CARD4/NOD1 is not involved in inflammatory bowel disease.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex genetic disorders. CARD15/NOD2, a member of the Ced4 superfamily which includes Apaf-1 and CARD4/NOD1, has recently been associated with genetic predisposition to CD but additional genetic factors remain to be identified. Because CARD4/NOD1 shares many structural and functional similarities with CARD15, we tested its putative role in IBD. PATIENTS AND METHODS: The 11 exons of CARD4 were screened for the presence of variants in 63 unrelated IBD patients. The only non-private genetic variation encoding for a substitution in the peptidic chain was genotyped in 381 IBD families (235 CD, 58 UC, 81 mixed, and seven indeterminate colitis families) using a polymerase chain reaction-restriction fragment length polymorphism procedure. Genotyping data were analysed by the transmission disequilibrium test. RESULTS: Five of nine sequence variations identified in the coding sequence of the gene encoded for non-conservative changes (E266K, D372N, R705Q, T787M, and T787K). Four were present in only one family. The remaining variant (E266K), which exhibited an allele frequency of 0.28, was not associated with CD, UC, or IBD. Furthermore, IBD patients carrying sequence variations in their CARD4 gene had a similar phenotype to those with a normal sequence. CONCLUSION: Our results suggest that CARD4 does not play a major role in genetic susceptibility to IBD.

Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.

Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.

Genetic refinement and physical mapping of a chromosome 16q candidate region for inflammatory bowel disease.

Crohn's disease (CD) is a complex genetic disorder for which a susceptibility gene, IBD1, has been mapped within the pericentromeric region of chromosome 16. In order to refine the location of IBD1, 77 multiplex CD families were genotyped for 26 microsatellite markers evenly spaced by approximately 1 cM. Nonparametric linkage analyses exhibited a maximum NPL score of 3.49 (P=2.37x10(-4)) in a region centred by markers D16S3136, D16S3117 and D16S770. Simulation studies showed that the probability for IBD1 to be located in a 5 cM region around these markers was 70%. A 2.5 Mb YAC and BAC contig map spanning this genetic region on chromosome band 16q12 was built. TDT analyses demonstrated suggestive association between the 207 bp allele of D16S3136 (P<0.05) and a new biallellic marker hb27g11f-end (P=0.01). These markers were located in the hb27g11 and hb87b10 BAC clones from the contig. Taken together, the present results provide a crucial preliminary step before an exhaustive linkage disequilibrium mapping of putatively transcribed regions to identify IBD1.

Effects of oral Saccharomyces boulardii on bacterial overgrowth, translocation, and intestinal adaptation after small-bowel resection in rats.

BACKGROUND: Small-bowel resection in animals results in alterations of the morphology and functional adaptation in the remaining intestine. The aim of our study was to study the effect of Saccharomyces boulardii versus placebo in rats after 50% small-bowel resection. METHODS: Sixty-three rats were assigned to one of three groups: small-bowel resection (n = 31), transected surgery controls (n = 16), or non-surgical controls (n = 16). Of the 31 rats with small-bowel resection, 15 were given S. boulardii (140 mg/dl), and 16 were given placebo. Intestinal markers measured included bacterial overgrowth (BO) on days 4 and 8 and translocation into mesenteric lymph nodes, liver, and spleen. Markers of small-bowel adaptation included histomorphology of the mucosa, protein content, and various brush-border enzymes (sucrase, glucoamylase, n-aminopeptidase). RESULTS: In the jejunal mucosal samples on day 8, S. boulardii-treated rats showed a significant increase in protein content (58.3 +/- 12 mg/10 cm) compared with placebo-treated rats (29.2 +/- 1.8) or non-surgery controls (18.3 +/- 1.2; P < 0.001). S. boulardii-treated rats also had significantly higher levels of all three brush-border enzymes. A significant increase of enzyme-specific activities was observed in the ileum of S. boulardii resected rats compared with the placebo resected group on day 4, and no significant differences were seen in the remnant ileum except an increase in protein content in S. boulardii-treated rats on day 8. Histomorphometric studies showed no differences in ileal villus height or translocation frequencies by day 8 in S. boulardii or placebo resected rats. CONCLUSIONS: These data indicate that, after resection, S. boulardii does not modify bacterial overgrowth or translocation frequency but does significantly enhance the functional adaptation of the remaining intestinal segments.

Trophic response of the intestinal mucosa to inflammation in rats injected with turpentine: a study using pair-fed controls.

