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Background and Purpose of Study: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association of DEFA1A3 with CD., Methods: Two-hundred and forty ethnic Danish CD patients were included in the study. Reverse transcriptase PCR assays determined DEFA1A3 expression in colonic tissue from a subset of patients. Immunohistochemical analysis identified alpha-defensin peptides in colonic tissue. Copy number of DEFA1A3 and individual alleles, DEFA1 and DEFA3, were compared with those for controls, by use of combined real-time quantitative PCR and pyrosequencing, and correlated with disease location., Results: Inflammatory-dependent mRNA expression of DEFA1A3 (P < 0.001), and the presence of alpha-defensin peptides, were observed in colonic tissue samples. Higher DEFA1A3 gene copy number (CD: mean copy number, 7.2 vs. controls 6.7; P < 0.001) and individual DEFA1 alleles (CD mean copy number 5.6 vs. controls 5.1; P < 0.01) were associated with CD, with strong association with colonic location (P < 0.001)., Conclusions: Alpha-defensins are involved in the inflammation of CD, with local mRNA and peptide expression. In combination with the findings that a high DEFA1A3 copy number is significantly linked to CD, these results suggest that a high DEFA1A3 copy number might be important in hindering the normal inflammatory response in CD, particularly colonic CD.

Familial disposition and mutations in the Caspase Recruitment Domain 15 (CARD15) have been associated with an increased risk for Crohn's disease (CD). This study investigated whether these risk factors correlate with colonic gene expression profiles generated by DNA-microarray technology. Tissue specimens from descending colon were obtained during colonoscopy from 45 CD patients (18 from areas with inflammation and 27 from noninflamed areas). Gene profiling analysis was performed using the Human Genome U133 Plus 2.0 GeneChip Array. Patients were classified according to their CARD15 status. Hybridization data were analyzed with dChip software. Nine patients with either one or two CARD15 mutations had no differentially expressed genes, compared to 36 patients with wild- type CARD15. There was only one differentially expressed EST between 8 patients who had familial disposition for inflammatory bowel disease (IBD) and 36 who did not, but hierarchical cluster analysis did not show group homogeneity. We conclude that gene expression profiling of mucosal biopsies from the descending colon of patients with CD could not be correlated with CARD15 status or with familial disposition for IBD.

Epithelial cell growth regulation has been reported to be altered in inflammatory bowel disease (IBD) patients. The cell growth regulatory factor, insulin-like growth factor binding protein 3 (IGFBP-3), may be partly responsible for this phenomenon. So far, IGFBP-3 levels have been assessed as values of total protein, which is a sum of bioactive intact 43- to 45-kDa protein and its inactive proteolytic cleavage fragments. We aimed to assess the levels of intact IGFBP-3 and its cleaving protease MMP-9 in IBD. Patients with IBD and controls were included. Total plasma IGFBP-3 concentration was measured in ELISA. Western blot analysis, which distinguishes between intact and cleaved IGFBP-3, was performed in order to determine the ratio of intact to total protein; this ratio was used to calculate the concentration of intact IGFBP-3. The profile of plasma proteases was evaluated in zymography and MMP-9 levels were determined in ELISA. The concentration of intact IGFBP-3 was significantly decreased in patients with moderate to severe IBD activity compared to those in remission or controls. Of note, a dramatic depletion of intact IGFBP-3 was found in 7.4% of patients with IBD. Zymography revealed that the dominant gelatinase was the pro-form of MMP-9. However, no differences in MMP-9 levels were noted between those with active disease and controls. The level of intact IGFBP-3 is decreased in IBD patients with moderate to severe disease activity. This decrease may be linked to altered IGFBP-3 production or to increased cleavage by proteases other than MMP-9.

A diminished tolerance to the normal gut bacterial flora has been suggested to be pathogenic in ulcerative colitis (UC) and the aim of this study was to evaluate the priming effect of selected bacterial wall products on UC neutrophil granulocytes. Neutrophils from 10 UC patients and 10 healthy controls were primed with bacterial lipoprotein (BLP) or lipopolysaccharide (LPS) and subsequently activated. Extracellular superoxide production was measured by the cytochrome c reduction assay. Priming neutrophils with BLP or LPS dose dependently increased the superoxide production in both UC and controls (P < 0.01), and BLP was more potent than LPS (P < 0.05). No differences were found between UC and controls. UC neutrophils do not seem to have an intrinsic abnormality with reduced tolerance to bacterial substances. However, bacterial wall products such as BLP modify neutrophil tissue-destruction mechanisms and might be pivotal for perpetuation of chronic colonic inflammation.

