Damage In Colitis Research Articles

Protective Effect of HT057 Treatment on Dextran Sulfate Sodium Induced Colitis in Mice

Inflammatory Bowel Disease (IBD) is a chronic and frequently disabling inflammatory disorder of the intestine. Atractylodes macrocephala and Taraxacum mongolicum have been used for various inflammatory diseases in traditional Korean medicine and can be used as food ingredients worldwide. In this study, protective effect of the herbal mixture of A. macrecephala and T. mongolicum (HT057) in dextran sulfate sodium (DSS) induced experimental colitis which mimics the clinical and histological features of IBD. Colitis was induced by 5% DSS with drinking water in balb/c mice. HT057 (30, 100 or 300 mg/kg) was orally administered to mice twice per day for 10 days. Colon length, mouse weight, disease activity index (DAI), myeloperoxidase (MPO) activity, and histological score were measured after administration. HT057 significantly improved the colon length (7.2%, p<0.01 at 100 mg/kg) which reflects the extent of colon damage in IBD as compared to control group. Mice with DSS‐induced colitis showed loss of body weight and DAI score which is a result of IBD along with diarrhea and blood loss compared to normal group, while mice treated with HT057 showed significantly protected the loss of body weight (7.8%, p<0.001 at 100 mg/kg) and DAI score (27.6%, p<0.001 at 300 mg/kg). HT057 also significantly reduced MPO activity, and histological score which reflect the severity and extent of inflammation. In conclusion, HT057 has protective effect in inflammatory intestinal mucosal injury against DSS induced colitis and these findings suggest that HT057 might be a promising functional food ingredient against IBD.

Plain magnetic resonance imaging as an alternative in evaluating inflammation and bowel damage in inflammatory bowel disease – a prospective comparison with conventional magnetic resonance follow-through

Objective. To compare prospectively the diagnostic accuracy of magnetic resonance imaging (MRI) without use of contrast medium orally or intravenously (plain MRI) with magnetic resonance follow-through (MRFT) in patients with inflammatory bowel disease (IBD). Material and methods. Plain MRI was carried out in addition to MRFT, to which the patients were referred. All patients underwent both examinations on the same day. For the evaluation, the bowel was divided into nine segments. Two radiologists, blinded to clinical findings, evaluated bowel wall thickness, diffusion weighted imaging (DWI), and other inflammatory changes in each bowel segments. Further, hyperenhancement of the bowel was also evaluated in MRFT. Results. A total of 100 patients (40 males and 60 females; median age: 38.5; range: 19–90) were enrolled; 44 with Crohn’s disease (CD), 25 with ulcerative colitis (UC), 24 with IBD unclassified (IBD-U), and 7 had other diagnosis. Sensitivity, specificity, and accuracy in CD ranged 50–86%, 93–94%, and 91–92% for wall thickening and 49–82%, 85–93%, and 84–89% for DWI, respectively. Sensitivity, specificity, and accuracy in UC range 0–40%, 87–100%, and 80–100% for wall thickening and 0–52%, 83–94% and 76–92% for DWI, respectively. The κ values for bowel wall thickening, DWI, and mural hyperenhancement were detected with fair agreement (κ = 0.26–0.39) at both MRI examinations, whereas only bowel wall thickening in MRFT were detected with moderate agreement (κ = 0.47) Conclusion. Plain MRI cannot currently replace MRFT in the workup of patients with IBD. Further research on plain MRI is needed to improve the protocol.

Alloferon Alleviates Dextran Sulfate Sodium-induced Colitis.

Dysfunction of gut immune regulation is involved in mucosal damage in inflammatory bowel disease (IBD). However, there is still no efficacious immune-regulator for the treatment of IBD. Alloferon is a novel immune-modulatory peptide that was originally isolated from infected insects. It shows anti-inflammatory effects by the regulation of cytokine production by immune cells and their activities. Therefore, we investigated the effect of alloferon in a mouse model of colitis using dextran sulfate sodium (DSS). Colitis was induced by administration of DSS in drinking water for 7 consecutive days. It was confirmed by the presence of weight loss, diarrhea, hematochezia, and colon contraction. Alloferon was injected 4 days after DSS administration. We found that alloferon improved the pathogenesis of IBD based on the reduced disease activity index (DAI) and colon contraction. Edema, epithelial erosion, and immune cell infiltration were found in mice administered DSS, but the phenomena were reduced following alloferon treatment. The plasma level of IL-6, a classical pro-inflammatory cytokine in colitis, was also decreased by alloferon. Moreover, alloferon inhibited the TNF-α-induced degradation and phosphorylation of IκB in Colo205 colon cancer cells. Taken together, these results show that alloferon has anti-inflammatory effects and attenuates DSS-induced colitis.

