S. G. M. Meuwissen

Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines

Oral administration of DSS has been reported to induce an acute and chronic colitis in mice. The aim of our study was to evaluate if the chronic phase of DSS‐induced colitis was characterized by a Th1/Th2 response and how this would relate to mucosal regeneration. Swiss Webster mice were fed 5% DSS in their drinking water for 7 days, followed by 2–5 weeks consumption of water. Control mice received only water. The animals were killed at 3 and 6 weeks after induction. Their colons were isolated for histology and immunohistochemistry, using specific MoAbs for T and B cells, macrophages, interferon‐gamma (IFN‐γ), IL‐4 and IL‐5. Colons were scored for inflammation, damage and regeneration. Two weeks after stopping DSS the colonic epithelium had only partially healed. Total colitis scores were still increased, especially in the distal colon, which was due to more inflammation, damage and less regeneration. In areas of incomplete colonic healing the basal parts of the lamina propria contained macrophages and CD4+ T cells. These CD4+ T cells showed a focal increase of IFN‐γ and IL‐4 staining compared with control animals. These findings were still observed 5 weeks after stopping DSS in some mice, albeit less extensive. Chronic DSS‐induced colitis is characterized by focal epithelial regeneration and a Th1 as well as Th2 cytokine profile. We postulate that chronic immune activation mediated by both populations of Th cells can interfere with colonic healing and can play a role in the pathogenesis of chronic colitis.

Secretion of Tumour Necrosis Factor α and Lymphotoxin α in Relation to Polymorphisms in the TNF Genes and HLA-DR Alleles. Relevance for Inflammatory Bowel Disease

The genes for tumour necrosis factor alpha (TNFα) and lymphotoxin alpha (LTα; TNFβ) are tandemly arranged in the central region of the MHC. They may, therefore, be of importance for the aetiology of MHC‐associated diseases. The authors have prospectively studied the secretion of TNFα and LTα in relation to polymorphisms at positions ‐308 and ‐238 in the TNFα gene (TNFA), and two polymorphisms in the first intron of the LTα gene (LTA), as well as HLA‐DR in 30 patients with chronic inflammatory bowel diseases (IBD) and 12 healthy controls. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF‐haplotypes: TNF‐C, ‐E, ‐H, ‐I, and ‐P. Significant associations between TNF haplotypes and TNFα and LTα secretion were found when PBMC were cultured with T‐cell activators, irrespective of disease. Mean TNFα secretion of individuals carrying the HLA‐DR3 associated TNF‐E haplotype was significantly higher, as compared to individuals without this haplotype (26 441 pg/ml versus 19 629 pg/ml; P = 0.014). Individuals carrying the TNF‐C haplotype produced the lowest amount of TNFα (17 408 pg/ml; P = 0.022). The TNF‐C and TNF‐E haplotypes differ only at position ‐308 in the promoter of TNFA. Individuals carrying the HLA‐DR1 associated TNF‐I haplotype produced significantly less LTα when compared to those who lack this haplotype (1979 pg/ml versus 3462 pg/ml; P = 0.006). As the TNF‐I haplotype is also associated with low TNFα secretion, this haplotype thus defines a ‘low secretor phenotype’. In conclusion, this is the first study to show associations between TNF haplotypes and TNFα and LTα secretion when T‐cell stimulators are used. These findings will contribute to define disease heterogeneity in IBD and may be of relevance for understanding the pathogenesis of autoimmune diseases.

