Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats.

We studied the presence of bacterial antigens in rat tissues. We produced a monoclonal antibody (MAb 2E9) directed against intestinal flora-derived peptidoglycan-polysaccharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed by immunohistology with a previously described MAb 2-4 directed to muramyl dipeptide. Other lymphoid organs did not stain positively with 2E9 and 2-4. Neonatal and young rats showed no staining of the spleen, but positivity could be induced by injecting peptidoglycan-polysaccharide complexes systemically. We conclude that bacterial fragments are present in splenic macrophages of conventional rats.

Degradation of intestinal glycoproteins by Bacteroides vulgatus.

Bacteroides vulgatus, isolated from a patient with Crohn's disease, produced in gnotobiotic rats 7 constitutive enzymes that might be concerned with the degradation of intestinal glycoproteins. Furthermore Bacteroides vulgatus caused an almost complete loss of blood group antigenicity of the intestinal glycoproteins. Enzymes with the potency to release toxic compounds from hepatic conjugates and plant glycosides, beta-glucuronidase and beta-glucosidase, respectively, were only detectable in small amounts. These findings indicate that Bacteroides vulgatus, which accounts for 40% of the total flora of patients with Crohn's disease, may play a role in the pathogenesis of Crohn's disease, by increasing the break-down of the mucus layer and therefore damaging its protective function.

Increased proteolysis and leucine aminopeptidase activity in faeces of patients with Crohn's disease.

Proteolytic and leucine aminopeptidase activity levels in faeces from patients with Crohn's disease and healthy subjects were compared and were found to be 2.5 and 6 times higher, respectively, in patients with Crohn's disease. Patients who had undergone partial bowel resection even showed a 12- and 40-fold increase of enzyme activities. A part of the elevated levels of faecal proteases probably results from ileal malabsorption. This was supported by studies in germ-free and conventional rats showing that the ileum is the major site of absorption of digestive proteolytic enzymes and leucine aminopeptidase. The other part of the increased protease activity was attributed to the abnormal anaerobic faecal flora of patients with Crohn's disease. Interactions between flora and enzymes were studied in rats. The resident flora of the caecum was found to stimulate absorption, or to inactivate the major part of the remaining enzymes. The elevated levels of faecal proteases in Crohn's disease may play a role in the damage of the intestinal mucosa.

Increased faecal glycosidases in patients with Crohn's disease.

The strictly anaerobic faecal flora of patients with Crohn's disease (CD) contains higher numbers of coccoid rods (Eubacterium, Peptostreptococcus and Coprococcus species) and gram-negative rods (Bacteroides vulgatus) than the flora of healthy subjects. The abnormal flora of patients with CD might affect levels of glycolytic enzymes and metabolic products and thus play some role in the pathogenesis of CD. Therefore, the activity of 23 glycosidases that can degrade mucus glycoproteins or plant polysaccharides, in faeces of patients with CD was compared with healthy subjects. Total enzyme activity in faecal samples of patients with CD was significantly higher than in healthy subjects, but statistically the activity of individual enzymes did not significantly differ. Faecal glycosidase activity is predominantly of bacterial origin since glycosidase activity was absent or very low in faeces of germ-free rats.

Inability of Lactobacillus casei strain GG, L. acidophilus, and Bifidobacterium bifidum to degrade intestinal mucus glycoproteins.

BACKGROUND: Lactic acid bacteria have been suggested for use in the prevention of relapse of ulcerative colitis and of recurrent pouchitis. These strains may not damage the protective intestinal mucus glycoproteins. METHODS: Lactobacillus casei strain GG and strains isolated from a commercial fermented product (Lactobacillus acidophilus, Bifidobacterium bifidum, and a mesophylic lactic culture) were cultured in vitro on hog gastric mucin and human intestinal glycoproteins. Furthermore, germ-free rats were mono-associated with Lactobacillus GG and poly-associated with the other strains. Glycoproteins were isolated from rat distal ileum, cecum, and colon. Mucus degradation was established by assaying carbohydrates (hexosamines, hexoses, pentoses), proteins, and blood group antigenicity. RESULTS: All strains colonized the intestinal mucus but were not found in the deep crypts. Degradation of mucus glycoproteins was observed neither in vitro nor in vivo. CONCLUSION: The tested strains do not break down intestinal mucus glycoproteins and thus far are safe to use for therapy.

Pouchitis: result of microbial imbalance?