Autoantibodies against neutrophil granulocytes are frequently observed in patients with ulcerative colitis, but a precise description of the autoantigens involved is lacking. Therefore, sera from 75 patients with ulcerative colitis were studied for antibody specificities by means of indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, and immunoblotting of extracts of neutrophils, neutrophil granules and lymphocytes. Fifty-six percent of the sera reacted in indirect immunofluorescence with ethanol-fixed neutrophils. On formalin-fixed cells most sera shifted fluorescence pattern, indicating a cytoplasmic origin of antigen(s). Only a few sera reacted with specific, known antigens. Immunoblots showed reactions with a broad panel of antigens with preferences for proteins around 55-65 kDa, which were present in azurophilic granules and in cytosol, an 80-kDa protein found in the specific granules, and a 110-kDa unknown cytosol component. In conclusion, neutrophil-specific IgG autoantibodies from ulcerative colitis patients react with several different antigens, most of which are of nonnuclear origin.

The pathogenic significance of cell adhesion molecules (CAMs) in ulcerative colitis (UC) is largely unknown. Colonic expression of E-selectin, sialyl Lewis X (sLe(x)), and macrophage inflammatory protein-1x (MIP-1alpha) as well as serum concentrations of E-selectin and MIP-1alpha in UC were studied. Thirty patients with UC, 10 patients with irritable bowel syndrome, and 10 healthy subjects were included. Colonic biopsies were stained immunohistochemically, and blood concentrations were measured with an ELISA technique. Soluble E-selectin did not correlate with diagnosis or disease activity. MIP-1alpha was below the detection limit. Epithelial cells expressed all three molecules, both on surface membranes and intracellularly. sLe(x) staining was weaker (P = 0.0002) and MIP-1alpha staining stronger (P = 0.014) in UC patients than in controls. Leukocyte MIP-alpha staining correlated with diagnosis (P = 0.021), sLe(x) staining (P = 0.023), and colonoscopy (P = 0.018). It is shown that E-selectin, sLe(x), and MIP-1alpha are synthesized and expressed by epithelial cells, indicating that CAMs are not only involved in leukocyte extravasation and migration, but also in the interaction between leukocytes and colonic epithelium. This knowledge might contribute to the development of improved treatments in UC.

Mononuclear cell proliferative response to IL-2 and functional response to IL-4 is disturbed in IBD. The aim of the present study was to verify whether altered expression of the common gamma chain (gamma c) might contribute to this abnormality. The gamma c expression on peripheral blood mononuclear cells was analyzed by flow cytometry using a monoclonal antibody to gamma c. IL-4 binding in association with gamma c expression was assessed using biotinylated IL-4 in two-color flow cytometry. Mean fluorescence of gamma c expression was significantly decreased in active CD patients as compared to inactive CD and healthy controls (P < 0.05). Cell activation as a possible reason for gamma c down-regulation was confirmed using the in vitro stimulated donor cells. In the same system it was shown that down-regulation of gamma c is accompanied by decreased numbers of IL-4 binding cells. In conclusion, a decreased expression of the gamma c on peripheral blood mononuclear cells from CD patients with active disease might have an important pathogenetic significance in this chronic intestinal disorder.

The pathogenesis of ulcerative colitis (UC) and Crohn's disease (CD) may be associated with a decreased production of cytokines suppressing macrophage and T-cell functions: interleukins (IL) -4 and IL-10. Serum concentrations of IL-4 and IL-10 were measured using an ELISA technique, and intestinal IL-4 and IL-10 mRNA was detected by a reverse transcriptase polymerase chain reaction (RT-PCR) in 34 patients with inflammatory bowel disease (IBD) (20 with UC and 14 with CD) and compared to 12 control subjects. The superoxide production was measured spectrophotometrically in activated PMNs initially incubated in the presence of IL-4 or IL-10. No differences were found in numbers of cells that might be potential IL-4 or IL-10 producers (T cells, macrophages, B cells, and mast cells) in biopsy specimens using immuno- and histochemistry. IL-4 mRNA was detectable in specimens from 77.8% of the UC patients (P > 0.05) and 0% of the CD patients (P < 0.05), as compared to 81.8 in controls, and was significantly different (P < 0.0001) between UC and CD patients. The IL-10 amplification product was detectable in specimens from 30.0% UC patients (P < 0.003), but not in CD patients (78.6%, P > 0.05) as compared to controls (91.7%). The circulating protein levels of IL-4 were below the detection limit in all groups (detection limit 4 pg/ml), while the median IL-10 concentration was 12.5 pg/ml in UC, 18.1 pg/ml in CD, and 19.5 pg/ml among controls (detection limit 3 pg/ml), which did not differ in any of the three groups (P > 0.05). Finally, the superoxide production was inhibited and delayed by the addition of IL-10 (P < 0.01), whereas IL-4 only delayed this parameter. In conclusion, apart from the well-known suppressive effect on proinflammatory cytokine production, IL-4 delays and IL-10 inhibits superoxide generation. IL-4 mRNA expression is decreased in intestinal tissue from CD patients, while IL-10 mRNA expression is decreased in majority of UC patients, suggesting different immunopathogenesis of the two diseases.

