Self-filling Blind Loops Research Articles

The gut-liver axis: impact of a mouse model of small-bowel bacterial overgrowth

BackgroundThe mechanisms by which intestinal bacteria impact liver diseases remain poorly understood. The aim of this study was to develop a mouse model of small-bowel bacterial overgrowth and to determine its impact on hepatobiliary injury. Materials and methodsA jejunal self-filling blind loop (SFBL) was created in C57BL/6 mice. Three weeks after surgery, the mice were euthanized, and bacterial cultures of luminal content of the loop and extraintestinal tissues were performed. Liver and jejunum were collected for histological grading of inflammation and injury. Serum liver biochemistry assays were performed. Hepatobiliary transporter mRNA expression in liver was measured by quantitative real-time polymerase chain reaction. Bile and blood were collected for measurement of total bile acids, phospholipid, and cholesterol. Mice undergoing jejunal transection and reanastomosis and laparotomy only served as control groups. ResultsSFBL induced a dramatic increase in intraluminal bacterial counts, mesenteric lymph node bacterial translocation, and evidence of jejunal and hepatobiliary injury. Significant reductions in hepatic expression of hepatobiliary transporters involved in biliary canalicular export and basolateral uptake were observed in SFBL mice. SFBL resulted in a significant increase in biliary total bile acid concentration, decreases in bile phospholipid and cholesterol output, and an increase in the bile acid/phospholipid ratio. ConclusionsWe have developed a reproducible mouse model of small-bowel bacterial overgrowth with evidence of liver inflammation, altered hepatobiliary transporter expression, and alterations in bile composition. This model may help to elucidate the mechanisms by which gut-derived bacterial factors impact the liver and contribute to the exacerbation of liver diseases and biliary injury.

Ileal Bacterial Stasis in Self-Filling Blind Loops is Associated With Increased TLR-2 and TLR-4 Expression in Wild-Type and IL-10 Knockout Mice

Ileal pouch-anal anastomosis is the standard surgical treatment for patients with refractory Ulcerative Colitis (UC). However, pouchitis occurs in roughly 50% of UC patients post-pouch creation, despite no previous small bowel inflammation. We hypothesize that pouchitis develops as a result of an abnormal intestinal response to bacterial stasis in a genetically susceptible host. As bacteria are present for a greater duration in stasis, we evaluated the expression of specific toll-like receptors (TLRs), which are major mechanisms for epithelial cells to sense bacteria.

Tracking of Green Fluorescent Protein Labeled Escherichia coli Confirms Bacterial Translocation in Blind Loop Rat

Previous investigators have documented small intestinal mucosal injury in blind loop rats. However, the definitive evidence of intestinal bacterial translocation in blind loop animals has been lacking. The purpose of this study was to confirm bacterial translocation in blind loop rats and to evaluate the preventive effect of glutamine on bacterial translocation caused by blind loops. Escherichia coli TG1 labeled with green fluorescent protein was used to track bacterial translocation by gavage to rats. Six groups (n = 10) of rats were studied: unoperated control rats; rats with self-emptying blind loop; rats with self-filling blind loop; unoperated control rats treated with glutamine, 400 mg/d; rats with self-emptying blind loop treated with glutamine, 400 mg/d; rats with self-filling blind loop treated with glutamine, 400 mg/d. Representative tissue specimens of the mesenteric lymph nodes, liver, spleen, and kidney were aseptically harvested for bacteria culture. Bacteria were detected in extraintestinal organs of rats with self-emptying blind loop, self-filling blind loop, and self-filling blind loop treated with glutamine. By fluorescence microscope and XbaI restriction digestion analysis, we elucidated that the bacteria isolated from extraintestinal organs were the same bacteria we gavaged to the rats. We confirmed bacterial translocation in self-filling blind loop and self-emptying blind loop rats. In addition, we also showed that glutamine prevents bacterial translocation in self-emptying blind loop rats.

