goblet cell vesicle immunostaining for MUC2 was found in the transitional mucosa adjacent to all types of adenoma. Although a trend to overexpression of MUC2 was observed with in situ hybridization this was not detected with immunohistology. De novo synthesis of MUC5AC, but not MUC5B or MUC6 mucin was seen in all adenomas and transitional mucosa using immunohistochemistry. There was no correlation of MUC2 or MUC5AC mucin with polyp size or the grade of dysplasia using the non-VNTR antibodies.This study demonstrates that anti-mucin non-VNTR antibodies reveal a different subcellular-localization in rectal adenomas compared with normal colorectal mucosa. Further, this pattern is in contrast to that reported for anti-mucin VNTR antibodies. Combined use of these reagents may benefit future assessment of these cancers.

The association between referral source and outcome in patients with colorectal cancer

AIM The colorectal two-week wait fast track (FT) referral system was nationally implemented in the UK in 2000 to ensure patients with colorectal cancer (CRC) received prompt access to specialized services. The aim of this study was to determine the association between the mechanism of referral to colorectal services and the 5-year outcomes for patients with CRC. METHODS Consecutive patients with newly diagnosed CRC presenting between October 2002 and September 2004 were identified retrospectively. Analysis for survival and recurrence of disease at 5 years from presentation was undertaken. Outcomes for patients were compared between fast track (FT), non-fast track (NFT) and emergency referral (ER) routes, using Kaplan-Meier survival estimates. RESULTS Out of 189 patients, 96 (51%) presented via the FT, 41 (22.5%) via the NFT and 52 (27.5%) via the ER referral route. The 5-year overall survival was 52.6% ± 5.1, 41.5% ± 7.7 and 38.5% ± 6.7 for the FT-, NFT- and ER groups respectively (p = 0.075). The 5-year cancer specific survival was 60.3% ± 5.2, 58.8% ± 5.3 and 43.5% ± 7.2 for the FT-, NFT- and ER groups respectively (p = 0.056). Patients referred as emergencies had worse 5-year overall survival; 49.3% ± 4.3 (FT&NFT) vs. 38.5% ± 6.7 (ER) (p = 0.042) and 5-year cancer specific survival 59.8% ± 4.4 (FT&NFT) vs. 43.5% ± 7.2 (ER) (p = 0.016). A total of 136 patients (FT n = 71, NFT n = 34, ER n = 31) underwent potentially curative surgery. Differences in 5-year survival did not reach statistical significance in these patients. CONCLUSION Referral route to specialist services for patients with CRC via the fast track pathway compared to non-fast track pathway was not associated with improved survival.

Fast-track barium enema: meeting the two-week wait rule for patients with suspected colorectal cancer.

Objectives  To meet the introduction of the two‐week wait (TWW) rule for patients with suspected colorectal cancer, a fast‐track barium enema (FTBE) service was set up. This study was conducted to evaluate the success of this approach in preparation for meeting the forthcoming targets on waiting times to treatment from referral and diagnosis.

Mucin expression in the ileoanal reservoir reflects incomplete mucosal adaptation.

Restorative proctocolectomy is regarded as a standard surgical procedure for patients who require a proctocolectomy for ulcerative colitis and familial adenomatous polyposis. The ileal mucosa undergoes colonic phenotypic change with time, but the extent and relevance of these changes to the long‐term safety of the ileoanal pouch are unclear. The aim of this study was to study the mucin biology of this adaptive process in order to assess its extent and possible impact on pouch safety. Ileoanal pouch biopsies from a cohort of patients and normal ileal and colonic controls were subjected to histological, biochemical, histochemical, and immunohistochemical mucin analysis. Mucin sulphation and sialic acid O‐acetylation were studied as parameters of colonic phenotypic change. Fifty‐one patients, 16 ileal, and 22 colonic controls were studied. Seventy percent of biopsies retained villous mucosal architecture, with no cases of dysplasia detected. Ileoanal pouch mucosal sulphation and sialic acid O‐acetylation did not reach colonic levels, thus indicating limited evidence for a more colonic phenotype. The data from this study suggest that colonic phenotypic change within the ileoanal reservoir is incomplete, with no cases of dysplasia detected. The degree of phenotypic change is less than in previous studies, which may support, butnot prove, our hypothesis that there may be a process of reversion to an ileal type mucosa in the ileoanal reservoir with time. Copyright

Complete resolution of intractable pouchitis in an obese patient following laparoscopic gastric banding

A 46-year-old woman with known severe ulcerative colitis underwent a total colectomy and ileal pouch anal anastomosis (IPAA) in 1991. Following surgery, she suffered chronic diarrhoea and abdominal pain. She required several admissions to hospital with these episodes and, following lower gastrointestinal endoscopy and biopsy, was diagnosed with recurrent pouchitis. Steroids, antibiotics and motility agents failed to control her symptoms over many years. Recurrent exacerbations resulted in up to 20 episodes of diarrhoea per day, severely affecting her social life and mental well being. She became dependent upon codeine phosphate to maintain reasonable control of her bowel function and had sought psychiatric help on many occasions. Given the longevity and severity of her symptoms, pouch excision and end ileostomy formation were under consideration. Frequent courses of steroids, for both pouchitis and previous ulcerative colitis, had led to a significant weight gain and a body mass index of 41. Dieting and medication (Orlistat) proved to be ineffectual in decreasing her weight and she was referred to the local bariatric surgery unit for assessment. Her pouchitis had preceded the use of Orlistat. In 2005, she successfully underwent laparoscopic adjustable gastric banding. This had an almost immediate effect on reducing the frequency of bowel movements from a mean of 14 motions per day to two motions per day. By 2 years, she had lost 55 kg in weight, had stopped anti-motility drugs and to date has had no further episodes of pouchitis or required hospital admission. Her social and psychological well being has improved considerably.