Cell surface adhesion molecules (CAM) are important promotors of the immunoinflammatory cascade. The circulating levels of soluble intercellular adhesion molecule 1 (ICAM-1) have previously been shown to correlate with disease activity in inflammatory bowel disease. The primary aim of this study was consequently to investigate if this also applies to mucosal levels of soluble ICAM-1. We measured soluble ICAM-1 levels in intestinal biopsy specimens and the endoscopic activity of 69 patients with ulcerative colitis (UC) and 14 controls and found that the median concentration of soluble ICAM-1 was significantly higher in patients with moderately or very active UC (15.0 ng/ml) as compared to slightly active (9.8 ng/ml) and inactive UC (9.5 ng/ml) as well as controls (6.5 ng/ml) (P < 0.005). To further elucidate the interactions, two other CAM [E-selectin and vascular cellular adhesion molecule 1 (VCAM-1)], together with interleukin-8 (IL-8), IL-2 receptor (IL-2R) alpha and beta chains, were also measured. A significant trend towards higher soluble E-selectin levels in biopsies with active UC (1.8 pg/ml) as compared to inactive UC (1.3 pg/ml) and to controls (< 1.0 pg/ml) (P < 0.01) was also found. In contrast, soluble VCAM-1 was barely detectable in biopsies from two UC patients. A significant correlation was found between soluble ICAM-1 and IL-8 concentrations (r = 0.46; P < 0.0001), and between sICAM-1 and sIL-2R alpha concentrations (r = 0.69; P < 0.0001), while sIL-2R beta was not detected. This study shows that intestinal ICAM-1 and E-selectin correlate with endoscopic activity of UC and with IL-8 and IL-2R alpha levels. These mediators may be useful in monitoring mucosal inflammation in studies exploring the therapeutical potential of targeting CAM. The lack of detectable VCAM-1, which is induced only in venous endothelium is interesting. It may suggest that intestinal inflammation mainly affects arterial endothelial cells and support the theory that intestinal vasculitis is involved in the pathogenesis of inflammatory bowel disease.

A simple filter-paper method for in vivo assessment of cytokines in intestinal mucosa of patients with ulcerative colitis (UC) was developed and evaluated. Twenty-eight patients were included. Twenty-three patients had ulcerative colitis (UC) and five irritable bowel syndrome (IBS). Inflammation was assessed endoscopically. Through a rectoscope, filter paper was applied to the macroscopically most inflamed area of the rectal mucosa until soaked. The filter paper was transferred to a buffer solution, and IL-1 beta and IL-1ra were assessed using ELISA. Positive correlations between endoscopic grading and In(IL-1 beta) (P < 0.0001) and In(IL-1ra) (P < 0.001) and a negative correlation between endoscopic grading and In(IL-1ra /IL-1 beta) (P < 0.02) were found. In measurements during and after a flare-up no significant change in IL-1ra but a significant decrease in IL-1 beta was detected, which is in agreement with investigations on biopsies. In conclusion, the filter-paper technique is an easily applicable, low-risk method that provides a means of monitoring cytokines in vivo in UC.

The adhesion molecule, lymphocyte function associated antigen (LFA-1) consisting of two subunits, CD11a and CD18, mediates lymphocyte migration into tissue and cell effector functions. Previous observations showed no differences in LFA-1 expression by circulating lymphocytes between inflammatory bowel disease patients and controls. The aim of the present work was to study subsets of circulating LFA-1+ lymphocytes in ulcerative colitis (UC) patients versus healthy controls. Peripheral blood mononuclear cells were obtained from 16 UC patients and 10 healthy volunteers. The percentages of CD11alo, CD11ahi, CD18lo, CD18hi T and B cells, as well as Cd25 expression on these cells were studied using double staining with monoclonal antibodies and panning procedures. The percentage of CD11hi and CD18hi T cells was significantly decreased in quiescent UC patients as compared to active disease patients and healthy controls (P < 0.05). The majority of CD25+ T cells were expressing CD11a and CD18 with low density. A detectable percentage, 2% (range 1-6%), of CD11ahiCD25+ (but not CD18hiCD25+) was found in UC patients with moderate to severe disease, but not in those with inactive UC to healthy controls. In conclusion, the percentage of CD11ahi+ and CD18hi+ T cells is decreased in peripheral blood of quiescent UC patients, which is probably associated with the effect of specific treatment. The percentage of CD11ahi+IL-2R alpha+ T cells is increased in peripheral blood of patients with active (moderate and severe) UC, which most likely reflects a sustained T-cell activation due to a persistent inflammatory process.

