Association between Polymorphisms in Antioxidant Genes and Inflammatory Bowel Disease.

Inflammation is the driving force in inflammatory bowel disease (IBD) and its link to oxidative stress and carcinogenesis has long been accepted. The antioxidant system of the intestinal mucosa in IBD is compromised resulting in increased oxidative injury. This defective antioxidant system may be the result of genetic variants in antioxidant genes, which can represent susceptibility factors for IBD, namely Crohn's disease (CD) and ulcerative colitis (UC). Single nucleotide polymorphisms (SNPs) in the antioxidant genes SOD2 (rs4880) and GPX1 (rs1050450) were genotyped in a Portuguese population comprising 436 Crohn's disease and 367 ulcerative colitis patients, and 434 healthy controls. We found that the AA genotype in GPX1 is associated with ulcerative colitis (OR = 1.93, adjusted P-value = 0.037). Moreover, we found nominal significant associations between SOD2 and Crohn's disease susceptibility and disease subphenotypes but these did not withstand the correction for multiple testing. These findings indicate a possible link between disease phenotypes and antioxidant genes. These results suggest a potential role for antioxidant genes in IBD pathogenesis and should be considered in future association studies.

DNA Damage and Oxidative DNA Damage in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammation has long been regarded as a major contributor to cellular oxidative damage and to be involved in the promotion of carcinogenesis. METHODS: We aimed to investigate the oxidative damage in inflammatory bowel disease [IBD] patients through a case-control and prospective study involving 344 IBD patients and 294 healthy controls. DNA damage and oxidative DNA damage were measured by comet assay techniques, and oxidative stress by plasmatic lipid peroxidation, protein carbonyls, and total antioxidant capacity. RESULTS: Higher DNA damage [p < 0.001] was found both in Crohn's disease [CD] (9.7 arbitrary units [AU]; interquartile range [IQR]: 6.2-14.0) and ulcerative colitis [UC] [7.1 AU; IQR: 4.4-11.7], when compared with controls [5.4 AU; IQR: 3.8-6.8], and this was also the case with oxidative DNA damage [p < 0.001] [CD: 3.6 AU; IQR: 1.8-6.8; UC: 4.6 AU; IQR: 2.4-8.1], when compared with controls: 2.3 AU; IQR: 1.2-4.2]. Stratifying patients into groups according to therapy (5-aminosalicylic acid [5-ASA], azathioprine, anti-TNF, and combined therapy [azathioprine and anti-TNF]) revealed significant between-group differences in the level of DNA damage, both in CD and UC, with the combined therapy exhibiting the highest DNA damage levels [11.6 AU; IQR: 9.5-14.3, and 12.4 AU; IQR: 10.6-15.0, respectively]. Among CD patients, disease behaviour [B1 and B2], and age at diagnosis over 40 years [A3] stand as risk factors for DNA damage. For UC patients, the risk factors found for DNA damage were disease activity, treatment, age at diagnosis under 40 years [A1 + A2] and disease locations [E2 and E3]. CONCLUSIONS: In IBD there is an increase in DNA damage, and treatment, age at diagnosis and inflammatory burden seem to be risk factors.

Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.

Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 µg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.

Oxidative Stress and DNA Damage: Implications in Inflammatory Bowel Disease.

This review will focus on published human studies on oxidative stress and DNA damage in inflammatory bowel disease (IBD), both ulcerative colitis and Crohn's disease, assessing their role in the pathophysiology of these diseases. Search was performed over PubMed and ScienceDirect databases to identify relevant bibliography, using keywords including "oxidative stress," "DNA damage," "IBD," and "oxidative DNA damage." Whether as cause or effect, mechanisms underlying oxidative stress have the potential to condition the course of various pathologies, particularly those driven by inflammatory scenarios. IBDs are chronic inflammatory relapsing conditions. Oxidative stress has been associated with some of the characteristic clinical features exhibited in IBD, namely tissue injury and fibrosis, and also to the ulcerative colitis-associated colorectal cancer. The possible influence of oxidative stress over therapeutic behavior and response, as well as their contribution to the oxidative burden and consequences, is also addressed. Due to the high prevalence and incidence of IBD worldwide, and also to its associated morbidity, complications, and disease and treatment costs, it is of paramount importance to better understand the pathophysiology of these diseases.

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.