The effects of restricted food intake and acute inflammation on the small bowel were studied, Wistar rats (250 g) were given subcutaneous injections of turpentine (TR) and compared to two control groups, at 18, 42 and 66 h. One was fed ad libitum (C), the other was pair fed (PF) with TR. The TR and PF rats showed hypoplasia of the jejunal mucosa with decreased protein and DNA contents at 42 h and 66 h. The hypoplasia resulted in a reduced villus height that was significantly different from the controls at 66 h (C: 468 +/- 17, TR[66] : 376 +/- 20, PF[66] : 258 +/- 2.9 microm, P<0.001). This decrease in villus height was significantly greater in the PF rats than in the TR rats at 66 h. The crypt height/villus height (C/V) ratio in the PF rats was greater than in the TR group at all times. However, the protein and DNA contents in the TR group were significantly higher than in the PF group at 42 h and 66 h (TR/PF[42] : 29.5 +/- 1.9 vs 20.5 +/- 2.0, P< 0.001, [66]: 25.8 +/- 2.0 vs 16.6 +/- 1.3 mg/10 cm, P,< 0.001). Disaccharidase activities (sucrase and glucoamylase) per 10 cm jejunum at 66 h were significantly lower in the PF group than in the control and TR groups (sucrase mU/10cm[66] C : 3090 +/- 144, TR 2683 +/- 479, PF 1969 +/- 144, P,< 0.001; glucoamylase mU/10 cm[66] 237 +/- 25, TR 169 +/- 40, PF 123 +/- 5, P< 0.01). The N-aminopeptidase patterns in the TR and PF groups were similar. These data suggest that dietary restriction during acute inflammation is the main factor causing hypoplasia of the jejunal mucosa. However, acute inflammation has a trophic effect on the morphological and function of the mucosa. This effect is probably due to inflammatory mediators, whose synthesis is stimulated by turpentine.

Effects of alimentary intact proteins and their oligopeptide hydrolysate on growth, nitrogen retention, and small bowel adaptation in inflammatory turpentine rat.

The effects of dietary proteins given as whole proteins (WP) or as a peptide hydrolysate (PH) on growth, nitrogen retention, and small bowel adaptation were assessed using two groups of male Wistar rats. Measurements were made 18, 42, and 66 h after acute inflammation induced by subcutaneous injections of 0.125 mL turpentine and in two control groups (n = 12). The two diets had the same caloric, nitrogen, vitamin, and mineral content. The WP diet resulted in better weight gain, nitrogen retention, and small intestinal adaptation by control rats than did the PH diet. Loss of body weight after 18 h of acute inflammation was significantly lower and nitrogen retention significantly higher in animals on the WP diet than in those on the PH diet. Small intestine morphology was maintained with the WP diet, whereas villus height was significantly lower after 66 h, and there were fewer mitoses per crypt in the rats on the PH diet. Glucoamylase activity at all times, and N-aminopeptidase activity at 18 h, were significantly higher in rats on the WP diet. The putrescine (at 42 h) and spermidine (at 18 h) concentrations in the mucosa were higher in the rats on the WP diet. These data suggest that synthetic diets should be tested for their nutritional value during acute inflammation before they are used in human nutrition.

Antigenicity and nutritional value of selected milk proteins and their hydrolysate in growing rats.

Two liquid diets containing selected milk proteins (SMP) or its small peptide hydrolysate (SPH) were fed to growing rats for 2 wk and the effects on growth, nitrogen balance, and small intestine adaptation were determined. Residual antigenicity of the SPH diet as measured by immunodot was reduced by 98.8%. Nitrogen intakes were not different. Weight gain was significantly higher in rats fed the SMP diet. In contrast, the absolute nitrogen balance was similar, suggesting that protein storage was identical with the two diets. A better nitrogen digestion-absorption rate with the SPH diet was observed as evidenced by the significantly increased fecal excretion with the SMP diet. Small intestine adaptation showed no difference between the two diets for mucosal weight, protein content/10 cm as well as for sucrase, glucoamylase, and N-aminopeptidase total activity/10 cm or specific activity (mU/mg protein). The DNA content of the mucosa/10 cm was significantly higher suggesting a mucosal hyperplasia in the SPH diet. The data suggest that in rats the SPH diet leads to nitrogen retention and small intestine adaptation similar to that of the SMP diet, despite better body weight gain by the latter.

Effects of two protein hydrolysates on growth, nitrogen balance and small intestine adaptation in growing rats.

The effects of feeding 2 protein hydrolysates, one prepared by controlled pepsin and pancreatic protease (including elastase II) hydrolysis of milk proteins (PPPH) and the other a di- and tripeptide bacterial protease hydrolysate of bovine albumin (DTPH), on the growth, nitrogen balance and small intestine adaptation of growing rats were analyzed. Two groups of 3-week-old rats (8 rats/group) were fed the liquid diets ad libitum for 2 weeks. The diets had the same caloric, nitrogen, carbohydrate and lipid contents. The amino acid compositions fulfilled the needs of growing rats. The diet differed in the original proteins, the hydrolysis technique used and the molecular weights of the peptides. Nitrogen intakes were similar. Although there was no difference in weight gain, nitrogen balance was significantly higher in the rats fed the PPPH diet (day 4-day 6:PPPH, 60 +/- 4%, DTPH, 25 +/- 5%; day 12-day 15: PPPH, 58 +/- 3%; DTPH, 30 +/- 5%). The stool nitrogens were identical, suggesting improved nitrogen storage in the rats fed the PPPH diet. Small intestine adaptation showed that the rats on the PPPH diet had significantly more protein (mg) and DNA (microgram) per 10 cm of the jejunum (PPPH, 25.6 +/- 2, 393 +/- 20; DTPH: 15.7 +/- 2, 258 +/- 23) and sucrase-specific activity and per microgram of DNA (PPPH, 133 +/- 5.7, 9.7 +/- 0.5; DTPH, 113 v 5, 7 +/- 1). The N-aminopeptidase-specific activity was the same in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Postobstructive enteropathy in infants with transient enterostomy: its consequences on the upper small intestinal functions.