Allelic polymorphism in IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) genes in inflammatory bowel disease

Recent reports have shown that allele 2 of the IL‐1 receptor antagonist (IL‐1Ra) gene is over‐represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL‐1Ra protein. Since the final outcome of the biological effects of IL‐1β may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL‐1β gene, which is relevant to IL‐1β protein production, may be involved in the genetic susceptibility to UC and Crohns disease (CD), in association with the established IL‐1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL‐1β and IL‐1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL‐1β gene and in intron 2 of the IL‐1Ra gene, were studied by polymerase chain reaction (PCR)‐based methods. The IL‐1β allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL‐1β gene polymorphism might participate synergistically with the IL‐1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non‐carriers of allele 2 of the genes encoding IL‐1β and IL‐1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fishers exact test, in the UC group (P= 0·023, OR = 2·81), as well as in the CD group (P= 0·01, OR = 3·79). Thus, non‐carriers of IL‐1β allele 2 were more often present in the subgroup of patients carrying the IL‐1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P= 1·00, OR = 0·92). These results suggest that the IL‐1β/IL‐1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases.

Distribution of four polymorphisms in the tumour necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD)

In 153 patients with IBD, 64 with Crohns disease (CD), and 89 with ulcerative colitis (UC), as well as in 54 healthy controls (HC), the frequencies of four known di‐allelic polymorphisms in the genes for TNF‐α and lymphotoxin alpha (LTα) were investigated. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF haplotypes: TNF‐C, ‐E, ‐H, ‐I, ‐P. Furthermore, the relation with the presence of perinuclear anti‐neutrophil cytoplasmic autoantibodies (P‐ANCA) was studied. A small, but statistically significant, association between the polymorphism at position ‐308 in the promoter region of the TNF‐α gene and UC was found. The frequency of the uncommon TNF‐α ‐308 allele 2 was found to be decreased in patients with UC compared with HC (allele frequency of allele 2 in UC patients 0±15 versus 0±25 in HC, P= 0±044). No significant differences in distribution of the TNF haplotypes were found between IBD patients and HC, although there was a tendency towards a higher frequency of the TNF‐C haplotype in UC patients compared with controls (haplotype frequency 22%versus 13%; P= 0±19). No statistically significant differences in distribution of the TNF haplotypes were observed between P‐ANCA‐positive and P‐ANCA‐negative UC patients. The strength of the associations indicates that TNF genes are not markers for the predisposition to suffer from IBD. They may, however, be markers of subsets of patients with UC and CD.

Hydrogen peroxide-enhanced transanal ultrasound in the assessment of fistula-in-ano

Appropriate classification of the fistulous tracts in patients with fistula-in-ano may be of value for the planning of proper surgery. Conventional transanal ultrasound has limited value in the visualization of fistulous tracts and their internal openings. Hydrogen peroxide can be used as a contrast medium for ultrasound to improve visualization of fistulas. PURPOSE: This prospective study evaluates hydrogen peroxide-enhanced ultrasound in comparison with physical examination, standard ultrasound, and surgery in the assessment of fistula-in-ano. METHODS: Twenty-one consecutive patients (4 women; mean age, 42 years) with fistula-in-ano were evaluated by local physical examination (inspection, probing, and digital examination), conventional ultrasound, and hydrogen peroxide-enhanced ultrasound before surgery. Ultrasound was performed using a B&K Diagnostic Ultrasound System™ with a 7-MHz rotating endoprobe. Hydrogen peroxide (3%) was infusedvia a small catheter into the fistula. The results of physical examination, ultrasound, and hydrogen peroxide-enhanced ultrasound were compared with surgical data as the criterion standard. The additive value of standard ultrasound and hydrogen peroxide-enhanced ultrasound compared with physical examination was also determined. RESULTS: At surgery, 8 intersphincteric and 11 transsphincteric fistulas and 2 sinus tracts (without an internal opening) were found. During physical examination, probing was incomplete in 13 patients, the diagnosis being correct in the other 8 patients (38%) as a low (intersphincteric or transsphincteric) fistula. With conventional ultrasound, the assessment of fistula-in-ano was correct in 13 patients (62%); defects in one or both sphincters could also be found (n=8). With hydrogen peroxide-enhanced ultrasound, the fistulous tract was classified correctly in 20 patients, the overall concordance with surgery being 95%. The internal opening was found at physical examination in 15 patients (71%), with hydrogen peroxide-enhanced ultrasound in 10 patients (48%), and during surgery in 19 patients (90%). Secondary extensions, confirmed during surgery, were found in five cases. In two patients, a secondary extension with hydrogen peroxide-enhanced ultrasound was not confirmed during surgery. Both patients developed a recurrent fistula. CONCLUSION: Hydrogen peroxide-enhanced ultrasound is superior to physical examination and standard ultrasound in delineating the anatomic course of perianal fistulas. It makes accurate preoperative assessment of the fistula possible and may be of value for the surgeon in planning therapeutic strategy.