The pathogenetic role of activated alpha beta and gamma delta T cells in inflammatory bowel disease (IBD) is not well defined. To elucidate this, interleukin-2 receptor (IL-2R) alpha and IL-2R beta single chain expression and coexpression by peripheral blood TCR alpha beta + cells and TCR gamma delta + cells was studied in 21 patients with ulcerative colitis (UC), 25 with Crohn's disease (CD), and 15 controls. The percentages of IL-2R alpha + beta-, IL-2R alpha-beta +, and IL-2R alpha + beta + TCR alpha beta + cells were increased in IBD patients with moderate and severe disease activity, as compared to controls (P < 0.01). In contrast, the percentages of IL-2R alpha-beta + and IL-2R alpha + beta + TCR gamma delta + cells were increased in patients with inactive UC (P < 0.01), but not in CD. The results suggest that activated alpha beta T cells are involved in the development of IBD. The differences in gamma delta T cell IL-2R expression between inactive UC and CD may correspond to a yet undefined etiopathogenetic difference between these two diseases.

Intercellular adhesion molecule (ICAM)-1 promotes the initial interaction between macrophages and T cells during immune activation. We have measured serum levels of soluble ICAM-1 (sICAM-1) by ELISA in 27 patients with ulcerative colitis (UC), 31 with Crohn's disease (CD), and 29 healthy subjects. The median sICAM-1 serum concentration was significantly increased in inflammatory bowel disease (IBD) patients (355 ng/ml, range 195-855) compared to controls (245 ng/ml, 155-580) (P = 0.001). Variance analysis for trend showed that sICAM-1 levels were significantly higher in patients with active CD and UC, compared to those with inactive disease and controls (P = 0.00002). The concentration of sICAM-1 was higher in CD patients (365 ng/ml 230-470) compared to UC (300 ng/ml 195-855) (P = 0.01). Furthermore, weak but significant correlations were found between serum levels of sICAM-1 and: soluble IL-2 receptors, orosomucoid, and C-reactive protein. It is suggested that increased circulating sICAM-1 levels may reflect increased adhesiveness and signal transmission across cells, probably as a result of shedding of the parent molecule during local cellular immunoresponses in vivo.

The possible effect of sulfasalazine, 5-aminosalicylic acid, and acetyl-5-aminosalicylic acid on endogenous arachidonic acid release and metabolism was studied in human polymorphonuclear leukocytes (PMNs). A new in vitro assay was used by which [1-14C]arachidonic acid is incorporated by purified peripheral PMNs until steady state was obtained (5 hr). After preincubation with the test drugs prior to activation with calcium ionophore A23187, the released eicosanoids were isolated by extraction and thin-layer chromatography (TLC) and quantitated by autoradiography and laser densitometry. Median drug concentrations needed for 50% inhibition of leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) release was 4-5 mM (range 1-9 mM) for both sulfasalazine and 5-aminosalicylic acid. The acetylated derivative of 5-aminosalicylic acid was ineffective. The present data suggest that inhibition of arachidonic acid lipoxygenation may be an essential action of sulfasalazine and its active metabolite, 5-aminosalicylic acid. Interference with lipoxygenase enzymes, rather than a steroid-like inhibition of arachidonic acid release from intracellular phospholipids, seems to be the mode of action.

A new method employing 99mTc-labeled pellets for determination of the gastric emptying rate and small bowel transit time is described. The participants were six normal subjects and 16 patients with irritable bowel syndrome (eight with diarrhea and eight with obstipation as the primary complaint). The gastric emptying rate was the same in the three groups. The patients in the obstipation group had a significantly longer small bowel transit time than the normals (P less than 0.02) and the patients in the diarrhea group (P less than 0.01). There was no demonstrable difference between the small bowel transit time in the normals and in the patients in the diarrhea group.