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HLA-DR4 subtypes carrying valine at residue 86 as a severity marker of ulcerative colitis
lmmlmology, Microbiology, and Inflmmmatory Disorders A1025
April 1998 • G4196 THE NEUTROPHIL ACTIVATING PROTEIN (NAP) OF HELICOBACTER PYLORI (H. PYLORI) IS A DPS HOMOLOGUE WITHOUT NEUTROPHIL ACTIVATING PROPERTIES. RPH Logan, GA Turner, D Thirwell, A Cockayne, P Jenks, CJ Hawkey, B Wren. Div of Gastroenterology & Institute of Infections & Immunity, University Hospital, Nottingham, NG7 2UH & Dept of Microbiology, St Bartholomew's Hospital, London. Introduction: Previous studies have suggested that soluble water extracts of H.pylori contain a 15kDa protein (NapA) which directly activates neutrophils (PMNs) and may thus be a factor in the aetiology of active gastritis. Aim: to establish the role of NapA by disruption and characterisation of a napA isogenic mutant Methods: napA gene fragments were isolated from H.pylori (strain N6) using degenerate primers and the full sequence of each gene fragment obtained from a H.pylori lambda(I)ZAP library. A defined mutant was constructed by insertional activation using a kanamycin cassette followed by allelic replacement (N6:nap-). Isolated clones were expanded and water extracts of parent and mutant strains prepared as previously described, pMN activation was assessed by flow cytometric shape change assay, expression of cdl lb/cdl8 and NBT dye reduction assay. Results: In comparison to parent strain N6, SDS-PAGE of N6:nap" showed loss of a 15 kDa protein indicating absence of NapA; disruption of nap was also confirmed by Southern blotting. In addition PAGE profiles revealed the differential expression of other proteins in N6:nap'. Serial dilutions (1:10) of water extracts from N6 and N6:nap- were equally able to activate PMNs as assessed by flow cytometry, cdllb/18 expression and NBT dye reduction assay. After bioinformatic re-analysis suggested close homology between nap and the regulatory DNA binding protein of E.coli (dps), the survival of N6 and N6:nap- under acid (ph4) and oxidative (H2O2, 20 and 120 mM) stress was determined (as colony forming units) and urease + catalase activities and protein profiles assessed. Triplicate assays were performed at least 5 times, and p values determined by Mann Whitney U. *=p<0.005, **=p<0.001, ***=p<0.0001
ph 4 ph 7 time: 30m 3h 30m 3h efu x 10s N6 6.4 0.46 19.4 8.5 N6:nap" 2.9* 0.07** 5.6 4.5
H202:
20mM 30m 3h 4.4 3.6
120mM 30m 3h
4.5 4.2 0.6*** 0.5**
3.4 0.3*
In N6:nap" acid and oxidative stress was also associated with reduced urease and catalase enzyme activities and changes in protein profiles. The ability of purified NapA to bind DNA was confirmed by gel shift assay. Conclusions: H.py/or/nap is a DNA binding (dps) homologue involved with acid and oxidative stress responses and is devoid of any neutrophil activating properties. Dr Logan and Dr Jenks are funded by the Wellcome Trust. G4197 HUMAN ANTI-THOMSEN-FRIEDENREICH (TF) ANTIBODIES, WHICH ARE UBIQUITOUS AND APPEAR ON WEANING, REACT WITH EPITOPES ON INTESTINAL BACTERIAL CELL WALLS. I London ], CA Hart2, Bo Jansson3, Jonathan M Rhodes 1. 1Department of Medicine, University of Liverpool, Liverpool, UK. 2Department of Medical Microbiology, Duncan Building, Royal Liverpool Hospital, Liverpool, UK. 3Biolnvent Therapeutic, Lund, Sweden. All human sera contain cold-reactive anti-TF antibodies, usually IgM or IgA subclasses, that appear in the serum on weaning. It has been suggested, but never proven, that they may reflect an immune response to "normal" bacterial antigens (Springer and Horton J. Clin. Invest. 1969; 48, 1280-1291). Interest in anti-TF antibodies has recently been heightened by the finding that some of these antibodies cause a dose-dependent proliferative effect on HT29 colorectal ceils that are known to express TF-antigen (Int. J. Cancer 1997; 73, 1-8 1997). Five human monoclonal anti-TF antibodies (TF-1 to 5) were supplied by Biolnvent (Lund, Sweden) for this study. These antibodies had been prepared following in-vitro stimulation by asialo-glycophorin of Leu-Leu-OMe4reated peripheral blood lymphocytes. Each antibody was screened against thirty-six diverse human bacterial isolates from a known library, including examples