Third degree obstetric perineal tears: risk factors and the preventive role of mediolateral episiotomy

Objective To determine risk factors for third degree obstetric perineal tears and to give recommendations for prevention.

Genetic markers in clinically well defined patients with ulcerative colitis (UC)

Results of genetic association studies in UC are conflicting. We propose that the power of candidate gene studies will increase when disease heterogeneity is taken into account. Phenotype frequencies of molecularly defined HLA‐DR alleles, polymorphisms in the tumour necrosis factor‐alpha (TNF‐α), lymphotoxin‐alpha (LT‐α), IL‐1 receptor antagonist (IL‐1Ra) and IL‐1β genes were determined in 98 clinically well characterized UC patients with a mean period of follow up of 10 years, and ethnically matched healthy controls (HC). The alleles HLA‐DRB1*0103 (phenotype frequency 6% versus 0.2%; P = 0.0002; odds ratio (OR) 27.6) and DRB1*15 (41% versus 26%; P = 0.001; OR = 2.0, compared with HC) were associated with overall disease susceptibility. Subgroup analysis revealed that DRB1*15 was only increased in females (53% versus 24%; P < 0.0001; OR = 3.5), but not in males. With regard to disease localization, all DRB1*0103+ patients had extensive disease (P < 0.002; OR = 33.5), and DRB1*15 was found in 59% of females with extensive colitis (P < 0.0001; OR = 4.4). DRB1*0103 was significantly increased in patients undergoing colectomy (P < 0.0002; OR = 84). No association between overall disease susceptibility and the cytokine gene polymorphisms were found. Subgroup analysis revealed several significant associations, but most did not retain significance when corrected for multiple comparisons. However, a noticeable finding was that haplotype TNF‐C was significantly associated with progression in extent of disease (P = 0.003, OR = 20.4). This study provides additional evidence for the role of DRB1 alleles in the susceptibility to UC, and supports the hypothesis that these alleles may determine the severity of the disease. The cytokine gene polymorphisms evaluated in this study do not seem to be strong risk factors for the overall disease susceptibility in UC, but may be involved in determining the severity of the disease.

Anal endosonography: Relationship with anal manometry and neurophysiologic tests

Thirty-seven patients were referred for evaluation of anal function; their clinical diagnoses were traumatic fecal incontinence (13), idiopathic (pudendal neuropathy) fecal incontinence (7), fecal soiling (9), and other (8). In all patients, anal endosonography (sphincter defects and internal sphincter thickness [IST]) and anal manometry (maximal basal pressure [MBP] and maximal squeeze pressure [MSP]) were performed. In 18 patients, neurophysiologic tests (EMG-maximal contraction pattern [MCP], single-fiber EMG [fiber density; FD], and pudendal nerve terminal motor latency [PNTML]) were also performed. Endosonography demonstrated in seven patients both an internal and external sphincter defect (Group 1), in seven patients an internal sphincter defect and in one patient an external sphincter defect (Group 2), and in 22 patients no sphincter defect (Group 3). There was a significant difference among these three groups for MBP and MCP, the lowest being in Group 1. Between the patients with traumatic fecal incontinence and idiopathic fecal incontinence, no differences in IST, MBP, MSP, MCP, FD, and PNTML were found. In two patients with a suspected obstetric trauma, there was an unexpected additional severe pudendal neuropathy. In one patient with a suspected obstetric trauma, no damage of the anal sphincters could be demonstrated. In one patient with suspected idiopathic fecal incontinence, there was an additional, unsuspected defect of the internal sphincter. There was concordance between endosonography and EMG in the mapping of the external sphincter. Clinical diagnoses can be misleading in differentiating between traumatic and idiopathic fecal incontinence; anal endosonography provides unsuspected and additional information about the sphincters; PNTML can reveal unsuspected neuropathy in traumatic fecal incontinence. Therefore, the combination of endosonography and PNTML is promising in selecting patients for surgery.