Abnormal accumulation in lipopolysaccharide in biliary epithelial cells of rats with self-filling blind loop

Primary sclerosing cholangitis (PSC) is known to be frequently associated with inflammatory bowel diseases. In a rat with self-filling blind loop (SFBL), a proposed animal model for PSC, hepatobiliary inflammation has previously been demonstrated. In this study, we assessed the involvement of lipopolysaccharide (LPS), a bacterial endotoxin, in the pathogenesis of hepatobiliary inflammation of the SFBL model. The hepatic localization of LPS was examined by immunohistochemistry using an anti-lipid A antibody. The portal blood concentration of LPS was measured by an endotoxin-specific chromogenic Limulus test (Endospecy test). LPS was localized in the biliary epithelial cells (BECs) of rats with SFBL, and the portal blood concentration of LPS was significantly higher than that of sham-operated rats. Development of hepatobiliary inflammation, peribiliary fibrosis, and injury to the intestinal mucosa were histologically confirmed. Constriction in the biliary trees was radiologically demonstrated. These findings suggested that abnormal accumulation of LPS, which may be derived from portal blood, in BECs was involved in the pathogenesis of hepatobiliary inflammation with intestinal injury.

Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats

Background & Aims: Recent data support an important role of resident luminal bacteria in experimental colitis. We determined how altered cecal bacterial loads influence colitis and gastritis. Methods: A cecal self-filling blind loop (SFBL) was created or the cecum was excluded from the fecal stream in specific pathogen–free HLA-B27 transgenic (TG) rats with early colitis and in nontransgenic (nonTG) littermates; controls underwent sham operation (SHAM). Luminal bacterial concentrations were determined by culture and counting chamber. Results: TG rats with SFBL had more severe cecal inflammation and leukocytosis than TG SHAM controls. TG excluded rats with low cecal bacterial loads had no cecal inflammation and less colitis and gastritis than SHAM controls, despite having normal distal colonic and gastric bacterial concentrations. Metronidazole attenuated cecal inflammation and eliminated Bacteroides in SFBL TG rats. NonTG SFBL rats had mild cecal inflammation and no gastritis and colitis. The ratio of total anaerobic to aerobic bacteria was 1000-fold greater in SFBL than in SHAM rats, with a 10,000-fold increased ratio of Bacteroides spp. to aerobes. Conclusions: The luminal bacterial load and composition determines the activity of cecal inflammation in genetically susceptible hosts. Lowering cecal bacterial concentrations can diminish inflammation in remote organs. GASTROENTEROLOGY 1999;116:310-319

Clinical models of intestinal adaptation.

Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na-glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self-filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na-glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor-alpha can specifically downregulate the tight junction, as indicated in the intestinal HT-29/B6 cell model.

Reactivation of arthritis induced by small bowel bacterial overgrowth in rats: role of cytokines, bacteria, and bacterial polymers

Arthritis is often associated with intestinal diseases, but the etiology is not known. We developed a rat model whereby arthritis was reactivated by experimental small bowel bacterial overgrowth (SBBO). Self-limited monoarticular arthritis was induced by intra-articular injection of 2 micrograms of rhamnose peptidoglycan-polysaccharide derived from group A streptococci into the ankle joints in female Lewis rats. Eleven days after intra-articular injection, when swelling was resolving, experimental SBBO induced by surgical creation of jejunal self-filling blind loops reactivated arthritis, but SBBO induced by creation of self-emptying blind loops, which minimally increases luminal bacteria, and sham operation did not (P < 0.001). Increased joint diameters in rats with self-filling blind loops persisted for at least 56 days after surgery. Reactivation of arthritis due to SBBO was prevented by anti-tumor necrosis factor alpha antiserum and interleukin 1 receptor antagonist (P < 0.001), indicating that these cytokines mediate joint swelling secondary to intestinal injury. Recombinant bactericidal/permeability-increasing protein, an agent which neutralizes endotoxin, and metronidazole, which is active against anaerobic bacteria, prevented arthritis (P < 0.001), but polymyxin B (which also neutralizes endotoxin) and gentamicin had no effect. Mutanolysin, an enzyme which degrades peptidoglycan-polysaccharide from group A streptococci, exacerbated arthritis for the first 6 days but then diminished joint swelling from 12 to 21 days after surgery (P < 0.001). These studies introduce a reproducible animal model of reactivation of arthritis secondary to intestinal injury and demonstrate a role for bacterial products from endogenous enteric organisms.