from the Enterobacteriaceae, Bacteroidaceae, Micrococcaceae, Neisseriaceae and Bacilliaceae families. Antibody binding to bacteria was assessed using a haemagglutination inhibition technique. All red ceils were obtained from a single donor and pre-treated with sialidase to reveal surface TF-anfigen expression. Of the initial thirty-six bacterial isolates screened against each of the five antiTF antibodies, Enterobacter sakazakii was alone able to inhibit haemagglutination by an anti-TF antibody. This effect was consistently seen (n=6) with the TF-1 anti-TF monoclonal antibody. Enterobacter spp. inhabit soil and water. Enterobacter sakazakii has been associated with neonatal sepsis and is a rare cause of nosocomal infection. This study supports the hypothesis that anti-TF antibodies may be produced in response to exposure to environmental bacteria.
• G4198 HLA-DR4 SUBTYPES CARRYING VALINE AT RESIDUE 86 AS A SEVERITY MARKER OF ULCERATIVE COLITIS. G. L6pez-Nava*, M. Fermlndez-Arquero, J. Garcfa-Paredes*, F. Vidal, P. Vigil, J.B.A. Crusius**, A.S. Pefia***, M. Dfaz-Rubio*, E. G. de la Concha.
Depts. of Immunology and Gastroenterology, San Carlos University Hospital, Madrid and Depts. of Gastroenterology, Sant Pau Hospital, Barcelona, Spain and Vrije Universiteit**, Amsterdam, The Netherlands. Genetic factors are known to play a role in the predisposition to ulcerative colitis (UC) and several HLA-DRB1 alleles have been shown to be either positively or negatively associated with the disease. Notably, a negative association with DR4 has been reported in UC white patients, although no study of the different DR4 subtypes has been reported. The aim of the present study was to investigate further the association of the HLA-DR4 group and its allelic subspecificities with susceptibility and disease phenotype in UC. We have studied 135 unrelated Spanish Caucasian patients with UC and 275 ethnically matched controls. The disease was classified as extensive (inflammation proximal to the splenic flexure) in 74 patients or distal in 61 patients. No association was found between UC and HLA-DR4, but when the patients were classified according to disease extension, a statistically significant positive association was found between DR4 and extensive disease (27% versus 8% in distal disease; p=0.005, OR=4.15). DR4 subtypes were analysed and the association of the extensive disease phenotype was found with DRBI*0404 (9% vs 0% in distal disease, p=0.012), as well as with all subtypes carrying valine at position 86 (23% vs 5% in distal disease, p=0.003, 0R=5.77). HLA-DR4 subtypes carrying valine at position 86 were also significantly increased in patients with age of onset of UC under 25 years (27% vs. 11% in distal disease, p=0.04, OR=2.82) Conclusions: This study shows that HLA-DR4 subtypes differ in their association with UC disease phenotype. It suggests that Val86 HLA-DR4 subtypes are severity markers of UC in the Spanish population. They predispose to an early onset and to the development of extensive colitis. The association of the HLA-DR4 group of alleles with UC will depend in the different geographic areas on the frequency of the different DR4 subtypes in the normal population. G4199 IMPACT OF ULCERATIVE COLITIS VS CROHN'S DISEASE ON HEALTH RELATED QUALITY OF LIFE. J. L6pez Vivancos*, F. Casellas. X. Badfa +, J. Vilaseca, J-R. Malagelada. Digestive System Research Unit. Hospital General Vail d'Hebron. *Internal Medicine Service, Hospital General de Catalunya. +Institut de Salut P~blica de