N. Della Valle

Glutathione supplementation improves oxidative damage in experimental colitis

BACKGROUND The pathogenesis of inflammatory bowel disease is due, in part, to enhanced free-radical production and reduced antioxidant potential in mucosa cells. AIM We evaluated in a rat model of trinitrobenzensulphonic acid (TNBS) colitis to see whether parenteral administration of glutathione is able to improve mucosal oxidative damage at onset (study A) and during chronic phases of colitis (study B). METHODS In study A, the rats were injected with a single dose of glutathione (200 mg/kg, i.p.) or saline (0,2 ml, i.p.) 1 h before colitis induction and killed 1 h later. In study B, rats with induced colitis were treated with daily injection of glutathione (50 mg/kg, i.p.) or saline (0,2 ml, i.p.), and killed at 1, 2, 4 and 8 weeks. We evaluated on mucosal samples the macroscopic and histological damage and the oxidative stress assessed by the mucosal levels of lipoperoxides, malonyldialdehyde, glutathione and cysteine. RESULTS In study A, colitis induction caused a significant increase to the total histological score (p<0.05), lipoperoxide and malonyldialdehyde levels (p<0.001), but did not affect glutathione and cysteine content. Glutathione pre-treatment decreased both total histological score (p<0.05) and lipoperoxide and malonyldialdehyde values (p<0.001). In study B, the extensive macroscopic and histological colonic damage induced by TNBS was accompanied by a reduction of glutathione and cysteine mucosal levels (p<0.01) and increased lipid peroxidation. Glutathione supplementation significantly improved colonic damage (p<0.01), restored glutathione and cysteine levels, and decreased, and even, if not totally, abolished lipid peroxidation (p<0.001). CONCLUSION This paper further supports the pathogenic role of the imbalance in oxidant/antioxidant content in inducing mucosal colonic damage.

Transglutaminases in Crohn's disease.

Transglutaminases are a family of Ca-dependent enzymes involved in various biological events. Circulating transglutaminase (factor XIIIa) is decreased in blood of patients with inflammatory bowel diseases. There is evidence that factor XIIIa and tissue type transglutaminase, present in cell cytosol, bind to various proteins of the extracellular matrix. This study examined the value of serum transglutaminase assay in the treatment and follow up of Crohns disease and then investigated the intestinal location of both forms of transglutaminases by immunohistochemistry in normal and abnormal tissues. Serum transglutaminase activity was assayed in 36 patients with active Crohns disease (CDAI > 150). Eighteen patients were studied prospectively from relapse into remission. A significant inverse correlation (p < 0.001) was found between circulating transglutaminase and Crohns disease activity index; a correlation was also found between serum transglutaminase and serum orosomucoid (p < 0.01) and C reactive protein (p < 0.01). Patients were prospectively studied until clinical remission showed improvement in both their CDAI score mean (SD) (230 (46) to 72 (34), p < 0.01) and transglutaminase activity mean (SD) (0.61 (0.12) to 0.93 (0.13) mU/ml, p < 0.01). The immunohistochemistry assessment showed a colocalisation of factor XIIIa and tissue transglutaminase to the extracellular matrix of damaged tissues. In conclusion, these data confirm the value of serum transglutaminase assay as marker of Crohns disease activity, extend the utility of serum transglutaminase assay to follow up of the disease, and emphasised the role of different types of transglutaminases in extracellular matrix assembly in the damaged tissues.

Differential expression of multiple transglutaminases in human colon: impaired keratinocyte transglutaminase expression in ulcerative colitis

Background and aims: Ulcerative colitis (UC) is characterised by refractory inflammatory ulceration and damage to the colon. The mechanisms underlying impaired healing have yet to be defined. As transglutaminase expression resulting in matrix protein cross linking is associated with increased wound healing in a rat model of colitis, we hypothesised that different types of transglutaminase might also play a role in UC. Patients and methods: Endoscopic and histological indices were studied in 26 patients with UC (10 active and 16 inactive) and in 20 normal controls undergoing colonoscopy. Transglutaminase activity was evaluated in plasma (factor XIIIa) by a radioenzymatic method. Factor XIIIa, tissue and keratinocyte transglutaminase protein content, and mRNA expression in the colon were evaluated by western blot analysis and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Colonic location of transglutaminases and their reaction products, the ε-(γ-glutamyl)lysine bonds, was evaluated by immunohistochemistry using specific monoclonal antibodies. Results: Transglutaminase activity was significantly lower in the plasma of patients with active UC (4.2 (2.4) mU/ml; p<0.05 v controls) than in those with inactive UC and controls (10.6 (2.2) and 12.1 (1.7) mU/ml). As shown by western blot, protein levels of tissue transglutaminase and factor XIIIa were unchanged in active UC compared with inactive disease and controls, while the keratinocyte form was reduced in active UC. Tissue transglutaminase and factor XIIIa immunostaining was strongly present in damaged areas colocalising with isopeptide bonds. In contrast, the keratinocyte form was almost absent in active UC and localised in the upper part of the crypts in normal subjects. RT-PCR showed upregulation of tissue transglutaminase mRNA in active UC (320% compared with controls) while keratinocyte transglutaminase gene expression was downregulated in active UC. Conclusions: The results of the present study support the concept that, in the damaged colon, transglutaminases are needed in response to chronic injury and underline the key role of these enzymes in mucosal healing.

OC.03.6 INFLIXIMAB THERAPY DOWNREGULATION OF BASIC FIBROBLAST GROWTH FACTOR/SYNDECAN 1 LINK: A POSSIBLE MOLECULAR PATHWAY OF MUCOSAL HEALING IN ULCERATIVE COLITIS

Background It is known that syndecan 1 in inflammatory bowel diseases is able to migrate from epithelial basolateral site to the stromal area and apical surface of epithelium with a consequent activation and modulation of basic fibroblast growth factor (bFGF), and this process sustains mucosal healing of ulcers. On the other hand, tumour necrosis factor (TNF) a mucosal levels are directly related to the entity of the damage in these disorders. Aim of the study A ‘post-hoc’ retrospective study was performed to estimate mucosal TNF a in rectal biopsies of subjects with ulcerative colitis (UC) before and after effective infliximab therapy and its relationship with syndecan 1, bFGF and endoscopic mucosal healing. Material and methods Paraffin-embedded rectal samples from 12 patients with UC responders to infliximab were analysed for TNF a, syndecan 1 and bFGF before and 6 months after therapy using a real-time reverse transcriptase polymersase chain reaction. Additionally, syndecan 1 location was evaluated by immunohistochemistry. Samples from 12 subjects with irritable bowel symptoms without endoscopic/ histological abnormalities represented the control group. Mucosal healing induced by the treatment was defined by an endoscopic Mayo subscore changing from 2e 3t o 0. ANOVA plus StudenteNewmaneKeuls was used for statistical analysis. Results The authors found that in the active disease, an increase in TNF a (p<0.001) is accompanied by raised levels of either syndecan 1 (p<0.005) and bFGF (p<0.005) compared with the control group 1