A microcholangiographic study of liver disease models in rats

Rats develop hepatobiliary injury due to small bowel bacterial overgrowth (SBBO) that, at specimen, resembles cholangiography sclerosing cholangitis. To better visualize the smaller bile ducts, we used microcholangiography to determine the spectrum of biliary lesions in this and five other models of liver disease. The models studied were as follows: (1) Surgically created jejunal, self-filling blind loops induce SBBO. (2) Intraperitoneal injection of a bacterial cell wall polymer, peptidoglycan-polysaccharide (PG-PS), causes granulomatous hepatitis. (3) Intraperitoneal injection of endotoxin (lipopolysaccharide) causes sinusoidal congestion and shock. (4) Bile duct ligation induces bile duct proliferation. (5) Alpha-naphthyl-isothiocyanate (ANIT) induces bile duct proliferation. (6) Carbon tetrachloride (CCl4) causes fibrosis and cirrhosis. Warmed barium sulfate, gelatin, and saline were injected in the extrahepatic bile duct. Liver slices (2 mm) underwent microradiographic techniques, and images were correlated with histology. Rats with SBBO had irregular and dilated extrahepatic bile ducts with thickened walls. Rats treated with endotoxin and CCl4 had normal microcholangiograms. Bile duct proliferation was identified following ANIT and bile duct ligation. Rats given PG-PS demonstrated irregular intrahepatic bile ducts. Microcholangiograms following SBBO and PG-PS showed similarities including focal ductal dilatation, narrowing, proliferation, and destruction. Various models of liver injury induce characteristic cholangiographic appearances. Microcholangiography is useful in examining biliary tract lesions and complements histology.

Experimental colitis in rats induces low-grade endotoxinemia without hepatobiliary abnormalities.

In three experimental models in rats, surgical construction of a self-filling blind loop (SFBL), trinitrobenzene sulfonic acid (TNB) -induced colitis, and the combination of SFBL and TNB, the hypothesis was studied that intestine-derived endotoxins play a role in the pathogenesis of hepatobiliary disorders in chronic inflammatory bowel disease (CIBD). After eight weeks of treatment, a mild increase in portal and systemic endotoxin levels and interleukin-6 concentrations was observed and the serum levels of alkaline phosphatase, bilirubin, and ALAT were only mildly increased in SFBL plus TNB rats. Histopathological examination of the liver showed hardly any abnormalities in all three rat models. These results show that low-grade portal and systemic endotoxinemia in rats, induced by bacterial overgrowth and/or chemical colitis, is not able to induce hepatobiliary alterations. To exclude definitively a possible role for portal endotoxinemia in the pathogenesis of CIBD-associated hepatobiliary abnormalities, however, an adequate animal model for CIBD is urgently needed.

Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific muralytic enzyme, we conclude that systemic uptake of PG-PS derived from endogenous enteric bacteria contributes to hepatobiliary injury induced by SBBO in susceptible rat strains.