Repeated or prolonged organic obstruction of the small intestine in the neonatal period can lead to severe refeeding problems, despite a transient ostomy. These problems are thought to result from a postobstructive enteropathy (POE) of the apparently normal small intestine segment above the obstruction. Ten infants with a POE, characterized by limited oral caloric and carbohydrate intakes and increased ostomy effluent, were compared with 8 controls with an enterostomy and a normal postoperative refeeding pattern. There was no statistical difference in the histomorphometric appearance of the mucosa or its digestive or absorptive capacity (brush-border hydrolases, glucose transport) between the two groups. The effluent and duodenal floras of the two groups were similar. However, all POE patients showed significant abnormal peristalsis characterized by barium and carmin transit times. This suggests that repeated or prolonged obstruction in the neonatal period could lead to a POE, caused by chronic motricity abnormalities of the small intestine above the obstruction. Although this POE is more frequent after small bowel atresia, it may also occur with other conditions causing prenatal and postnatal intestinal obstruction.

Effects of dietary whey proteins, their peptides or amino-acids on the ileal mucosa of normally fed and starved rats.

The effects of three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; trypsic whey protein hydrolysate, WPH, and amino-acid mixture, AAM) were studied on the mucosa morphology and brush border hydrolase (BBH) activities (disaccharidases, peptidases) of the ileum of normally fed male Wistar rats (controls) and during refeeding of rats starved for 72h. All three diets produced repair of the fasting induced mucosal atrophy; the AAM diet gave the most rapid response and highest villus height (p < 0.01). This was correlated with an increase in crypt mitoses (p < 0.01). Similar results were obtained in controls with AAM. The sucrase (S) and acid amino peptidase (AAP) specific activities of controls were higher (p < 0.01) on the WPH diet; neutral amino peptidase (NAP) was unaffected. Dipeptidyl peptidase IV (DDP) was lowest on AAM while glucoamylase (G) highest on WP. Fasting increased S and DDP activity, and produced no change in the other BBH. Large variations in BBH occurred during refeeding except for NAP which remained stable. Control values were restored at 96h, except for AAP. The results show that BBH and mucosa morphology of the ileum in the rat can be modified by the molecular form of the nitrogen source and that the nutritional status interferes with this adaptation. These data could have implications for the therapy of small bowel disease.

[Effect of fasting and refeeding on the adaptation of the small intestine in rats. A model for physiopathologic studies]. / Effet du jeûne et de la réalimentation sur l'adaptation de l'intestin grêle chez le rat. Un modèle d'études physio-pathologiques.

A chronological study was carried out on 50 male Wistar rats (350 g) to determine the effects of 3 days of fasting and 16 h to 9 days of refeeding on the morphology of jejunal and ileal mucosa (villus, crypt and enterocyte heights; number of mitosis), on some aspects of their functional adaptation (sucrase, maltase, protein) and on nitrogen and lipid absorptions. Three days of fasting resulted in weight loss (12 p. 100), in a jejunal mucosa atrophy (villus height: 376 +/- 18 vs. 492 +/- 4 microns in controls; enterocyte height: 31 +/- 2 vs. 41 +/- 0.3 micron in controls) and a decrease in disaccharidases activities (sucrase: 927 +/- 90 vs. 3,363 +/- 21 mU/10 cm length in controls). No change in ileal mucosa morphometry was noticed. Ad libitum refeeding caused a rapid and progressive weight gain, a jejunal morphometric regrowth identical to control values at 16 h (villus height: 521 +/- 20, enterocyte height 42 +/- 0.9 microns), and maximum at 40 h of refeeding (villus height: 601 +/- 5 microns). Disaccharidases adaptation was delayed, with a maximum at 64 h of refeeding (sucrase: 3,524 +/- 56 mU/10 cm length). Despite a 30 p. 100 increase of food consumption over the whole study (45 p. 100 during the first 16 h of refeeding), nitrogen and lipid absorption coefficients remained identical to those found in controls with an increased nitrogen balance of 70 p. 100 at 16 h and 54 p. 100 at 40 h refeeding, as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)