- Email: [email protected]
Bacterial colonization in juice and biopsies of the achlorhydric stomach
April 1995
• HAPLOTYPE RESTRICTED DIFFERENCES IN IN VITRO PRODUCTION OF TUMOR NECROSIS FACTOR ALPHA AND LYMPHOTOXIN ALPHA IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE AND HEALTHY CONTROLS. G. Bouma1, JBA Cmsius', M Oudkerk Pool 1, JJ Kolkman~, BME yon Blomberg~, SGM Meuwissen~, and AS PefiaL Departments of Gastroenterology1 and Pathology2, Free University Hospital, Amsterdam. Although the aetiology of the chronic inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) is still unknown, it is established that disease occurs in a genetically susceptible subject. Tumor necrosis factor alpha (TNFcx) and lymphotoxin alpha (LTc0 are potent proinflammatory and immunoregulatory eytokines. The genes for these cytokines are tandemly arranged in the central region nfthe MHC, between the HLA-B and HLA-D locus, at the short arm of chromosome 6. This location has prompted much speculation about the role of TNFtx and LTtx in the aetiology of MHC-associated diseases. In order to study the effect of gene polymorphisms on the production of TNFct and LTa, we performed a prospective study in which we determined the production of these cytokines in relation to bi-allelic polymorphisms in the TNFc~ and LTc~ genes in 30 patients with IBD and 12 healthy controls. Individuals were typed for two polymorphisms in the TNF~ gone, and two polymorphisms in the LTc~ gene, resulting in 4 haplotypes (TNF-C, -E, -I and -P). Peripheral blood mononuclear cells were cultured in the presence of the T-cell activators anti-CD3 and antiCD28, and TNF~ and LTtx production was measured by commercially available ELISA's. No statistically significant differences in TNFtx and LTa secretion were observed between UC patients, CD patients and controls. However, large differences in production were found in relation to the TNF haplotypes; Individuals carrying the haplotype E are the highest producers of TNFc~ (p=.008, as compared to individuals without this haplotype). The lowest production was found in individuals carrying the C haplotype (p=.012 as compared to individuals without this haplotype). The only difference between the C and E haplotypes is the nucleotide position at position -308 in the promoter region of the TNFcz gene. The production of LTc~ in relation to the genotypes showed that individuals carrying the TNF-I haplotype showed a statistically very significant lower production of LTt~ as compared to individuals who lack this haplotype (p =.002). In conclusion, we have shown strong correlations between TNF haplotypes and TNFct and LTct secretion. These findings may contribute to define the genetic heterogeneity of IBD.
• IMMUNOPEROXIDASE TISSUE STAINING TO DIAGNOSE E. COLI O157:H7 INFECTION: A CAUSE OF ISCHEMIC COLITIS? L.J. Brandt. C. Su, E. Alt, S. Sigal, K. Patterson, P.I. Tarr. Montefiore Medical Center, & Bronx Municipal Hosp., Bronx, N¥, Children's Hospital and Medical Center, Seattle, W~sh. E. coli O157:H7 is increasingly recognized to cause diarrhea and hemorrhagic colitis. E . c o l i O157:H7 is diagnosed by finding Shiga-like toxins in the stool or non-sorbitol fermenting colonies on stool culture using MacConkey-Sorbitol agar. We attempted to develop an immunohistochemical technique to diagnose E. coli O157:H7 organisms on colon biopsy or resection specimens and to determine if an association exists between E. coli O157:H7 and idiopathic "ischemic colitis". 22 specimens of colon were studied: E. coli 0157:H7 colitis (4);ischemic colitis (6); ulcerative colitis (8);pseudomembranous colitis (3) and normal colon (1). Formalin-fixed, paraffin-embedded tissue was deparaffinized, rehydrated, incubated with 3% peroxide in methanol and rinsed with phosphate buffered saline (PBS). Sections were then immunolabeled with peroxidase-labeled affinitypurified antibodies (BacTrace ®) to E. coli O157:H7 (Kirkegaard & Perry Labs, Gaithersburg, MD), stained with peroxidase chromagen reagent (Biomeda Corp., Foster City, CA) and counterstained with aqueous hematoxylin. Each staining procedure included positive and negative controls. Results: Only the 4 k n o w n E . c o l i colitis and 1 of 6 ischemic colitis specimens stained positively. Conclusion: This study shows that immunohistochemistry is useful to diagnose enteric infection with E. coli O157:H7 in humans. Commercially available antibodies to E. coli O157:H7 can be applied to colon tissue samples to detect this organism. In addition, this study suggests a causative role for E. coli 0157:H7 in ischemic colitis.