Biliary tract disease in rats with experimental small bowel bacterial overgrowth

Small bowel bacterial overgrowth, which develops in surgically created jejunal self-filling blind loops, is associated with hepatic injury in susceptible rat strains. The histological findings are portal tract inflammation and bile duct proliferation and destruction. In this study, evidence of injury to the extrahepatic and intrahepatic bile ducts in susceptible and resistant inbred rats with self-filling blind loops was determined by bile flow rates, histological appearance of cross-sections of bile ducts and cholangiography. Lewis and Wistar rats with self-filling blind loops had thickening of the cross-sectional area of the extrahepatic bile ducts compared with controls and nonsusceptible Buffalo rats (p < 0.001). Daily metronidazole therapy significantly reduced thickening of bile ducts in Lewis and Wistar rats with self-filling blind loops. Bile flow was increased in Lewis and Wistar rats with blind loops compared with controls (p < 0.05). Cholangiograms of Lewis and Wistar rats with self-filling blind loops demonstrated extrahepatic ductal dilatation and ectasia with irregular, beaded, rapidly tapering and tortuous intrahepatic ducts. Blinded cholangiographical scores of susceptible rat strains with self-filling blind loops were higher (score = 5.9 ± 2.3) than those of controls (1.2 ± 0.4), rats with blind loops treated with metronidazole (0.8 ± 1.3) and nonsusceptible Buffalo rats with blind loops (0.6 ± 0.9) (p < 0.01). Biliary histological and cholangiographical abnormalities found in this rat model resemble features of primary sclerosing cholangitis. This animal model will be useful in exploring mechanisms of the association of hepatobiliary inflammation with intestinal injury and investigating potential therapeutic agents. (HEPATOLOGY 1990;13:766–772.)

Biliary tract disease in rats with experimental small bowel bacterial overgrowth

Small bowel bacterial overgrowth, which develops in surgically created jejunal self-filling blind loops, is associated with hepatic injury in susceptible rat strains. The histological findings are portal tract inflammation and bile duct proliferation and destruction. In this study, evidence of injury to the extrahepatic and intrahepatic bile ducts in susceptible and resistant inbred rats with self-filling blind loops was determined by bile flow rates, histological appearance of cross-sections of bile ducts and cholangiography. Lewis and Wistar rats with self-filling blind loops had thickening of the cross-sectional area of the extrahepatic bile ducts compared with controls and nonsusceptible Buffalo rats (p less than 0.001). Daily metronidazole therapy significantly reduced thickening of bile ducts in Lewis and Wistar rats with self-filling blind loops. Bile flow was increased in Lewis and Wistar rats with blind loops compared with controls (p less than 0.05). Cholangiograms of Lewis and Wistar rats with self-filling blind loops demonstrated extrahepatic ductal dilatation and ectasia with irregular, beaded, rapidly tapering and tortuous intrahepatic ducts. Blinded cholangiographical scores of susceptible rat strains with self-filling blind loops were higher (score = 5.9 +/- 2.3) than those of controls (1.2 +/- 0.4), rats with blind loops treated with metronidazole (0.8 +/- 1.3) and nonsusceptible Buffalo rats with blind loops (0.6 +/- 0.9) (p less than 0.01). Biliary histological and cholangiographical abnormalities found in this rat model resemble features of primary sclerosing cholangitis. This animal model will be useful in exploring mechanisms of the association of hepatobiliary inflammation with intestinal injury and investigating potential therapeutic agents.

Hepatic injury associated with small bowel bacterial overgrowth in rats is prevented by metronidazole and tetracycline