Electrocardiographic and Echocardiographic Endpoints: Insights into Cardiovascular Damage in IBD

Purpose: The lack of reliable conventional predictive cardiovascular (CV) co-morbidities among inflammatory bowel disease (IBD) patients and the absence of literature describing the results of CV testing in this population support the present investigation to identify whether electrocardiographic (ECG) and echocardiographic (ECHO) abnormalities are more prevalent among inflammatory bowel disease (IBD) patients than controls, and to characterize the ECG and ECHO anomalies among IBD patients with coronary artery disease (CAD). Methods: This retrospective cohort study included adult IBD patients seen in the outpatient gastroenterology clinic at Jackson Memorial Hospital (Miami, FL). Predictive variables for traditional cardiovascular disease such as hypertension, dyslipidemia, diabetes, obesity, and smoking were recorded. The primary outcomes were abnormal ECG and ECHO findings. Abnormalities on ECG were classified as major or minor, as per the Minnesota code criteria. Pathology with ECHO was defined as reduced left ventricular ejection fraction [EF] ≤ 45%, valvulopathy, wall motion abnormalities, and diastolic dysfunction, as determined by an attending cardiologist. Results: The study comprised 102 IBD patients and 385 age- and gender-matched controls. The baseline characteristics of the study population are shown in Table 1. Although clinical heart failure and CAD were observed in IBD patients more than controls, dyslipidemia and obesity were less common. IBD patients and controls had similar ECG findings (Table 2). A trend toward increase in major ECG changes in the IBD patients with CAD was observed, limited by small numbers and primarily driven by evidence of ischemia or infarction and intra-ventricular blocks (Table 2). On ECHO, an increased prevalence of mitral regurgitation was observed in the IBD group: see Table 2. This finding may be secondary to low intravascular volume from diarrhea in IBD patients.Table 1: Baseline characteristics of IBD cases and controlsTable 2: ECG and ECHO findings of IBD cases and controlsConclusion: CV disease in IBD patients is associated with substantial ECG abnormalities, underlining the importance of identifying the non-traditional IBD-specific risk factors for CVD and monitoring for its clinical consequences.

English

Ulcerative colitis and Crohn’s disease are chronic recurrent inflammatory bowel disease (IBD) of unknown origin. Oxidative stress is believed to be a key factor in the pathogenesis of the mucosal damage in IBD. The aqueous extract of Hyphaena thebaica fruits showed an antioxidant activity; this is due to the substantial amount of their water-soluble phenolic contents. This study was undertaken to study the effect of H. thebaica in experimentally induced IBD. Sprague Dawley rats were pretreated orally for 5 days with distilled water in control, H. thebaica 500 mg/kg in acetic acid + H. thebaica group and 5-aminosalisylic acid (5-ASA) 100 mg/kg in acetic acid + 5ASA groups. Colitis was induced by transrectal administration of 4% acetic acid on 5th day. All the animals were sacrificed with ether overdose 48 h after colitis induction, and 10 cm colon segment was resected from distal end. Colon was weighed (for disease activity index) and scored macroscopically and microscopically after histological staining. Biochemical assessments included myeloperoxidase (MPO) and tissue catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD) measurements. H. thebaica showed significant prevention of increase in colon weight and disease activity index along with decrease in macroscopic and microscopic lesion score as compared to control group. Significant improvement was observed in the levels of MPO, CAT and SOD, except GSH. However, the effect of H. thebaica was significantly less than 5-ASA. H. thebaica at 500 mg/kg showed significant amelioration of experimentally induced IBD, which may be attributed to its antioxidant and anti-inflammatory properties.   Key words: Colitis, antioxidant, Hyphaena thebaica, myeloperoxidase, catalase, superoxide.

