Amado Salvador Peña

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.

A TaqI polymorphism in the 3′UTR of the IL-12 p40 gene correlates with increased IL-12 secretion

Interleukin-12 (IL-12) is a key cytokine for the induction of Th1 immune responses. We evaluated whether a TaqI polymorphism in the 3′UTR of the IL-12 p40 gene affects secretion of IL-12 in vitro, and whether this polymorphism is associated with susceptibility to Crohns disease (CD). IL-12 p40 and p70 secretion by monocytes in relation to genotype was determined in 63 healthy donors. Genotype and allele frequencies of the TaqI polymorphism in 150 CD patients were compared with 145 ethnically matched healthy controls (HC). No significant association was found between genotype and IL-12 p40 secretion after stimulation of monocytes with SAC+IFNγ. In contrast, increasing IL-12 p70 secretion was found across the categories of non-carriers, heterozygotes and homozygotes for the variant allele (median values±SEM: 147±27, 282±51 and 482±34 pg/ml, respectively; P<0.005). The allele and genotype frequencies of this polymorphism in patients with CD did not differ statistically significantly from HC. The presence of a TaqI polymorphism in the IL12 p40 3′UTR correlates with increased in vitro IL-12 p70, but not p40 secretion. While this polymorphism does not appear to be correlated with susceptibility to CD in the limited population of patients tested here, it could influence the occurrence of the disease in certain subsets of patients.

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.

The polymorphic IL-1B and IL-1RN genes in the aetiopathogenesis of peptic ulcer

Besides environmental factors, the genetic background of an individual may contribute to the development and final outcome of peptic ulcer disease. Interleukin‐1β (IL‐1β) and the interleukin‐1 receptor antagonist (IL‐1ra) are cytokines that play a key role in modulating the inflammatory response in the gastrointestinal mucosa. This study aimed to investigate whether polymorphisms in the IL‐1B and IL‐RN genes are involved in the susceptibility to and final outcome of peptic ulcer disease. DNA from 179 unrelated Spanish Caucasian patients with peptic ulcer diseases and 99 ethnically matched healthy controls was typed for the TaqI polymorphism at position + 3954 in the IL‐1B gene and the variable number of tandem repeats polymorphism in intron 2 of the IL‐1RN gene. The determination of Helicobacter pylori status and non‐steroidal anti‐inflammatory drug (NSAIDs) use was studied in all patients and in controls. H. pylori infection and NSAID use were more frequent in ulcer patients than in controls. There were no significant differences in carriage rate, genotype and allele frequencies of the IL‐1RN and the IL‐1B+3954 gene polymorphisms between peptic ulcer patients and controls. However, a strong allelic association between IL‐1B and IL‐1RN genes was found in duodenal ulcer patients (P < 0·0006). Logistic regression identified H. pylori infection and NSAIDs use as independent risk factors for peptic ulcer diseases whereas the simultaneous carriage of IL‐1B+3954 allele 2 and IL‐1RN allele 2 was associated with reduced risk for duodenal ulcer disease (OR: 0·37, 95% CI = 0·14–0·9). Our data suggest that IL‐1B and IL‐1RN genes in addition to bacterial and environmental factors play a key role in determining the final outcome of peptic ulcer disease.

Prevalence of mutations of the NOD2/CARD15 gene and relation to phenotype in Spanish patients with Crohn disease