Susceptible rat strains develop hepatobiliary injury following the surgical creation of self-filling blind loops that cause small bowel bacterial overgrowth. Luminal bacteria or their cell wall polymers were implicated in the pathogenesis of the lesions because sham-operated rats and rats with self-emptying blind loops, having only slightly increased bacterial counts, did not develop hepatic injury. In this study, antibiotics with different spectra of activities were continuously administered starting 1 day or 22 days after surgery to determine which intestinal flora may be responsible for the development of hepatic injury in rats with small bowel bacterial overgrowth. Four weeks following surgery, Lewis rats with self-filling blind loops receiving no antibiotics had elevated liver histology scores (8.2 ± 1.3 vs. 0.7 ± 0.4) and plasma aspartate aminotransferase levels (269 ±171 vs. 84 ± 24) compared with sham-operated rats, P < 0.001. Oral gentamicin as well as oral and intraperitoneal polymyxin B, which binds endotoxin, did not prevent hepatic injury in rats with self-filling blind loops. However, oral metronidazole and tetracycline therapy continuously administered beginning 1 day after surgery diminished hepatic injury (histology score 3.0 ± 1.8, 2.9 ± 1.1; aspartate aminotransferase 87 ± 25, 98 ± 34; respectively P < 0.001 compared with self-filling blind loops receiving no antibiotics). Metronidazole also protected Wistar rats that require 12 weeks to develop hepatic injury following experimentally induced small bowel bacterial overgrowth compared with rats with self-filling blind loops that received no antibiotic treatment (histology score 10.4 ± 1.3 vs. 0.7 ± 1.1 and aspartate aminotransferase 273 ± 239 vs. 76 ± 20, P < 0.001). When rats started metronidazole therapy 22 days after self-filling blind loop surgery, elevated aspartate aminotransferase values decreased to normal during the next 77 days and final histology scores were normal. All rats with self-filling blind loops had negative peritoneal, liver, spleen, and blood cultures but approximately 75% of mesenteric lymph node cultures were positive irrespective of antibiotic treatment. Because Bacteroides species have been implicated in causing vitamin B12 and disaccharidase deficiencies in rats with self-filling blind loops, we documented the presence or absence of these organisms from blind loops using selective culture techniques. Metronidazole and tetracycline eliminated Bacteroides sp. from blind loops, but polymyxin B and gentamicin did not. These results suggest that Bacteroides sp. and possibly other anaerobic bacteria play an important role in the pathogenesis of hepatic injury associated with small bowel bacterial overgrowth.

Evidence for peptidoglycan absorption in rats with experimental small bowel bacterial overgrowth

Surgical creation of jejunal self-filling blind loops (SFBL) causes small bowel bacterial overgrowth which is associated with hepatobiliary inflammation in the susceptible Lewis and Wistar rat strains. Since hepatic injury occurs when small bowel anaerobic bacterial concentrations are increased 4 to 6 log10 units per ml and hepatic bacterial cultures are negative, we postulate that the inflammation is caused by absorption of phlogistic cell wall polymers originating from bacteria within the loop. To demonstrate absorption of bacterial cell wall polymers, we measured plasma and hepatic levels of immunoreactive peptidoglycan-polysaccharide (PG-PS) following intraluminal injection as well as anti-PG antibodies as an indirect measure of absorption and/or accumulation of endogenous PG. PG-PS purified from group A streptococci was detected in plasma by enzyme-linked immunosorbent assay after intraluminal injection; rats with SFBL showed significantly more uptake into plasma and the liver than sham-operated rats or SFBL rats which were treated with metronidazole (P less than 0.025). Total plasma immunoglobulin A (IgA), IgG, and IgM levels did not differ among sham-operated rats and those with self-emptying blind loops or SFBL, but plasma anti-PG IgA (P less than 0.05), IgG, and IgM (P less than 0.01) levels were increased in rats with SFBL. Metronidazole and tetracycline prevented the elevation of anti-PG antibody, but gentamicin and polymyxin B did not. Anti-lipid A, anti-soy protein, and anti-chow antibodies in plasma were not consistently increased in rats with SFBL indicating the lack of a generalized antibody response to luminal antigens. These data suggest that PG from normal flora bacteria is absorbed from the intestinal lumen and that mucosal injury and/or increased luminal concentrations of PG, such as those induced by small bowel bacterial overgrowth, lead to enhanced absorption of potentially inflammatory bacterial polymers.