Inflammatory Bowel Disease: Mechanisms, Redox Considerations, and Therapeutic Targets

Oxidative stress is thought to play a key role in the development of intestinal damage in inflammatory bowel disease (IBD), because of its primary involvement in intestinal cells' aberrant immune and inflammatory responses to dietary antigens and to the commensal bacteria. During the active disease phase, activated leukocytes generate not only a wide spectrum of pro-inflammatory cytokines, but also excess oxidative reactions, which markedly alter the redox equilibrium within the gut mucosa, and maintain inflammation by inducing redox-sensitive signaling pathways and transcription factors. Moreover, several inflammatory molecules generate further oxidation products, leading to a self-sustaining and auto-amplifying vicious circle, which eventually impairs the gut barrier. The current treatment of IBD consists of long-term conventional anti-inflammatory therapy and often leads to drug refractoriness or intolerance, limiting patients' quality of life. Immune modulators or anti-tumor necrosis factor α antibodies have recently been used, but all carry the risk of significant side effects and a poor treatment response. Recent developments in molecular medicine point to the possibility of treating the oxidative stress associated with IBD, by designing a proper supplementation of specific lipids to induce local production of anti-inflammatory derivatives, as well as by developing biological therapies that target selective molecules (i.e., nuclear factor-κB, NADPH oxidase, prohibitins, or inflammasomes) involved in redox signaling. The clinical significance of oxidative stress in IBD is now becoming clear, and may soon lead to important new therapeutic options to lessen intestinal damage in this disease.

Genetic variations in matrix metalloproteinases may be associated with increased risk of ulcerative colitis

Increased production of matrix metalloproteinases (MMPs) plays an important role in tissue damage in inflammatory bowel disease (IBD). Genetically encoded variation between individuals in MMP production may therefore contribute to disease onset, type, or severity. We undertook an extensive candidate gene single nucleotide polymorphism (SNP) study of MMP-1, -2, -3, -7, -8, -9, -10, -12, -13, and -14 and tissue inhibitor of metalloproteinases (TIMPs)-1, -3, and -4 in ulcerative colitis (UC). We identified tagging SNPs across these genes, and genotyped these SNPs in a Caucasian New Zealand dataset consisting of 419 UC patients and 907 controls. SNPs in a number of MMP genes were associated with UC. After correcting for multiple testing SNPs in MMP-3, MMP-8, MMP-10, and MMP-14 remained significant in their associations with UC. In a second study, using samples from a Dutch cohort, most of the significant findings in the New Zealand cohort were not replicated. However, data from an international meta-analysis provide some support for the initial findings. In conclusion, this study provides preliminary evidence to suggest that genetic variation in the MMPs may play a role in interindividual differences in UC susceptibility and clinical outcome. Further studies are needed in other cohorts to determine the robustness of these observations in different populations.

Pistacia lentiscus Resin Regulates Intestinal Damage and Inflammation in Trinitrobenzene Sulfonic Acid–Induced Colitis

Mastic (Pistacia lentiscus) of the Anacardiaceae family has exhibited anti-inflammatory and antioxidant properties in patients with Crohn's disease. This study was based on the hypothesis that mastic inhibits intestinal damage in inflammatory bowel disease, regulating inflammation and oxidative stress in intestinal epithelium. Four different dosages of P. lentiscus powder in the form of powder were administered orally to trinitrobenzene sulfonic acid-induced colitic rats. Eighty-four male Wistar rats were randomly assigned to seven groups: A, control; B, colitic; C-F, colitic rats daily supplemented with P. lentiscus powder at (C) 50 mg/kg, (D) 100 mg/kg, (E) 200 mg/kg, and (F) 300 mg/kg of body weight; and G, colitic rats treated daily with cortisone (25 μg/kg of body weight). Colonic damage was assessed microscopically. The cytokines tumor necrosis factor-α, intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-6, IL-8, and IL-10 and malonaldehyde were measured in colonic specimens. Results were expressed as mean ± SE values. Histological amelioration of colitis (P≤.001) and significant differences in colonic indices occurred after 3 days of treatment. Daily administration of 100 mg of P. lentiscus powder/kg of body weight decreased all inflammatory cytokines (P≤.05), whereas 50 mg of P. lentiscus powder/kg of body weight and cortisone treatment reduced only ICAM-1 (P≤.05 and P≤.01, respectively). Malonaldehyde was significantly suppressed in all treated groups (P≤.01). IL-10 remained unchanged. Cytokines and malonaldehyde remained unaltered after 6 days of treatment. Thus P. lentiscus powder could possibly have a therapeutic role in Crohn's disease, regulating oxidant/antioxidant balance and modulating inflammation.