Background: We assessed the prevalence of R702W, G908R, and L1007fs coding mutations in the NOD2/CARD15 gene and the genotype–phenotype relation in Spanish patients with Crohn disease. Methods: A cohort of 204 unrelated patients with Crohn disease and 140 healthy controls were studied. The phenotype was established before commencement of genotyping. Genotyping of the R702W, G908R, and L1007fs gene polymorphisms of NOD2/CARD15 was performed by two independent laboratories using different techniques. In the case of discordant results, specific sequencing of DNA strands was performed. Results: At least one mutation was present in 32.8% of patients compared to 10.7% in controls (OR = 4.08, 95% CI 2.21 to 7.50). In patients with Crohn disease, the frequency of R702W, G908R, and L1007fs carriers was 13.7%, 8.3%, and 14.2%, respectively. Compound heterozygotes and homozygotes occurred in 3.4% and 2.9% of patients and in none of the controls. The correlation of genotype–Vienna classification showed a significant association with ileal disease (RR = 1.61, 95% CI 1.21–2.15, P = 0.001) and an inverse association with colonic localization (RR = 0.29, 95% CI 0.11–0.80, P = 0.007). There was a significant association between G908R carriership and previous appendectomy, surgical interventions, and stricturing behavior. A gene‐dosage effect on phenotypic characteristics was not observed. Conclusions: In a Spanish population from Madrid, mutations of the NOD2/CARD15 gene were a marker of susceptibility to Crohn disease and were associated with ileal disease. Carriers of the G908R mutation showed a stricturing disease behavior, history of appendectomy, and surgical interventions over the course of the disease.

TNF and LTA gene polymorphisms reveal different risk in gastric and duodenal ulcer patients

A small proportion of patients infected with Helicobacter pylori or using non-steroidal anti-inflammatory drugs (NSAIDs) develops peptic ulcer disease. Since family studies have shown the importance of the genetic background of the host in the development of gastric and duodenal ulcers, immunogenetic factors involved in the regulation of inflammation deserve further study. Polymorphisms in the genes encoding tumour necrosis factor (TNF) and lymphotoxin-α (LTA) have been shown to contribute to the severity of infectious disease. Our aim was to study four bi-allelic polymorphisms in the TNF and LTA genes, which occur as five haplotypes, in patients with peptic ulcer disease. A total of 130 patients with duodenal ulcer, 50 with gastric ulcer and 102 ethnically-matched Spanish Caucasian healthy controls were studied. H. pylori infection was determined by invasive and non-invasive tests. Odds ratios were obtained by logistic regression analysis. H. pylori was detected in 91.8% of peptic ulcer patients and in 73.3% of controls (P < 0.001). Patients with gastric ulcer had a lower frequency of the TNF−308 allele 2 and a higher frequency of the LTANcoI 2.2 genotype when compared with duodenal ulcer patients (P < 0.01 and P = 0.03, respectively). Carriers of haplotype TNF-I were more frequent in gastric ulcer patients (49%) than in controls (28%) (P < 0.05) and the haplotype TNF-E was significantly more frequent in duodenal ulcers than in gastric ulcers (27% vs 8.2%; P < 0.01). Logistic regression analysis identified haplotype TNF-I carrier status as an independent risk factor for peptic ulceration in H. pylori-infected patients (OR: 4.2; 95%CI: 1.7–10.2). These results suggest that TNF and LTA gene polymorphisms are related to the development of gastric and duodenal ulcer and may determine disease outcome in H. pylori infection.

HLA-DRB1*03, BUT NOT THE TNFA-308 PROMOTER GENE POLYMORPHISM, CONFERS PROTECTION AGAINST FISTULISING CROHN'S DISEASE

Abstract Crohn’s disease (CD) appears in forms so diverse that it has been hypothesized CD might be a syndrome, with different pathogenic mechanisms leading to the various clinical phenotypes. This may plausibly explain the conflicting and inconclusive results with regard to HLA associations in unselected groups of patients. The power of these association studies may increase when disease heterogeneity is taken into account. As fistulising CD has been proposed as a separate subgroup of patients with CD, we studied the carrier frequencies (CF) of the DRB1 alleles in 35 unrelated Caucasian Dutch CD patients with proven peri-anal fistulas. A striking decrease in the frequency of the DRB1*03 allele was found in those patients with peri-anal fistulas when compared with a panel of 2400 healthy controls (HC) (3% vs 25%; P = 0.005; Odds Ratio [OR] = 0.09). The DRB1*03 allele is in strong linkage disequilibrium with a polymorphism at position –308 in the promoter region of the gene encoding TNFα (TNFA-308*2). We investigated whether this allele frequency was decreased as well. Surprisingly, the CF of TNFA-308*2 was 29%, not different from the CF of 98 HC (34%; P = 0.7; OR = 0.8). This study is the first showing a significant negative association between DRB1*03 and a particular subgroup of CD patients. Thus, patient selection may largely determine the outcome of genetic association studies in CD, as we previously observed no association with this allele in an unselected population of CD patients. As DRB1*03 frequency, but not the closely linked TNFA-308*2, was decreased, this suggests recombination between the DRB1 and TNFA loci in this group of patients, and may help to define the biological basis of fistula formation.