Tight junction regulation during impaired ion transport in blind loops of rat jejunum

Epithelial cell tight junction structure in self-filling blind loops of rat jejunum, a model for blind loop syndrome in humans, was analyzed morphometrically along the crypt-villus axis. In control jejunum, the number of strands and junctional depth, including meshwork depth, decreased from crypt to villus tip. In the blind loop, aberrant strands appeared below the meshwork, particularly in crypt cells. Consequently, total junctional depth was greater than in controls. Furthermore, strand number and junctional meshwork depth were increased in blind loops at the villus tip. It is that site along the crypt-villus axis which showed the most shallow junction in control jejunum. This structural change is paralleled by a three-fold increase in epithelial resistance as previously measured by alternating current impedance analysis. Relative Na over Cl permeability (PNa:Cl) was obtained from dilution potential measurements. PNa:Cl was 1.50:1 in control jejunum and 1.35:1 in the blind loop (n.s.). Considering the cation selectivity of the tight junction, the increase in epithelial resistance in blind loops cannot be attributed to a collapse of the lateral intercellular space but is due to changes in tight junctional permeability resulting from structural alteration. The blind loop syndrome represents a further example of diminished epithelial ion transport and concomitant decrease in tight junction permeability, thus supporting the general concept of regulation of the tight junction in response to active transport activity.

Hepatic inflammation in rats with experimental small intestinal bacterial overgrowth

Hepatobiliary inflammation and other extraintestinal manifestations accompany certain intestinal disorders, perhaps because of proliferation or enhanced transport of luminal bacteria or their phlogistic cell-wall components. Using jejunal self-filling blind loops to create small bowel bacterial overgrowth, we compared biochemical and histological evidence of hepatic inflammation in 3 rat strains chosen for their variable inflammatory responses to bacterial cell wall polymers. Lewis and Wistar rats developed weight loss, hepatomegaly, and hepatic inflammation 4 and 12 wk, respectively, after creation of SFBL. Plasma aspartate aminotransferase levels in Lewis rats 4 wk (578 ± 77 U/L) and Wistar rats 12 wk (220 ± 35 U/L) after self-filling blind loops were significantly greater than in rats with self-emptying blind loops (112 ± 24 U/L, p < 0.001; 104 ± 22, p < 0.05) or sham-operated Lewis (84 ± 24, p < 0.001) or Wistar (78 ± 10, p < 0.001) rats. Randomized comparison using a histology grading score showed abnormalities that parallelled aminotransferase values. Lewis and Wistar rats with self-filling blind loops had hepatic injury with bile duct proliferation, fibrosis and acute and chronic periportal and focal parenchymal inflammation. Lewis and Wistar rats with self-emptying blind loops developed occasional mild histologic lesions. 50% of Lewis rats with self-filling blind loops for 4 wk died compared with only 15% in other groups. However, Buffalo rats with self-filling blind loops developed no weight loss, hepatomegaly, or hepatic injury. Anaerobic cultures of blood, peritoneum and liver were negative in all strains. Diet-restricted, sham-operated Wistar rats with weights similar to the Wistar rats with self-filling blind loops did not develop histologic abnormalities or elevated aminotransferase levels (76 ± 31 U/L). These results show that experimental small bowel bacterial overgrowth causes significant hepatic inflammation leading to fibrosis in susceptible rat strains. Caloric deprivation and hepatic bacterial invasion are not etiologically responsible. We suggest that bacterial cell wall polymers or other bacterial toxins from the blind loop cause hepatic lesions in genetically susceptible hosts.