Immunology, Microbiology, and Inflammatory Disorders A787
BACTERIAL COLONIZATION IN JUICE AND BIOPSIES OF THE ACHLORHYDRIC STOMACH G.Brandi~ G.Biasco, B.Biavati~, GM.Paganelli, C.Masci, R Santuoci, C.Calabrese, G.Di Febo, M.Miglioli, L.Barbara. Istituto di Clinica Atedica e Gastroenterologia, Policlinico S.Orsola, llstituto di Mwrobiologia, Facoltd di Agraria, Universitdt di Bologna Aehlorhydria allows bacterial growth in the gastric juice. Little is known about the relationship between microflora in the lumen of the stomach and colonization of the mucosa. This study evaluates for the first time the bacterial concentration and microbiological profile of gastric juice and biopsies of patients with ACG and subjocta treated with omeprazole. Patients and methods: 8 subjects with non-ulcer dyspepsia (NUD) (5m, m.a. 36); 6 pts. (4m; ma. 39) with ACG; 12 pts. (7m; m.a. 49) with reflux oesophagitis (6) or duodenal ulcer (6) treated with omeprazole (Ome) 20 rag/day for 2 months 0ast dose: 24h prior to endoscopy). During endoscopy (8 a.m.) were taken in sterility 5-10 ml of gastric juice and normal mucosal biopsies (antrum x 4, body x 4). The pH of gastric juice was evaluated with pH-meter. The juice was diluted in saline and sowed in non-selective media (BHA) incubated in air for the count of aerobes (Aer) and in a jar for anaerobes (Ana). Selective media were used for Lattobaeilli (L); Enterobacteria (E); Streptococci (Str); Staphilococci (Stp). Dominant strains were identified with electrophoresis of surface proteins, VFA assay, and API system. Biopsies were weighted, homogenized in sterility and diluted with a ratio 1 g/9 ml, then processed as was gastric juice. Nitrates reducing activities (Nra) in the juice and in the biopsies were evaluated in nitrates broth with Griess-llosvay reaction. Results (n ° CFU/ml; means+SD): NUD AGC Ome pH 1.35 + 0.32 7.48 ± 0.63 6.90 _+ 1.29 J, A, EI J A B J A B Stp NO 3.6+1.1 4.4_+0.8 4.6±0.8 2.7_+1.2 4.0-2-0.4 4.0±0.4~ Str NG 6.9_+0.7 6.3_+0.3 6.5_+0.7 5.2±1.5a 5.1±1.1 a 4.9±1.0° Aer NG 7.4±0.5 6.5_+0.4 6.9_+0.9 5.9_+1.1a 5.4±1.0° 5.4_+1.0° Aria NG 7.4_+0.5 7.0_+0.4 7.1~0.7 6.2+_1.1,b 5.5±1.19 5.8-+0.9a L NG 6.3_+0.7 5.8-+1.3 5.9-+1.1 4.8±1.5o 3.9±1.4b 4.2±1.0b E NG 2.7_+1;0 4.0_+0.2 4.1±0.5 2.0±0.2 3.9±0.1 3.9±0.1 Nra NG 5.7±2.0 5.0±1.1 5.6±0.9 4.6±1.8 4A±l.l 4.6±1.7 [a=p<0.01, b=p<0.05 AGC vs Ome]. Conclusions: in achlothydric subjects (ACG, Ome) there is an important microbial proliferation, with only slight differences between bacterial concentration in the juice and in the biopsies. Not all the strains present in the juice were found also in the biopsies. The growth of all microbial strains in the omeprazole group was significantly lower than ACG group. The lack of difi%rences of morning pH between groups is probably due to circadian variations of gastric acidity during therapy. Nitrates-reducing activities are h i ~ and not different in the two groups of achlorhydric patients.