Effect of fruit extract of Fragaria vesca L. on experimentally induced inflammatory bowel disease in albino rats

Aim:Ulcerative colitis and Crohn’s disease are chronic recurrent inflammatory bowel disease (IBD) of unknown origin. Oxidative stress is believed to be a key factor in the pathogenesis and perpetuation of the mucosal damage in IBD.Materials and Methods:Ethanolic extract of Fragaria vesca (EFFV) fruits was prepared by percolation method and subjected to oral toxicity testing using OECD guidelines. Albino rats were pretreated orally for 5 days with 3% gum acacia in control, EFFV 500 mg/kg in test and 5-aminosalisylic acid (5-ASA) 100 mg/kg in standard groups. Colitis was induced by transrectal administration of 4% acetic acid on 5th day. All the animals were sacrificed with ether overdose 48 hours after colitis induction, and 10 cm colon segment was resected from proximal end. Colon was weighed (for disease activity index) and scored macroscopically and microscopically after histological staining. Biochemical assessments included myeloperoxidase (MPO) and tissue catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD) measurements. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Dunnett’s “t” test.Results:EFFV showed significant (P < 0.05) prevention of increase in colon weight and disease activity index along with decrease in macroscopic and microscopic lesion score as compared to control group. Significant improvement was observed in the levels of MPO, CAT and SOD, except GSH (P < 0.05). However, the effect of EFFV was significantly less than 5-ASA (P < 0.05).Conclusions:EFFV at 500 mg/kg showed significant amelioration of experimentally induced IBD, which may be attributed to its antioxidant and anti-inflammatory properties.

Renal manifestations and complications of inflammatory bowel disease

Renal manifestations and complications are not rare in patients with inflammatory bowel disease (IBD) and may present as nephrolithiasis, amyloidosis, tubulointerstitial nephritis, and glomerulonephritis. Symptoms of renal impairment are not always specific and since the underlying bowel disease is preponderant, renal function deterioration may be underestimated. Additionally, medical treatment of patients with IBD such as aminosalicylates, cyclosporine, and tumor necrosis factor-α inhibitors can cause renal complications, although direct correlation to bowel disease is not always clear. The well-documented renal manifestations and complications of IBD, as well as the possible renal side effects of new drugs, emphasize the need for periodic evaluation of renal function. New markers of renal function may facilitate early diagnosis and unravel the complex mechanisms responsible for kidney damage. The purpose of this review is to summarize the renal manifestations and complications as well as the markers of renal function utilized in IBD, attempting to shed more light on the pathophysiology of renal damage in IBD.

The absence of functional PI3Kγ prevents leukocyte recruitment and ameliorates DSS-induced colitis in mice

Phosphatidylinositol-3-kinase gamma (PI3Kγ) is the major PI3K that is activated in response to chemoattractants. It is responsible for the migration of leukocytes from the bloodstream to sites of injury or infection. Constant migration of new leukocytes to the intestinal mucosa may be an important factor in maintenance of inflammation and tissue damage in inflammatory bowel disease (IBD). Reducing this influx, for example by inhibition of PI3Kγ, might therefore be a potential goal for therapy. Here we investigated the role of PI3Kγ in the migration of leukocytes to sites of intestinal inflammation. We induced colitis in mice with a point mutation that inactivates PI3Kγ enzymatic activity (‘kinase-dead’) by oral administration of dextran sodium sulphate (DSS). Mice were treated with 1.5% DSS for 1 week and effects on cytokine production, leukocyte recruitment and disease severity were examined. Both clinical and histological parameters showed that the severity of colitis was significantly reduced in PI3Kγ-kinase-dead mice compared to controls. Although mutant mice had a less severe colitis than controls they produced significantly more pro-inflammatory Th1 cytokines such as Il-12, Tnfα and Ifnγ and more Il-10. PI3Kγ mutant mice showed increased numbers of resident macrophages and T cells in the colonic lamina propria in an unstressed condition but failed to recruit new leukocytes to the mucosa upon treatment with DSS despite the increased cytokine levels. These results suggest that PI3Kγ plays a critical role in lamina propria leukocyte trafficking and that loss of PI3Kγ-activity ameliorates DSS-induced colitis in mice.

Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR).

Cytokines serve a central function as key factors in the regulation of the intestinal immune response and mediation of tissue damage in inflammatory bowel disease (IBD). Abnormalities in the expression of immunoregulatory cytokines such as IL-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. Therefore, cytokine mRNA concentrations were determined in the mucosa of patients with IBD at sites of active (n = 13) and inactive (n = 12) ulcerative colitis (UC), active (n = 11) and inactive (n = 11) Crohn's disease (CD) and in control patients (n = 14) using quantitative RT-PCR. IL-10 mRNA concentrations were significantly increased in patients with both active UC (P < 0.001) and active CD (P < 0.005) compared with control patients. IFN-gamma mRNA concentrations were also significantly increased both in patients with active UC (P < 0.02) and active CD (P < 0.05) compared with control patients, whereas IL-2 mRNA levels were significantly (P < 0.02) increased only in active CD. IL-4 mRNA expression in the intestinal mucosa was frequently below the detection limit. Our results demonstrate that chronic intestinal inflammation in patients with CD is characterized by an increase of Th1-like cytokines. Furthermore, the increased IL-10 mRNA expression at sites of active IBD suggests that IL-10 is an important regulatory component involved in the control of the inflammatory response in inflammatory bowel disease.

(GT)n Dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is not associated with inflammatory bowel disease risk or disease course

Haem 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 5'-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.

Cathepsin D is up-regulated in inflammatory bowel disease macrophages.

Down-regulation of receptors involved in the recognition or transmission of inflammatory signals and a reduced responsiveness support the concept that macrophages are 'desensitized' during their differentiation in the intestinal mucosa. During inflammatory bowel disease (IBD) intestinal macrophages (IMACs) change to a reactive or 'aggressive' type. After having established a method of isolation and purification of IMACs, message for cathepsin D was one of the mRNAs we found to be up-regulated in a subtractive hybridization of Crohn's disease (CD) macrophages versus IMACs from control mucosa. The expression of cathepsin D in intestinal mucosa was analysed by immunohistochemistry in biopsies from IBD and control patients and in a mouse model of dextran sulphate sodium (DSS)-induced acute and chronic colitis. IMACs were isolated and purified from normal and inflamed mucosa by immunomagnetic beads armed with a CD33 antibody. RT-PCR was performed for cathepsin D mRNA. Results were confirmed by Northern blot and flow cytometrical analysis. Immunohistochemistry revealed a significant increase in the cathepsin D protein expression in inflamed intestinal mucosa from IBD patients compared to non-inflamed mucosa. No cathepsin D polymerase chain reaction (PCR) product could be obtained with mRNA from CD33-positive IMACs from normal mucosa. Reverse transcription (RT)-PCR showed an induction of mRNA for cathepsin D in purified IMACs from IBD patients. Northern blot and flow cytometry analysis confirmed these results. Cathepsin D protein was also found in intestinal mucosa in acute and chronic DSS-colitis but was absent in normal mucosa. This study shows that expression of cathepsin D is induced in inflammation-associated IMACs. The presence of cathepsin D might contribute to the mucosal damage in IBD.

A primary dysregulation in the immunoregulatory role of the intestinal mucosal epithelial cell in inflammatory bowel disease pathogenesis?: Biology of inflammatory response as tissue pattern entities in Crohn's versus ulcerative colitis