The Role of Gut-Glucagon-Like Immunoreactants in the Control of Gastrointestinal Epithelial Cell Renewal

In vitro and in vivo studies have provided considerable information on the possible physiologic function of circulating gastrointestinal hormones as well as locally acting regulatory peptides in the multifactorial control of adaptive gastrointestinal epithelial cell proliferation and cell renewal. It has been suggested by circumstantial evidences that enteroglucagon (EG; G-GLI I) may act as a trophic factor on the intestinal mucosa which may account for adaptive changes of the small intestine following various stimuli. However, we have shown that there are experimental conditions (germ-free rats after conventionalisation; jejunal self-filling blind loops) in which intestinal hyperplasia does not correspond to an increase in the concentrations of enteroglucagon in plasma or intestinal mucosa. Furthermore, despite a continuous immunoneutralisation of circulating endogenous enteroglucagon by monoclonal antibodies there was an adaptive, hyperplastic response of the ileal remnants after a 70% proximal small bowel resection which was of the same magnitude as in the control group but was even greater considering the increased number of mitoses per crypt. In order to gain additional insight into the putative role of enteroglucagon as an enterotrophic regulatory peptide, an in vitro model was used to investigate the effect of highly purified rat G-GLI I on the proliferative response of primary small intestinal epithelial cells of fetal rats. Whereas there was a well known growth-promoting action of EGF, the proliferation of rat fetal intestinal epithelial cells was inhibited by the addition of purified G-GLI I. These results indicate that enteroglucagon does not act as an enterotrophic factor but provide the first direct evidence consistent with an antitrophic role of enteroglucagon in the small intestine.

Effect of dietary restriction on total and bacterium-specific mucosal secretory immunoglobulin A in bile-diverted intestinal self-filling blind loops.

The effect of starvation on the mucosal secretory immunoglobulin A (sIgA) response to bacterial antigens was studied in bile-free rat self-filling blind loops constructed at the end of a Roux-en-Y branch of jejunum. Rats were fed a 50% restricted diet for 1 to 4 weeks after surgery. sIgA was measured in the mucosa and lumen by an enzyme-linked immunosorbent assay. Dietary restriction caused a final rise of luminal sIgA which was less than 50% of that of normally fed controls Luminal bacteria counts were not different in the two groups. The percentage of total sIgA precipitated with intestinal bacteria was not significantly affected by dietary restriction, and there was no change in the specific binding of sIgA to several bacterial species. Nonprecipitated sIgA exhibited a low but significant specific binding to bacteria in both diet-restricted and fed rats. Diet restriction therefore reduced the total sIgA response to luminal bacteria, but the specific bacterial binding capacity per microgram of sIgA was not altered. In these short-term experiments diet-restricted animals appeared to be capable of secreting sIgA in excess of requirement, since the nonprecipitable luminal fraction contained free sIgA with binding capacity for bacteria. The ability of sIgA to react with specific antigens may therefore be of more significance as an indicator of bacterial susceptibility than the measurement of total sIgA.

Regional Differences in Attachment of Enteroadherent Escherichia coli Strain RDEC‐1 to Rabbit Intestine