• SPONTANEOUSLY COLITIC C3H/HeJBir MICE DEMONSTRATE ANTIBODY REACTIVITY TO ISOLATED C O L O N I E S O F E N T E R I C B A C T E R I A . S.L. Brandwein, R.P. McCabe, B.U. Ridwan, K.B. Waites, E.H. Birkenmeier, J.P. Sundberg, C.O. Elson, Division of Gastroenterology and Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294 and The Jackson Laboratory, Bar Harbor ME 04609 C3H/HeJBir mice, a substrain of C3H/HeJ, develop spontaneous colitis early in life at the time of bacterial colonization. We have previously demonstrated that these colitic mice produce serum antibodies to unselected cecal bacteria but not to epithelial cells. The purpose of this study was to evaluate the antibody reactivity of these mice against isolated colonies of enteric bacteria which are important constituents of normal flora. Methods: Enhanced chemiluminescence Western blotting was performed to examine the serum responses of colitic C3H/HeJBir and non-colitic C3H/HeJ mice against bacterial antigens. Isolated bacterial colonies were obtained from American Type Culture Collection and the clinical m i c r o b i o l o g y laboratory. Bacteria were prepared using mechanical lysis and other fractionation techniques. Results: The serum from C3H/HeJBir mice detected one or more protein bands in most bacterial colonies, while the C3H/HeJ mice detected none. Of the colonies tested overall, the aerobic bacteria had more protein antigens recognized than the anaerobes. One of the most reactive anaerobic bacteria was Bacteroides which had several strongly reactive bands. Aerobic bacteria, including gram positive bacteria such as Enterococcus and gram negative bacteria such as Enterobacteriaceae, each had multiple protein bands detected. When Enterobacteriaceae such as Escherichia coli, Proteus mirabilis, and Salmonella typhimurium were fractionated, the antigens detected by the colitic serum were localized to the outer membrane. Purified outer membrane components including endotoxin and porins such as OmpC, OmpD, and O m p F had no reactivity with colitic serum. None of the isolated bacteria tested reproduced the pattern of serum reactivity to unselected cecal bacteria. Conclusions: Colitic C3H/HeJBir mice produce antibodies to multiple isolated colonies of bacteria from the normal enteric flora. In the Enterobacteriaceae, the outer membrane proteins were the principal proteins detected by the serum from the colitic mice. We speculate that there are specific antigenically dominant colonies of enteric bacteria to which the serum reactivity is directed. These antibodies may reflect an immune dysregulation resulting in colonic inflammation in the C3H/HeJBir mice. (Supported by NIH grants P01 DK44240 and T32 AIO7051).
• HAPLOTYPE RESTRICTED DIFFERENCES IN IN VITRO PRODUCTION OF TUMOR NECROSIS FACTOR ALPHA AND LYMPHOTOXIN ALPHA IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE AND HEALTHY CONTROLS. G. Bouma1, JBA Cmsius', M Oudkerk Pool 1, JJ Kolkman~, BME yon Blomberg~, SGM Meuwissen~, and AS PefiaL Departments of Gastroenterology1 and Pathology2, Free University Hospital, Amsterdam. Although the aetiology of the chronic inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) is still unknown, it is established that disease occurs in a genetically susceptible subject. Tumor necrosis factor alpha (TNFcx) and lymphotoxin alpha (LTc0 are potent proinflammatory and immunoregulatory eytokines. The genes for these cytokines are tandemly arranged in the central region nfthe MHC, between the HLA-B and HLA-D locus, at the short arm of chromosome 6. This location has prompted much speculation about the role of TNFtx and LTtx in the aetiology of MHC-associated diseases. In order to study the effect of gene polymorphisms on the production of TNFct and LTa, we performed a prospective study in which we determined the production of these cytokines in relation to bi-allelic polymorphisms in the TNFc~ and LTc~ genes in 30 patients with IBD and 12 healthy controls. Individuals were typed for two polymorphisms in the TNF~ gone, and two polymorphisms in the LTc~ gene, resulting in 4 haplotypes (TNF-C, -E, -I and -P). Peripheral blood mononuclear cells were cultured in the presence of the T-cell activators anti-CD3 and antiCD28, and TNF~ and LTtx production was measured by commercially available ELISA's. No statistically significant differences in TNFtx and LTa secretion were observed between UC patients, CD patients and controls. However, large differences in production were found in relation to the TNF haplotypes; Individuals carrying the haplotype E are the highest producers of TNFc~ (p=.008, as compared to individuals without this haplotype). The lowest production was found in individuals carrying the C haplotype (p=.012 as compared to individuals without this haplotype). The only difference between the C and E haplotypes is the nucleotide position at position -308 in the promoter region of the TNFcz gene. The production of LTc~ in relation to the genotypes showed that individuals carrying the TNF-I haplotype showed a statistically very significant lower production of LTt~ as compared to individuals who lack this haplotype (p =.002). In conclusion, we have shown strong correlations between TNF haplotypes and TNFct and LTct secretion. These findings may contribute to define the genetic heterogeneity of IBD.