The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic localisation of Crohn's disease without a major role for the Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway

It is with great interest that we read the paper by Frachimont and colleagues ( Gut 2004; 53 :987–92) in which they describe a novel association of the toll-like receptor 4 (TLR4) +896 A>G polymorphism with both Crohn’s disease (CD) and ulcerative colitis (UC), supporting the genetic influence of pattern recognition receptors (PRRs) in triggering inflammatory bowel disease (IBD). PRRs are sensors of pattern associated molecular patterns of microorganisms in the intestinal flora. Independently, we performed a similar study. However, special attention to the presence of anti- Saccharomyces cerevisiae antibody (ASCA) was taken, as Tada and colleagues1 have recently reported that the S cerevisiae mannan-LBP complex is recognised by CD14 on monocytes and signalling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by lipopolysaccharide (LPS). Patients and controls were recruited from the Outpatient Department of Gastroenterology, VU University Medical Centre, Amsterdam, the Netherlands. The group consisted of 112 CD patients and …

Association of interleukin 1 gene family polymorphisms with duodenal ulcer disease.

Cytokine genes taking part in the immunological response to Helicobacter pylori infection are good candidates to study for genetic predisposition to duodenal ulcer disease (DU). Among cytokines, interleukin (IL)‐1β and its natural specific inhibitor, the interleukin‐1 receptor antagonist, are cytokines that play a key role in regulating gastric acid secretion and modulating the immune response in the gastrointestinal mucosa. We aimed to investigate whether polymorphisms in the IL‐1B and IL‐1RN genes are involved in the susceptibility to duodenal ulcer. DNA from 131 unrelated Spanish Caucasian patients with DU and 105 ethnically matched healthy controls was typed for the IL‐1B‐511, IL‐1B‐31, and IL‐1B + 3954 gene polymorphisms, and the VNTR polymorphism in intron 2 of the IL‐1RN gene by polymerase chain reaction (PCR)‐based methods and TaqMan assays. H. pylori status and non‐steroidal anti‐inflammatory drugs (NSAIDs) use was determined in all patients and controls. Logistic regression analysis identified H. pylori infection (OR: 9·74; 95%CI = 3·53–26·89) and NSAIDs use (OR: 8·82; 95%CI = 3·51–22·17) as independent risk factors for DU. In addition, the simultaneous carriage of IL‐1RN*2, IL‐1B‐511*C, IL‐1B‐31*T and IL‐1B + 3954*C alleles was a genetic risk factor for DU in patients with H. pylori infection (OR: 3·22; 95%CI = 1·09–9·47). No significant differences in IL‐1RN and IL‐1B genotypes were found when patients were categorized according to gender, age of onset, smoking habit, NSAIDs use, type of complication and positive family history. Our results provide further evidence that host genetic factors play a key role in the pathogenesis of duodenal ulcer.

CARD15 gene mutations are not associated with ankylosing spondylitis.

An insertion mutation at nucleotide 3020 (3020insC) and a missense mutation G2722C in the CARD15 gene on chromosome 16p have been reported to be associated with Crohns disease (CD). The protein encoded by the CARD15 gene is expressed in peripheral monocytes and regulates apoptosis and NF-κB activation, factors which play an important role in inflammation. Since CD and ankylosing spondylitis (AS) are interrelated disorders, we have investigated whether these mutations in the CARD15 gene are also associated with AS. We studied 113 unrelated AS patients and 152 unrelated healthy controls. No significant differences were found between patients and controls in the prevalence of the insertion 3020insC mutation and the G2722C missense mutation, OR = 1.36, 95% CI: 0.27–6.84, P = 0.70 and OR = 0.58; 95% CI: 0.18–1.94; P = 0.38, respectively. We conclude that the insertion 3020insC mutation and the G2722C missense mutation in the CARD15 gene are not involved in the susceptibility to AS.