SummaryRDEC‐1 (serotype 015:NM) is an enteropathogenic Escherichia coli that adheres to mucosal epithelial cells in the ileum, cecum, and colon, but not in the jejunum, of rabbits during diarrheal illness. As RDEC‐1 can adhere to jejunal brush borders in vitro, rapid distal clearing of the enteropathogen from the upper gastrointestinal tract may explain the absence of in vivo adherence of RDEC‐1 to rabbit jejunum. To test this hypothesis, self‐filling blind loops (SFBL) were created in the jejunum of rabbits that were then inoculated with RDEC‐1 to determine if stasis would promote increased luminal colonization and increased mucosal attachment of RDEC‐1 in the proximal small intestine. Eight rabbits with SFBL and seven nonoperated littermates were inoculated with RDEC‐1 4 weeks after surgery. Semiquantitative estimation of fecal shedding of RDEC‐1 and the presence and severity of diarrhea were monitored for 7 days. Rabbits were then killed, and colonization of jejunum and ileum by anaerobic bacteria and luminal colonization of jejunum, ileum, and cecum by RDEC‐1 was quantitated. Mucosal adherence of RDEC‐1 to jejunum, ileum, and cecum was determined by Giemsa staining of intestinal sections obtained from rabbits with and without SFBL. In addition, brush border membranes (BBM) were prepared from blind loop jejunum, nonoperated jejunum, and ileum. In vitro adherence of RDEC‐1 to brush borders was quantitated under phase contrast microscopy. Maximal fecal shedding of RDEC‐1 began 3–4 days after infection in all rabbits, but diarrhea was more severe in rabbits with SFBL. As anticipated, the total number of anaerobic bacteria in jejunal blind loops (12.28 ± 2.01 log10 CFU/cm) and adjacent afferent segments (11.77 ± 0.88) was elevated, compared with levels in the jejunum of nonoperated rabbits (6.14 ± 1.76). Luminal colonization with RDEC‐1 developed in both jejunal SFBL (6.38 ± 0.80) and in adjacent jejunal segments only to the same degree as in the jejunum of nonoperated rabbits (5.27 ± 0.97). Despite stasis and luminal colonization of SFBL jejunum with RDEC‐1 (6.38 ± 0.80) in levels indistinguishable from those found in the ileum (6.43 ± 0.78), RDEC‐1 was not adherent to SFBL jejunum but was adherent to rabbit ileum. In vitro studies indicated that RDEC‐1 could adhere to BBM prepared from both non‐operated (6.0 ± 1.0 RDEC‐1/BBM) and SFBL jejunum (6.5 ± 1.8), as well as to ileal brush borders (8.0 ± 2.0). We conclude that although RDEC‐1 colonize the lumen of the upper jejunum during in vivo infection of rabbits, the organisms do not adhere to jejunal enterocytes even in a region of stasis such as the SFBL. Stasis was not sufficient to promote mucosal adherence of RDEC‐1, nor did the presence of anaerobic flora in the jejunum alter the intestinal mucosa in such a way as to promote enteroadherence of the pathogen. As RDEC‐1 could adhere to SFBL and normal jejunal brush borders in vitro, these studies suggest that factors other than intestinal transit prevent attachment of RDEC‐1 to enterocytes in the jejunum in vivo.

Regional differences in attachment of enteroadherent Escherichia coli strain RDEC-1 to rabbit intestine: luminal colonization but lack of mucosal adherence in jejunal self-filling blind loops.

RDEC-1 (serotype 015:NM) is an enteropathogenic Escherichia coli that adheres to mucosal epithelial cells in the ileum, cecum, and colon, but not in the jejunum, of rabbits during diarrheal illness. As RDEC-1 can adhere to jejunal brush borders in vitro, rapid distal clearing of the enteropathogen from the upper gastrointestinal tract may explain the absence of in vivo adherence of RDEC-1 to rabbit jejunum. To test this hypothesis, self-filling blind loops (SFBL) were created in the jejunum of rabbits that were then inoculated with RDEC-1 to determine if stasis would promote increased luminal colonization and increased mucosal attachment of RDEC-1 in the proximal small intestine. Eight rabbits with SFBL and seven nonoperated littermates were inoculated with RDEC-1 4 weeks after surgery. Semiquantitative estimation of fecal shedding of RDEC-1 and the presence and severity of diarrhea were monitored for 7 days. Rabbits were then killed, and colonization of jejunum and ileum by anaerobic bacteria and luminal colonization of jejunum, ileum, and cecum by RDEC-1 was quantitated. Mucosal adherence of RDEC-1 to jejunum, ileum, and cecum was determined by Giemsa staining of intestinal sections obtained from rabbits with and without SFBL. In addition, brush border membranes (BBM) were prepared from blind loop jejunum, nonoperated jejunum, and ileum. In vitro adherence of RDEC-1 to brush borders was quantitated under phase contrast microscopy. Maximal fecal shedding of RDEC-1 began 3-4 days after infection in all rabbits, but diarrhea was more severe in rabbits with SFBL.(ABSTRACT TRUNCATED AT 250 WORDS)