Secreted enteric antimicrobial activity localises to the mucus surface layer.

OBJECTIVES: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the alpha-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn's ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. METHODS: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. RESULTS: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes, 48 (4)/44 (4)/8 (8); Enterococcus faecalis, 44 (10)/49 (3)/7 (7); Bacterium megaterium, 56 (4)/42 (3)/2 (1); Streptococcus pyogenes, 48 (4)/46 (3)/6 (6); Escherichia coli, 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. CONCLUSION: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.

Early changes in rectal nitric oxide and mucosal inflammatory mediators in Crohn's colitis in response to infliximab treatment.

BACKGROUND: Treatment with tumor necrosis factor-alpha monoclonal antibody (infliximab) reduces clinical activity and intestinal inflammation in Crohn's disease. AIM: To study the time-course of the effects of infliximab with reference to mucosal cytokine and inducible nitric oxide synthase expression. METHODS: Thirty-two patients with Crohn's disease were treated with single dose infliximab (5 mg/kg). Disease activity was assessed days 1, 3, 7 and 28 using Harvey-Bradshaw index. Rectal nitric oxide levels were determined and rectal biopsies collected before treatment, 1 h after infusion and on days 3, 7 and 28. Immunohistochemical staining against inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-1beta and interferon-gamma were performed. RESULTS: Clinical response was seen in 14 patients with down-regulation of global immunohistochemistry expression, reaching nadir day 3. Rectal nitric oxide was increased at baseline (3578 +/- 1199 parts per billion, ppb) compared with controls (89 +/- 13 ppb) (P < 0.001). In patients with clinical response, rectal nitric oxide decreased from 3926 +/- 1687 ppb to 1050 +/- 428 ppb day 28 (P < 0.05). CONCLUSIONS: Down-regulation of mucosal inflammatory mediators occurs after infliximab. Rectal nitric oxide levels parallel down-regulation of inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-1beta and interferon-gamma and may serve as a quantitative biomarker of intestinal inflammation.

Germ-free and colonized mice generate the same products from enteric prodefensins.

The use of germ-free mice offers the possibility to study antibacterial components in a gut uncolonized by bacteria. We have developed a method to extract and high pressure liquid chromatography-fractionate the antibacterial factors present in the small intestine of a single mouse. By mass spectrometry and sequence analyses of fractions exhibiting antimicrobial activity, we identified and characterized the defensin region in germ-free mice as well as in colonized mice. Defensins made up around 15% of the total antibacterial activity both in germ-free and colonized mice. The intestine of germ-free mice exhibited the same set of mature enteric defensins (defensins 1, 2, 3, 4, and 6) as mice colonized by a normal microflora. Mature defensins are generated through processing of larger precursors by enzymatic removal of a signal peptide and a propiece. We found that all prodefensins were cleaved at a Ser/Ala-Leu bond, giving 34-residue propiece peptides and only trace amounts of the predicted 39-residue peptide. This first step must be followed by the removal of a residual peptide to render the mature defensins, indicating that the processing is more complex than previously anticipated. The same propieces were found in both germ-free and colonized mice, suggesting that the same processing operates independent of bacterial presence in the intestine.

Experimental colitis induced by dextran sulphate sodium in mice: beneficial effects of sulphasalazine and olsalazine.

BACKGROUND: Animal models of inflammatory bowel disease are artificial and more or less representative of human disease. However, the dextran sulphate sodium (DSS) induced intestinal inflammation model has recently been shown to fulfil some pathological criteria for an adequate experimental model. AIM: To determine whether this form of experimental intestinal inflammation responds to established therapy used for human inflammatory bowel disease. METHODS: DSS was used to induce intestinal inflammation in conventional Balb/c mice and athymic nu/nu CD-1(BR) mice, and the well-documented 5-aminosalicylic acid (5-ASA) based anticolitis drugs sulphasalazine (SASP) and olsalazine (OLZ) were used to study therapeutic effects. Parameters which have been shown to reflect DSS-induced intestinal inflammation (body weight, colon length, spleen weight, diarrhoea, and rectal bleeding) were measured in the Balb/c mice. RESULTS: Significant amelioration was seen on these parameters after different treatment protocols. Survival in nu/nu CD-1 mice was studied, and after 16 days a death rate of 50% was noted in the DSS group. SASP (100 mg/kg/day) and OLZ (50 mg/kg/day) significantly prolonged the survival to 29 and 38 days, respectively. SASP and OLZ showed a dose-dependent effect in the range between 10 and 100 mg/kg/day, doses closely corresponding to those used in humans. CONCLUSIONS: SASP and OLZ are able to ameliorate the DSS-induced intestinal inflammation. The dose-response patterns suggested that the active therapeutic moiety for the two drugs appears to be mainly the liberated 5-ASA molecule.

Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: effects in CD4(+) -cell depleted, athymic and NK-cell depleted SCID mice.