To elucidate the role of microbiological factors in pouchitis, this study investigated the composition of ileal reservoir microflora, the mucus degrading capacity of bacterial enzymes as well as the pH and the proteolytic activity of pouch effluent. Stool samples were collected from five patients with pouchitis and nine patients without pouchitis. The flora of patients with pouchitis had an increased number of aerobes, a decreased ratio anaerobes to aerobes, less bifidobacteria and anaerobic lactobacilli, more Clostridium perfringens, and several species that were not found in control patients (for example, fungi). Furthermore the pH was significantly higher in patients with pouchitis (median value 6.5) than in control patients (5.4). To find out if the pH might influence the breakdown of intestinal mucus glycoproteins, the activity of glycosidases and proteases, and the degradation of hog gastric mucin by the pouch flora was tested at pH 5.2-7.6. Some glycosidases were inhibited, others were stimulated by a low pH, however, in each sample the proteolytic activity was inhibited for 75% at pH 5.2 compared with pH 6.8 and 7.6. Degradation of hog gastric mucin by the pouch flora was an active process at pH 7.2: within two to four hours of incubation more than half of the mucin was degraded. At pH 5.2 it took twice as long. It is concluded that pouchitis possibly results from instability of the flora in the pouch, which causes homeostasis to disappear (dysbiosis), and the protection of the pouch epithelium by the mucus layer becomes affected by increased activity of bacterial and host derived enzymes.

Changes in bacterial enzymes and PCR profiles of fecal bacteria from a patient with ulcerative colitis before and after antimicrobial treatments.

A 26-year-old female patient, suffering from recurrent attacks of ulcerative colitis accompanied by extraintestinal symptoms (erythema nodosum and pyoderma gangrenosum), was evaluated for the effect of antibacterial agents on the intestinal bacteria and their enzymatic activities. The enzymes were assayed both at the onset of disease symptoms and after treatment with each of five drug regimens (fluconazole and cefadroxil, cefuroxime axetil and cestriaxone sodium, ciprofloxacin and cestriaxone sodium, ciprofloxacin alone, and ciprofloxacin, metronidazole, and cephalexin). The activities of azoreductase, nitroreductase, oxidoreductase, glucuronidase, and sulfatase were generally lower following all of the treatments, especially when ciprofloxacin was included. The DNA from each sample was amplified by PCR, using random primers. Profiles of amplified DNA on agarose gels showed different patterns, indicating differences in the microflora before and after the antimicrobial treatments. The clinical response to antibacterial therapy was consistent with the decreased bacterial enzymatic activities and changes in the microbial population. Ciprofloxacin, which was associated with the most dramatic falls in enzymatic activity, also had the best clinical results. We conclude that intestinal bacteria and their enzymes play important roles in ulcerative colitis and that population changes can be monitored using PCR profiles.

Changes in bacterial composition and enzymatic activity in ileostomy and ileal reservoir during intermittent occlusion: a study using dogs.

Bacterial flora, activities of 10 potential mucus- and dietary polysaccharide-degrading enzymes, blood group antigenicity of the intestinal glycoproteins, and proteolytic activity in the output from experimentally colectomized dogs with conventional ileostomies and dogs with valveless ileal reservoirs (pouches) were determined. The ileostomies of dogs with conventional surgery (group II) and with pouches (group III) were occluded intermittently during a 6-week period. The duration of occlusion was progressively increased. Group I, five dogs with conventional ileostomies, served as a control group. After occlusion of the ileal pouch for 7 h, total numbers of bacteria increased threefold, glycosidase activity increased fivefold, and blood group antigenicity of the intestinal glycoproteins, which was high in the output from the nonoccluded pouch, was no longer detectable. Proteolytic activity was not influenced by occlusion of the pouch. Significantly lower numbers of bacteria, only minor glycosidase activity, high blood group antigenicities of the intestinal glycoproteins, and higher proteolytic activity were found in ileostomy effluents from groups I and II. Histopathological examination showed chronic inflammation and changes in crypt-villus ratio in all dogs with ileal reservoirs; the ileal mucosa from the dogs with conventional ileostomies did not show any abnormalities. Consequences of the flora-related enzyme activities for the ileal mucosa are discussed.

Induction in rats of serum agglutinins to Eubacterium, Peptostreptococcus and Coprococcus species by the faecal flora from patients with Crohn's disease and healthy subjects.

Sera from patients with Crohn's disease more often than those from other subjects contain agglutinins against anaerobic coccoid rods, identified as Peptostreptococcus productus, Eubacterium contortum (two strains) and Coprococcus comes. The presence of antigens of the four coccoid rods in faeces of patients with Crohn's disease and healthy subjects was investigated by inoculation of rats with faecal suspensions. Antigens of the coccoid rods were detected in faeces from both patients with Crohn's disease and healthy subjects.

Isolation and characterization of the viscous, high-molecular-mass microbial carbohydrate fraction from faeces of healthy subjects and patients with Crohn's disease and the consequences for a therapeutic approach.