• IMMUNOPEROXIDASE TISSUE STAINING TO DIAGNOSE E. COLI O157:H7 INFECTION: A CAUSE OF ISCHEMIC COLITIS? L.J. Brandt. C. Su, E. Alt, S. Sigal, K. Patterson, P.I. Tarr. Montefiore Medical Center, & Bronx Municipal Hosp., Bronx, N¥, Children's Hospital and Medical Center, Seattle, W~sh. E. coli O157:H7 is increasingly recognized to cause diarrhea and hemorrhagic colitis. E . c o l i O157:H7 is diagnosed by finding Shiga-like toxins in the stool or non-sorbitol fermenting colonies on stool culture using MacConkey-Sorbitol agar. We attempted to develop an immunohistochemical technique to diagnose E. coli O157:H7 organisms on colon biopsy or resection specimens and to determine if an association exists between E. coli O157:H7 and idiopathic "ischemic colitis". 22 specimens of colon were studied: E. coli 0157:H7 colitis (4);ischemic colitis (6); ulcerative colitis (8);pseudomembranous colitis (3) and normal colon (1). Formalin-fixed, paraffin-embedded tissue was deparaffinized, rehydrated, incubated with 3% peroxide in methanol and rinsed with phosphate buffered saline (PBS). Sections were then immunolabeled with peroxidase-labeled affinitypurified antibodies (BacTrace ®) to E. coli O157:H7 (Kirkegaard & Perry Labs, Gaithersburg, MD), stained with peroxidase chromagen reagent (Biomeda Corp., Foster City, CA) and counterstained with aqueous hematoxylin. Each staining procedure included positive and negative controls. Results: Only the 4 k n o w n E . c o l i colitis and 1 of 6 ischemic colitis specimens stained positively. Conclusion: This study shows that immunohistochemistry is useful to diagnose enteric infection with E. coli O157:H7 in humans. Commercially available antibodies to E. coli O157:H7 can be applied to colon tissue samples to detect this organism. In addition, this study suggests a causative role for E. coli 0157:H7 in ischemic colitis.
Immunology, Microbiology, and Inflammatory Disorders A787
BACTERIAL COLONIZATION IN JUICE AND BIOPSIES OF THE ACHLORHYDRIC STOMACH G.Brandi~ G.Biasco, B.Biavati~, GM.Paganelli, C.Masci, R Santuoci, C.Calabrese, G.Di Febo, M.Miglioli, L.Barbara. Istituto di Clinica Atedica e Gastroenterologia, Policlinico S.Orsola, llstituto di Mwrobiologia, Facoltd di Agraria, Universitdt di Bologna Aehlorhydria allows bacterial growth in the gastric juice. Little is known about the relationship between microflora in the lumen of the stomach and colonization of the mucosa. This study evaluates for the first time the bacterial concentration and microbiological profile of gastric juice and biopsies of patients with ACG and subjocta treated with omeprazole. Patients and methods: 8 subjects with non-ulcer dyspepsia (NUD) (5m, m.a. 36); 6 pts. (4m; ma. 39) with ACG; 12 pts. (7m; m.a. 49) with reflux oesophagitis (6) or duodenal ulcer (6) treated with omeprazole (Ome) 20 rag/day for 2 months 0ast dose: 24h prior to endoscopy). During endoscopy (8 a.m.) were taken in sterility 5-10 ml of gastric juice and normal mucosal biopsies (antrum x 4, body x 4). The pH of gastric juice was evaluated with pH-meter. The juice was diluted in saline and sowed in non-selective media (BHA) incubated in air for the count of aerobes (Aer) and in a jar for anaerobes (Ana). Selective media were used for Lattobaeilli (L); Enterobacteria (E); Streptococci (Str); Staphilococci (Stp). Dominant strains were identified with electrophoresis of surface proteins, VFA assay, and API system. Biopsies were weighted, homogenized in sterility and diluted with a ratio 1 g/9 ml, then processed as was gastric juice. Nitrates reducing activities (Nra) in the juice and in the biopsies were evaluated in nitrates broth with Griess-llosvay reaction. Results (n ° CFU/ml; means+SD): NUD AGC Ome pH 1.35 + 0.32 7.48 ± 0.63 6.90 _+ 1.29 J, A, EI