Within a framework of dual involvement of mucosa and submucosa on the one hand, and of the muscularis propria of the bowel wall on the other, it might be valid to consider involvement of the vascular supply as the essential means in itself of not only causing the morphologic lesions in inflammatory bowel disease, but also especially in accounting for persisting patterns of inflammatory response both in ulcerative colitis and in Crohn's disease. Inflammatory bowel disease as a group constitutes a spectrum of biologic and pathobiologic manifestations in terms not only of inflammatory involvement of the bowel wall but also in terms of how the bowel in its turn deals with inflammation as a pathologic lesion in its own right. Parameters of inflammatory bowel activity transcend simple concepts of etiology and pathogenesis as applicable to category disorders such as infections or bowel ischemia. Indeed, the strictly characterized initiation of the inflammatory bowel response as a function of defective regulation of the antigenicity of the luminal contents on the one hand, and on interactions between nitric oxide and free oxygen radicals on the other, might help determine a persistence of tissue damage in inflammatory bowel disease that is either relapsing/remitting or chronic in progression. In a final analysis, perhaps, there might be involved a single central form of pathway induction of dysregulated immune reactivity arising from an early disturbance in activation patterns as induced by the onset of luminal antigenicity at an early or specific-stage, further characterized perhaps by specific forms of intestinal epithelial defects of the bowel mucosa in patients subsequently developing inflammatory bowel disease. Specific genetic markers for disease susceptibility and for therapeutic responsiveness are particularly of interest. The Nucleotide binding oligomerization Domain 2 (NOD2) would recognize microbial lipopolysaccharide or else mark systemic responses to pathogens that are pathogenic to evolving inflammatory bowel disease.

Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization, and association with mucosal antioxidants.

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress-related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite-mediated protein nitration (3-NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide-producing enzyme xanthine oxidase (XO). In addition, bi- and multi-variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohn's disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3-NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3-NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3-NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3-NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite-independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothionein and with Mn-superoxide dismutase activity, suggesting the involvement of hydroxyl radicals and superoxide anions. In UC, however, the amount of MDA was associated with epithelial catalase expression and neutrophilic myeloperoxidase activity, suggesting a hydrogen peroxide- and/or hypochlorous acid-mediated mechanism. The present study underlines the importance of oxidative stress in the pathogenesis of IBD and provides clues regarding the (anti)oxidants involved which indicate that this process evolves through diverging pathways in CD and UC.

Cathepsin D contributes to mucosal damage in inflammatory bowel disease

female mice by intra-rectal administration of DNB. Anti-neutrophil serum (ANS) was administered by intra-peritoneal injection 24-hr prior to induction of colitis and then daily until sacrifice. Colonic tissue was retrieved at various time points for assessment of myeloperoxidase (MPO) activity, histology, and macroscopic damage score. MP neuronal cell numbers were examined using HuOHuD (a neuronal marker) immunostaining and cell apoptosis was determined by TUNEL, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) immunostaining and immunoblotting. Results: Colitis was associated with mucosal neutrophil infiltration at 4 hr with transmural extension by 12 hr. MPO activity increased significantly above control at 6 hr, 12 hr and 24 hr (4, 22, and 5 fold increase respectively). Although ANS failed to alter the overall histologic damage score, far fewer neutrophils were evident, epithelial damage and apoptosis was less severe, and MPO activity was reduced to levels similar to control. Apoptosis in the MP as determined by TUNEL was evident at 4 hr (8x control) and maximal at 12hr (65x control). The peak in apoptosis was confirmed by significant cleaved caspase-3 and cleaved PARP immunostaining and the appearance of the pl 8 fragment associated with a reduction in neuronal cell numbers (45% of control). ANS treatment significantly reduced the degree of caspase-3 dependent apoptosis and significantly attenuated the loss of MP neurons. Conclusion: Acute colitis was associated with caspase dependent MP neuronal cell death that was attenuated by ANS. These resuks suggest that neutrophil infiltration into the mucosa is an important determinant of MP cell injury. Moreover, this data suggests the potential for the development of therapies that specifically target neutrophils. Supported by CCFC (Canada).

Mechanisms of tissue damage in inflammatory bowel disease

Crohn disease and ulcerative colitis are caused by an excessive immune-inflammatory reaction in the intestinal wall. Analysis of the types of immune response ongoing in the inflamed intestine has revealed that in Crohn disease there is predominantly a T helper cell type 1 response, with exaggerated production of interleukin (IL)-12 and interferon-gamma, whereas in ulcerative colitis the lesion seems to be more of an antibody-mediated hypersensitivity reaction. Despite these differences, downstream inflammatory events are probably similar in both conditions. In both Crohn disease and ulcerative colitis there is an increased synthesis of proinflammatory cytokines, including IL-1beta, IL-6, IL-8, IL-16, and tumor necrosis factor-alpha accompanying the influx of nonspecific inflammatory cells into the mucosa. These cytokines contribute to the tissue damage either directly or indirectly by enhancing the production of matrix metalloproteinases and growth factors, which produce ulceration as well as mucosal repair.