1. An earlier study by our group revealed that the viscosity of faeces from patients with Crohn's disease is significantly lower than that of healthy subjects. This is due to low concentrations of a high-molecular-mass carbohydrate, probably of bacterial origin. The cause of this phenomenon might be the impaired barrier function of the gut mucosa. Low viscosity may allow close contact of intestinal contents (bacterial products and toxins) with the intestinal wall. This could play a role in the maintenance of the disease.2. The first aim of this study was to investigate the high-molecular-mass carbohydrate fraction, responsible for viscosity, in detail. We also tried (in a pilot study) to raise the intestinal viscosity of patients with Crohn's disease with the undegradable food additive hydroxypropylcellulose (E463), in an attempt to alleviate clinical symptoms.3. The high-molecular-mass fraction (>300 kDa) responsible for faecal viscosity was sensitive to lysozyme and contained high levels of muramic acid. It was concluded that this material consisted mainly of peptidoglycan polysaccharides and was consequently of bacterial origin. The muramic acid in material from patients with Crohn's disease was 7.5 (1.5-13.9)%, which was less than in healthy subjects [11.4 (8.5-24.1)%; P=0.0004]. Furthermore, viscosity in material from patients with Crohn's disease was found to be half [14.9 (1.0-33.6) cP] of that found in healthy subjects [35.0 (2.7-90.7) cP; P=0.004].4.A daily dose of 1 g of hydroxypropylcellulose caused an increase in faecal viscosity in patients with Crohn's disease (from 1.4 to 2.3 cP) and in healthy subjects (from 4.9 to 7.5 cP). Faecal consistency improved in patients with Crohn's disease (from watery and loose to formed) and the defecation frequency decreased from 3-4 to about 2 times a day. No changes in defecation patterns were found in healthy subjects.5. These data indicate that the high-molecular-mass fraction that is responsible for faecal viscosity is peptidoglycan. Furthermore, a daily dose of a hydroxypropylcellulose solution to increase the viscosity of the intestinal contents of patients with Crohn's disease might be beneficial. This approach merits further study.

Antibodies to Coprococcus comes in sera of patients with Crohn's disease. Isolation and purification of the agglutinating antigen tested with an ELISA technique.

Previous studies showed that agglutinating antibodies to Coprococcus comes, an anaerobic Gram-positive coccoid rod isolated from the faecal flora of patients with Crohn's disease, are more frequently found in sera of Crohn patients than in ulcerative colitis patients and healthy subjects. Isolation of the antigen may be useful in developing a more sensitive and specific diagnostic test. The present study describes first a method to improve the presentation of the relevant agglutinating antigen by the bacterium and second, the purification by column chromatography of a relatively crude antigen extract of C. comes described previously by Hazenberg et al. (1). Comparative results with the agglutination reactions and ELISA technique of extensive series of patients with Crohn's disease and healthy subjects have shown that the agglutinating antigen of C. comes has been isolated. Although the present ELISA technique cannot replace the simple and reliable agglutination reaction for screening purposes, the purified antigen will allow further immunological studies and it is to be hoped that a deeper insight into pathogenesis of the disease will be gained.

Potato tuber proteins efficiently inhibit human faecal proteolytic activity: implications for treatment of peri-anal dermatitis.

BACKGROUND: Frequent diarrhoea after intestinal resections and faecal incontinence in healthy infants may lead to perianal injury. A causative agent may be a high concentration of pancreatic proteases in faeces. The aim of the present study was to assess whether protease inhibitors are applicable for treating and preventing peri-anal dermatitis by inhibiting the initial cause of the inflammation, the faecal proteases. DESIGN: Proteolytic activity was estimated in faeces of subjects frequently suffering from peri-anal dermatitis: patients with intestinal resections and healthy infants. The development of perianal dermatitis was studied after the construction of a reservoir with ileoanal anastomosis. The inhibitory effect of crude and partly purified potato juice on proteolytic activity of faecal output from patients with intestinal resections and healthy infants was investigated in vitro and in vivo (skin tests). RESULTS: Faecal protease activity in faeces from patients with intestinal resections and healthy infants was found to be significantly higher than in healthy adults. After the construction of an ileum reservoir, 46 of 48 patients developed a protease-related peri-anal dermatitis. The partly purified protein fraction from potatoes inhibited the larger part of faecal proteases in vitro and completely prevented skin irritation by pancreatic proteases dissolved in sterilized faecal fluid, in a 24-h skin test, on the back of healthy human volunteers. CONCLUSIONS: Potato proteins contain protease inhibitors, which suppress almost the complete proteolytic activity in faeces. Topical application of potato protease inhibitors might be a novel approach in preventing protease-induced peri-anal dermatitis, and therapeutic studies are needed to confirm our results.

Induction of arthritis in rats by soluble peptidoglycan-polysaccharide complexes produced by human intestinal flora.

Peptidoglycan-polysaccharide complexes were isolated from feces of a healthy subject and from ileostomy fluid from 5 patients. Peptidoglycan-polysaccharide complexes were tested for arthritogenicity in a rat model, by subcutaneous injection in Freund's incomplete adjuvant. Complexes from the healthy subject did not induce arthritis, but those from ileostomy fluid of 1 of the patients induced severe, chronic joint inflammation. We concluded that peptidoglycan-polysaccharide complexes from intestinal flora are potentially arthritogenic in rats. This arthritogenicity may be influenced by the content of muramic acid and rhamnose in these complexes.