J A B J A B Stp NO 3.6+1.1 4.4_+0.8 4.6±0.8 2.7_+1.2 4.0-2-0.4 4.0±0.4~ Str NG 6.9_+0.7 6.3_+0.3 6.5_+0.7 5.2±1.5a 5.1±1.1 a 4.9±1.0° Aer NG 7.4±0.5 6.5_+0.4 6.9_+0.9 5.9_+1.1a 5.4±1.0° 5.4_+1.0° Aria NG 7.4_+0.5 7.0_+0.4 7.1~0.7 6.2+_1.1,b 5.5±1.19 5.8-+0.9a L NG 6.3_+0.7 5.8-+1.3 5.9-+1.1 4.8±1.5o 3.9±1.4b 4.2±1.0b E NG 2.7_+1;0 4.0_+0.2 4.1±0.5 2.0±0.2 3.9±0.1 3.9±0.1 Nra NG 5.7±2.0 5.0±1.1 5.6±0.9 4.6±1.8 4A±l.l 4.6±1.7 [a=p<0.01, b=p<0.05 AGC vs Ome]. Conclusions: in achlothydric subjects (ACG, Ome) there is an important microbial proliferation, with only slight differences between bacterial concentration in the juice and in the biopsies. Not all the strains present in the juice were found also in the biopsies. The growth of all microbial strains in the omeprazole group was significantly lower than ACG group. The lack of difi%rences of morning pH between groups is probably due to circadian variations of gastric acidity during therapy. Nitrates-reducing activities are h i ~ and not different in the two groups of achlorhydric patients.
• SPONTANEOUSLY COLITIC C3H/HeJBir MICE DEMONSTRATE ANTIBODY REACTIVITY TO ISOLATED C O L O N I E S O F E N T E R I C B A C T E R I A . S.L. Brandwein, R.P. McCabe, B.U. Ridwan, K.B. Waites, E.H. Birkenmeier, J.P. Sundberg, C.O. Elson, Division of Gastroenterology and Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35294 and The Jackson Laboratory, Bar Harbor ME 04609 C3H/HeJBir mice, a substrain of C3H/HeJ, develop spontaneous colitis early in life at the time of bacterial colonization. We have previously demonstrated that these colitic mice produce serum antibodies to unselected cecal bacteria but not to epithelial cells. The purpose of this study was to evaluate the antibody reactivity of these mice against isolated colonies of enteric bacteria which are important constituents of normal flora. Methods: Enhanced chemiluminescence Western blotting was performed to examine the serum responses of colitic C3H/HeJBir and non-colitic C3H/HeJ mice against bacterial antigens. Isolated bacterial colonies were obtained from American Type Culture Collection and the clinical m i c r o b i o l o g y laboratory. Bacteria were prepared using mechanical lysis and other fractionation techniques. Results: The serum from C3H/HeJBir mice detected one or more protein bands in most bacterial colonies, while the C3H/HeJ mice detected none. Of the colonies tested overall, the aerobic bacteria had more protein antigens recognized than the anaerobes. One of the most reactive anaerobic bacteria was Bacteroides which had several strongly reactive bands. Aerobic bacteria, including gram positive bacteria such as Enterococcus and gram negative bacteria such as Enterobacteriaceae, each had multiple protein bands detected. When Enterobacteriaceae such as Escherichia coli, Proteus mirabilis, and Salmonella typhimurium were fractionated, the antigens detected by the colitic serum were localized to the outer membrane. Purified outer membrane components including endotoxin and porins such as OmpC, OmpD, and O m p F had no reactivity with colitic serum. None of the isolated bacteria tested reproduced the pattern of serum reactivity to unselected cecal bacteria. Conclusions: Colitic C3H/HeJBir mice produce antibodies to multiple isolated colonies of bacteria from the normal enteric flora. In the Enterobacteriaceae, the outer membrane proteins were the principal proteins detected by the serum from the colitic mice. We speculate that there are specific antigenically dominant colonies of enteric bacteria to which the serum reactivity is directed. These antibodies may reflect an immune dysregulation resulting in colonic inflammation in the C3H/HeJBir mice. (Supported by NIH grants P01 DK44240 and T32 AIO7051).