Reduced Plasma Antioxidant Concentrations and Increased Oxidative DNA Damage in Inflammatory Bowel Disease

Background: Oxidative stress is believed to play a key role in the pathogenesis of inflammatory bowel disease (IBD)-related intestinal damage. Circulating antioxidants may have a role to play in preventing free radical-mediated tissue injury. Methods: Plasma vitamin A, E and carotenoid concentrations, leukocytic genomic damage and 8-hydroxy-deoxy-guanosine (8-OHdG) concentration were determined in 46 ulcerative colitis (UC) patients, 37 Crohn disease (CD) patients and 386 controls. A 20 ml blood sample was taken from each subject for antioxidant and 8-OHdG measurements. A food frequency questionnaire was administered to a sample of subjects from each group to evaluate daily intake of dietary compounds. Results: Antioxidant concentration was significantly reduced in IBD patients, particularly in those with active disease, with respect to controls ( P < 0.0001). 8-OHdG concentrations were significantly increased in IBD patients compared to controls, independent of disease activity ( P < 0.05). No correlation was found between antioxidant and 8-OHdG concentrations. Carotenoid concentrations were significantly reduced in malnourished IBD patients (0.89 ± 0.14 μmol/l) compared to patients with normal or high body mass index (1.83 ± 0.12 μmol/l; P < 0.05), independent of disease activity or extension. Protein, fruit and vegetable intakes of IBD patients were significantly lower than those of controls. Conclusions: Depletion of antioxidants is likely to be important in the pathophysiology of IBD: UC and CD patients show increased free radical peripheral leukocyte DNA damage and decreased plasma antioxidant defenses. These results indicate the necessity of further studies to establish whether optimal vitamin status may improve the clinical course of UC and CD.

Altered ascorbic acid status in the mucosa from inflammatory bowel disease patients.

Attempts to establish the presence of oxidant stress and tissue damage in inflammatory bowel disease (IBD) have relied on determining the capacity of peripheral blood inflammatory cells to produce reactive oxygen species (ROS) and other indirect indices. These approaches have failed to address whether or not there are adequate chemical antioxidant defences to prevent oxidative injury in the inflamed mucosa. In this investigation we have determined the mucosal concentrations of reduced and total ascorbic acid and the redox status in paired non-inflamed and inflamed mucosa using colonic biopsies from IBD patients. In inflamed mucosa from Crohn's disease (CD) patients, reduced and total ascorbic acid content decreased by 35% (p = 0.014 and p = 0.009, respectively). In ulcerative colitis (UC) patients, mucosal total ascorbic acid content decreased by 73% (p = 0.069) and reduced ascorbic acid by 41% (p = 0.014). The proportion of total ascorbic acid present in its reduced form in histologically normal mucosa from CD patients was unusually low at approximately 30%. In the paired-inflamed mucosa from CD patients, the redox ratio was also approximately 30% despite the loss of 35% of total ascorbate. In UC patients, the ascorbate redox ratio in the non-inflamed mucosa was 23% which increased to 51% in paired inflamed mucosa. This increase reflected the loss (73%) of total ascorbate. Reduction of dehydroascorbic acid by GSH/NADPH dependent dehydroascorbic acid reductase decreased significantly (p = 0.046) in inflamed mucosa from UC patients, suggesting that the capacity of the inflamed mucosa to maintain the concentration of reduced ascorbic acid is also diminished. HPLC analysis of mucosal preparations for diketogulonic acid, the decomposition product of dehydroascorbic acid, did not account for the loss of total ascorbate in the inflamed mucosa suggesting that ascorbate equivalents underwent further decomposition reactions or were excreted to the colonic lumen. We conclude that the normal luminal environment is strongly oxidising in character and that oxidant stress derived from inflammatory cells contributes to the loss of 35-73% total and reduced ascorbate. In absolute terms, the overall loss of this antioxidant buffering capacity would decrease the capacity of the inflamed mucosa to prevent oxidative tissue damage and hinder recovery of the inflamed mucosa.