Serum Nitrate Levels in Ulcerative Colitis and Crohn's Disease

BACKGROUND Nitric oxide is an important mediator in inflammatory and autoimmune-mediated tissue destruction and may be of pathophysiologic importance in inflammatory bowel disease. We studied whether serum levels of nitrate, the stable end-product of nitric oxide, are increased in active Crohns disease or ulcerative colitis, in comparison with quiescent disease and healthy controls. The setting was the gastroenterology unit of the Free University Hospital, Amsterdam. METHODS In 146 patients--75 with ulcerative colitis and 71 with Crohns disease--and 33 controls serum nitrate was measured by the Griess reaction after enzymatic conversion of nitrate to nitrite with nitrate reductase. RESULTS Median serum nitrate concentrations did not differ statistically significantly between ulcerative colitis (median, 34.2 mumol/l; range, 15.6-229.4 mumol/l), Crohns disease (median 32.3 mumol; range 13.2-143.2 mumol/l), and healthy controls (median, 28.7 mumol/l; range, 13.0-108.4 mumol/l). However, when active ulcerative colitis patients (median, 44 mumol/l; range, 29.1-229.4 mumol/l were compared with inactive ulcerative colitis patients (median, 31.2 mumol/l; range, 15.6-59.7 mumol/l), a significant difference in nitrate concentration was found (p < 0.0001). A significant positive correlation was found between serum nitrate levels in ulcerative colitis and erythrocyte sedimentation rate (ESR) (r = 0.30, p - 0.01), leucocyte count (r = 0.27, p = 0.02), and thrombocyte count (r = 0.24, p = 0.04). Comparing active Crohns disease patients (median, 37.5 mumol/l; range, 13.2-143.2 mumol/l) with inactive Crohns disease patients (median, 31.3 mumol/l; range, 14.5-92.3 mumol/l) also showed a significant difference in serum nitrate concentration (p < 0.009). Serum nitrate levels correlated with the ESR (r = 0.26, p = 0.028) and serum albumin (r = 0.38, p = 0.004) as well. CONCLUSION Nitric oxide production is increased in both active ulcerative colitis and Crohns disease and may be implicated in the pathogenesis of inflammatory bowel disease.

Defecography in patients with anorectal disorders

To evaluate the results and clinical impact of defecography in patients with anorectal disorders, 100 results of defecographic examinations from 92 patients were reviewed. The defecographic results were screened for the anorectal angle, defined both at rest and during straining, perineal descent, and abnormalities of the rectal configuration during straining. Anal manometry, saline infusion test, rectal capacity measurement, and anal electromyography (EMG) were also performed. There was a significant difference (P<0.001) both at rest (22°) and during straining (12°) between the two anorectal angle measurements. Incontinent patients had a larger anorectal angle, both at rest and during straining, than continent patients (P<0.04), but with a large overlap. The anorectal angle was not influenced by gender or age. An abnormal rectal configuration was found in 62 defecographic examinations. From the 8 patients with rectopexy performed for a large rectocele or intussusception, incontinent patients with an intussusception had the best results. In four patients, anal EMG showed an increased activity of the external sphincter during straining. Two of these four patients had abnormal defecograhic results. No correlations were found between anorectal angle and the other function tests. In conclusion, the anorectal angle lacks clinical relevance. In patients with defecation problems, defecography may be indicated whenever other investigations (physical examination, anal manometry, anal EMG) have excluded local pathology or a spastic pelvic floor syndrome. In these situations, defecography could detect an intussusception, which could easily be treated with rectopexy.