Administration of dextran sulfate to mice, given in the drinking water results in acute or subacute colonic inflammation, depending on the administration protocol. This colonic inflammation exhibits ulceration, healing and repair, and a therapeutic response that makes it valuable for the study of mechanisms that could act in the pathogenesis of human ulcerative colitis, a disease thought to have an immunologically dependent pathogenesis. To investigate if immunological mechanisms were involved in the induction of colonic inflammation in this model, mice with different degrees of immunodeficiency were used. It was shown that dextran sulfate induced colitis could be induced in Balb/c mice depleted of CD4(+) helper T cells by treatment with monoclonal antibodies preceded by adult thymectomy. The depletion of CD4(+) was verified by flow cytometric analysis. Furthermore, the colonic inflammation could equally be induced in athymic CD-1 nu/nu mice lacking thymus-derived T cells, in T and B-cell deficient SCID mice, and also in SCID mice depleted of NK cells by treatment with anti-asialo GM1 antibodies. The NK-cell depletion was verified by measuring spleen NK-cell activity. The resulting colonic inflammation in all these types of deficient mice was qualitatively comparable, as shown by clinical and histological appearance. These results indicate that the presence of functional T, B and NK cells is not crucial for the induction of dextran sulfate colitis in mice.

Immune complex induced experimental colitis: beneficial effect of sulphasalazine in rabbits.

Experimental colitis was induced in rabbits by exposing the colon mucosa to 1% formalin followed by i.v. injections of soluble immune complexes made with antigen in excess. The animals were preimmunized with Escherichia coli O14:K7:H--inducing antibodies cross-reactive to intestinal epithelium. Animals with this colitis were divided in two groups. One group was treated with sulphasalazine and the other was given vehicle only. Sulphasalazine was administered daily at 125.5 mumol (50 mg) per kg body weight. The administration was started at the same day as the colitis was initiated. At Day 6, 13 and 30 following induction of colitis, biopsies were sampled and histologically evaluated. Inflammation was assessed by scores for inflammatory cells, crypt distortion, decreased crypt number and presence of crypt abscesses, thus corresponding to the picture seen in humans. A statistically significant lower score of inflammation was seen on Day 6 and 13 (P less than 0.01) and on Day 30 (P less than 0.05) following induction of colitis in animals treated with sulphasalazine.

Characteristics of immune-complex-induced chronic experimental colitis in rats with a therapeutic effect of sulphasalazine.

Experimental colitis was induced in rats by topical irritation of the colonic mucosa with 1 ml of 1% formalin followed by intravenous injection of 0.5 ml soluble immune complexes (IC) made in vitro in antigen excess and having characterized precipitation and complement activation profiles. The rats had been preimmunized with Escherichia coli O14:K7:H- to produce antibodies cross-reactive with colonic mucosa, thus aggravating the colitis to chronicity. Histologic evaluation of inflammation in the colon was performed on days 6, 12, and 18 by determining the number of phagocytic cells. The colitis was inhibited by sulphasalazine therapy given daily, 125.5 mumols (50 mg)/kg body weight, starting on the day when the inflammation was produced with IC and formalin. Sulphasalazine therapy significantly (p less than 0.05) decreased the number of phagocytic cells in the mucosa on days 12 and 18 but not on day 6. The results may give a clue to the beneficial pharmacologic effects of sulphasalazine in the treatment of ulcerative colitis.

A fluorescence method for the visualization of inflammatory cells in intestinal mucosa.

Treatment of paraffin or cryostat sections from rat, rabbit and man, with fluorescein isothiocyanate (FITC) in buffer (1 microgram/l), resulted in visualization of highly fluorescent leukocytes with a granular staining in light microscopy. FITC staining of paraffin-embedded colonic tissue was compared with three other staining methods, i.e., (1) FITC-conjugated anti-neutrophil antibodies, (2) endogenous peroxidase with 3,3-diaminobenzidine, and (3) FITC-conjugated Lotus tetragonolobus lectin. Good correlations between FITC staining and the three other methods were found (r:0.97, 0.98 and 0.98; P less than 0.001). Inhibition of receptors for the FITC conjugates prior staining, by preincubation with unconjugated anti-neutrophil antibodies or L-fucose, abolished the staining with antibodies and lectin. This study shows that staining with unlabeled FITC represents a quick and convenient method for the estimation of phagocytic cells (polymorphonuclear cells and macrophages) in histological specimens.

Statistical aspects of cell motility determinations with a modified chemotaxis assembly for multiwell filter assays.

A multiple chamber assembly for determination of neutrophil migration consisting of disposable lower plastic chambers and an easily cleaned and inert upper chamber made of stainless steel was constructed and used in experimental work. Using results of chemotaxis determinations in the assembly and a statistical model the consequences of various ways to present cell migration data are discussed. With any method for multiple measurements of chemotaxis of cells from a given sample, there are occasional extreme results ('outliers') deviating considerably from other determinations. We show that the medians of chemotaxis determinations performed in triplicate are less influenced by such outliers than the mean values. The risk of an outlier affecting the median value was in our assembly about 0.2%, compared with a 10% chance of influencing the mean value.