SummaryThe cathepsins D (CTSD), B (CTSB) and L (CTSL) are important for the intracellular degradation of proteins. Increased cathepsin expression is associated with inflammatory diseases. We have shown previously an induction of CTSD expression in intestinal macrophages (IMAC) in inflamed mucosa of patients with inflammatory bowel disease (IBD). Here we investigated the regulation of CTSB and CTSL in IMAC during IBD and effects of CTSD and CTSB/CTSL inhibition in vivo. Human IMAC were isolated from normal and inflamed mucosa. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for CTSB and CTSL mRNA. Immunostaining was used to confirm PCR results. Cathepsin inhibition was investigated in the dextran-sulphate-sodium (DSS) colitis model in mice with application of pepstatin A (CTSD inhibitor), CA-074 (CTSB inhibitor) and Z-Phe-Tyraldehyde (CTSL inhibitor). CTSL mRNA was significantly up-regulated in IMAC isolated from IBD mucosa. Up-regulated protein expression was found mainly in areas of mucosal damage by immunostaining. Inhibition of CTSD in mouse DSS colitis was followed by an amelioration of the disease. Inhibitor-treated mice showed a significant lower histological score (HS) and less colon reduction in comparison to controls. Similarly, simultaneous inhibition of CTSB/CTSL was followed by a significant amelioration of colitis. Expression of tissue-degrading cathepsins is increased in IMAC in IBD. Inhibition of CTSD as well as CTSB/CTSL is followed by an amelioration of experimental colitis. The prevention of mucosal damage by cathepsin inhibition could represent a new approach for the therapy of IBD.

SummaryRecently we demonstrated that in inflammatory bowel disease (IBD) macrophage-oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti-oxidative potential. Using the dextran sulphate sodium (DSS)-induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 mg/ mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti-CD3 antibody in the presence of interleukin (IL)-2 (final concentration 10 U/ml). After incubation for 24 h, IL-1b, IL-6, IL-12 tumour necrosis factor (TNF)-a and interferon (IFN)-g levels in supernatants were analysed by the beadlyte® cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3·2 with acteoside versus 5·2 with phosphate-buffered saline (PBS) (P < 0·02). In chronic colitis both 120 mg (3·3 versus 5·2) or 600 mg acteoside (3·0 versus 5·2) significantly ameliorated colitis (both P < 0·02). Stimulated MLN from mice with chronic DSS-induced colitis treated with acteoside showed a significant down-regulation of IFN-g secretion (195 pg/ml with 600 mg acteoside versus 612 pg/ml with PBS, P < 0·02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.

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SummaryHaem oxygenase-1 (HO-1) up-regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up-regulation of HO-1 expression in patients with Crohn's disease (CD) and ulcerative colitis (UC) was demonstrated. A HO-1 gene promoter microsatellite (GT)n dinucleotide repeat polymorphism was associated with regulation of HO-1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO-1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5Ј-flanking region of the HO-1 gene containing the (GT)n dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)n repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short-, middle-and long-sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)n dinucleotide repeats of the HO-1 promotor region have no significance for the pathophysiology and disease course of IBD.

We therefore determined genetic variations of NAT1 in patients with UC and looked for a possible association with the clinical response to 5-ASA. METHODS: DNA was obtained from 78 patients with UC. 77 % of the patients were in remission during 5-ASA treatment, whereas 23 % suffered from active disease. NAT1 genotyping was performed for 23 known alleles using RFLP and sequence analysis. Clinical response to 5-ASA was determined by medical record review and associated with NAT1 genotypes. RESULTS: Utilising PCR we amplified a 570-bp coding region of the human NAT1 gene in addition to 240 bp in the 3'-untranslated region (UTR). 4 NAT1 alleles previously known as NAT1*3, *4, *10 and *11 were recovered. 31 % of the patients were heterozygous and 4 % homozygous for the NAT1*10 allele. 6 % were heterozygous for the NAT1*3 allele. 6 % were heterozygous for the NAT1*11 allele. No association was found between NAT1 genotype and clinical response as well as side effects to 5-ASA in patients with UC. CONCLUSIONS: NAT1 genotypes do not predict response or side effects to mesalamine in patients with UC. Variations caused by non-genomic effects may be associated with the clinical response to 5-ASA.