John H. Schwab

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 less than 0.001; 104 +/- 22, p less than 0.05) or sham-operated Lewis (84 +/- 24, p less than 0.001) or Wistar (78 +/- 10, p less than 0.001) rats. Randomized comparison using a histology grading score showed abnormalities that paralleled 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.

Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments

This study was designed to determine if poorly biodegradable bacterial cell wall components can produce chronic intestinal inflammation. A sterile aqueous suspension of sonically disrupted group A or group D streptococcal cell wall fragments was injected intramurally into the small intestine and cecum of 100 rats. Gross findings in rats killed at intervals of 1 day to 6 mo included intestinal thickening, adhesions, and mesenteric contraction. Acute histologic inflammation subsided by 2 wk, but chronic granulomatous inflammation persisted for 6 mo in the rats injected with group A streptococcal cell wall fragments and 3 mo in the rats injected with group D streptococcal cell wall fragments. Ninety-six control rats identically injected with human serum albumin or phosphate-buffered saline demonstrated mild acute inflammation that resolved, with only 1 rat having chronic intestinal inflammation. Granulomas in the intestine, mesentery, and mesenteric lymph nodes developed in 46% of the rats injected with group A fragments and 45% of the rats injected with group D streptococcal cell wall fragments, compared with 20% of the controls injected with albumin and 4% of the controls injected with phosphate-buffered saline. Group A streptococcal antigen was detected by immunofluorescence at the site of inflammation for 4 mo, and possible reactivation of acute inflammation was seen up to 6 mo after injection. We conclude that bacterial cell wall fragments are capable of producing chronic granulomatous inflammation in the intestinal wall if present in appropriate particle size and concentration. We speculate that cell walls from the enteric microflora may leak across a permeable mucosa in chronic inflammatory bowel disease to initiate and sustain local and systemic inflammation.

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 less than 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 less than 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat : Role for glycine-gated chloride channel

ABSTRACT Peptidoglycan polysaccharide (PG-PS) is a primary structural component of bacterial cell walls and causes rheumatoid-like arthritis in rats. Recently, glycine has been shown to be a potential immunomodulator; therefore, the purpose of this study was to determine if glycine would be protective in a PG-PS model of arthritis in vivo. In rats injected with PG-PS intra-articularly, ankle swelling increased 21% in 24 to 48 h and recovered in about 2 weeks. Three days prior to reactivation with PG-PS given intravenously (i.v.), rats were divided into two groups and fed a glycine-containing or nitrogen-balanced control diet. After i.v. PG-PS treatment joint swelling increased 2.1 ± 0.3 mm in controls but only 1.0 ± 0.2 mm in rats fed glycine. Infiltration of inflammatory cells, edema, and synovial hyperplasia in the joint were significantly attenuated by dietary glycine. Tumor necrosis factor alpha (TNF-α) mRNA was detected in ankle homogenates from rats fed the control diet but not in ankles from rats fed glycine. Moreover, intracellular calcium was increased significantly in splenic macrophages treated with PG-PS; however, glycine blunted the increase about 50%. The inhibitory effect of glycine was reversed by low concentrations of strychnine or chloride-free buffer, and it increased radiolabeled chloride influx nearly fourfold, an effect also inhibited by strychnine. In isolated splenic macrophages, glycine blunted translocation of the p65 subunit of NF-κB into the nucleus, superoxide generation, and TNF-α production caused by PG-PS. Further, mRNA for the beta subunit of the glycine receptor was detected in splenic macrophages. This work supports the hypothesis that glycine prevents reactive arthritis by blunting cytokine release from macrophages by increasing chloride influx via a glycine-gated chloride channel.

IMMUNOLOGICAL PROPERTIES OF BACTERIAL CELL WALL MUCOPEPTIDES.

Summary Specific antibodies to cell wall mucopeptide can be obtained which should be useful in elucidating and comparing the structure among microorganisms. Also significant is the observation that nearly all animal sera studied react with most mucopeptide preparations, regardless of source. This represents a “natural” immunological response against an ubiquitous antigenic moiety common to a variety of microbial muco-peptides. In addition to this response, serum from immunized rabbits contains antibodies which react with groupings of smaller available concentration on the intact mucopeptide and hence requires addition of 10-20 times as much antigen for maximal precipitation.

Immunosuppressant from group A streptococci.

A cytoplasmic component of group A streptococci suppresses both 19S and 7S antibody responses of mice to sheep erythrocytes. Partial purification is achieved by differential centrifugation and gel filtration. When the direct and indirect hemolytic plaque techniques are used, a single injection of this group A material given before injection of erythrocytes produces more than 90-percent suppression of either primary or secondary immune response.

Studies on preparation of bacterial cell walls and criteria of homogeneity

Abstract Group A streptococci can be disrupted in a sonic vibrator in the presence of glass beads to yield large cell wall fragments. A technique is described, involving sucrose zone centrifugation, for collecting cell walls with a high degree of purity, and in good yield, suitable for studies on biological properties. It is concluded that cell walls prepared by the technique of sucrose zone centrifugation display a relatively high degree of homogeneity as indicated by electron microscopy and moving boundary electrophoresis. Immunological studies show the cell wall preparations to be antigenically complex although not necessarily contaminated with non-cell wall antigens.

Antibacterial action of PMN lysosomal cationic proteins resolved by density gradient electrophoresis.

Summary The antibacterial and enzymatic cationic proteins of acid extracts of PMN lysosomes were resolved on a preparative scale by density gradient electrophoresis. The 32P releasing activity was closely associated with antibacterial action. Lysosomal ribonuclease was electrophoretically heterogeneous.

Differential susceptibility of mouse lymphocytes to an immunosuppressant from group A streptococci

Abstract Cytoplasmic components (SF) of group A streptococcus have been shown to suppress the immune response. Lymphocytes exhibit a differential susceptibility to these components when several functional criteria are applied. PFC appear to be more susceptible than RFC suggesting that the long-lived, undifferentiated lymphocytes that form rosettes escape the effects of SF, but antibody-forming cells are suppressed. Similarly, thymic dependent functions such as PHA responsiveness, mixed lymphocye interaction, and synergism with bone marrow are unaffected. Bone marrow functions, such as hematopoiesis and the ability to synergize with normal thymus, however, are suppressed by SF pretreatment. SF, furthermore, causes a loss of lymphocytic cells from marrow, and a depression of in vitro proliferative capacity. These results indicate that SF is immunosuppressive due to a selective depression of bone marrow function.