Catalunya. Barcelona, Spain. Ulcerative colitis (CU) and Crohn's disease (CD) share some clinical manifestations but have distinct physiopathological, epidemiological, morphological and clinical characteristics. Both conditions diminish health related quality of life (HRQOL), but whether impact on HRQOL and on different dimensions of life is different between UC or CD is unknown. Thus, we interviewed prospectively 116 patients with UC (clinically active in 29 and inactive in 87) and 95 with CD (active in 30 and inactive in 65). HRQOL was assessed using a validated spanish version of the Inflammatory Bowel Disease Questionnaire (IBDQ). Analysis included five dimensions: intestinal symptoms, general symptoms, emotional, social and functional. Two general HRQOL questionnaires (EUROOOL and Psychological General Well Being Index -PGWBI-) of HRQOL were also administered. RESULTS: Global IBDQ score was similar in UC and CD, for both inactive (median [95% CI]: 6.3 [5.9-6.2]) vs (6.2 [5.8-6.1]) and active (4.1 [3.6-4.4]) vs (4.1 [3.7-4.5]) disease, although the score was significantly lower in active disease (p<0.01). For each of the 5 dimensions assessed, scores were lower in active than in inactive disease, but again differences between UC and CD were not statistically significant. [ UC ACTIVE
UC INACTWE
CD ACTIVE [ CD INACTIVE
EUROQO[ dimensions visual scale
0.6 [0.6-0.7]* 50 [49-63]
1.0 [0.8-0.9] 80 [78-83]
0.6 [0.6-0.7]* 50 [45-62]*
0.9[0.8-0.9] 80 [76-83]
PGWBI
78 [70-82]*
107 [99-107]
74 [68-86]*
102 [92-103]
(* = p < 0.001, active vs inactive disease) In conclusion, the impact of the disease on global and each dimensional HRQOL score is similar in ulcerative colitis and Crohn's disease.
April 1998 • G4196 THE NEUTROPHIL ACTIVATING PROTEIN (NAP) OF HELICOBACTER PYLORI (H. PYLORI) IS A DPS HOMOLOGUE WITHOUT NEUTROPHIL ACTIVATING PROPERTIES. RPH Logan, GA Turner, D Thirwell, A Cockayne, P Jenks, CJ Hawkey, B Wren. Div of Gastroenterology & Institute of Infections & Immunity, University Hospital, Nottingham, NG7 2UH & Dept of Microbiology, St Bartholomew's Hospital, London. Introduction: Previous studies have suggested that soluble water extracts of H.pylori contain a 15kDa protein (NapA) which directly activates neutrophils (PMNs) and may thus be a factor in the aetiology of active gastritis. Aim: to establish the role of NapA by disruption and characterisation of a napA isogenic mutant Methods: napA gene fragments were isolated from H.pylori (strain N6) using degenerate primers and the full sequence of each gene fragment obtained from a H.pylori lambda(I)ZAP library. A defined mutant was constructed by insertional activation using a kanamycin cassette followed by allelic replacement (N6:nap-). Isolated clones were expanded and water extracts of parent and mutant strains prepared as previously described, pMN activation was assessed by flow cytometric shape change assay, expression of cdl lb/cdl8 and NBT dye reduction assay. Results: In comparison to parent strain N6, SDS-PAGE of N6:nap" showed loss of a 15 kDa protein indicating absence of NapA; disruption of nap was also confirmed by Southern blotting. In addition PAGE profiles revealed the differential expression of other proteins in N6:nap'. Serial dilutions (1:10) of water extracts from N6 and N6:nap- were equally able to activate PMNs as assessed by flow cytometry, cdllb/18 expression and NBT dye reduction assay. After bioinformatic re-analysis suggested close homology between nap and the regulatory DNA binding protein of E.coli (dps), the survival of N6 and N6:nap- under acid (ph4) and oxidative (H2O2, 20 and 120 mM) stress was determined (as colony forming units) and urease + catalase activities and protein profiles assessed. Triplicate assays were performed at least 5 times, and p values determined by Mann Whitney U. *=p<0.005, **=p<0.001, ***=p<0.0001
ph 4 ph 7 time: 30m 3h 30m 3h efu x 10s N6 6.4 0.46 19.4 8.5 N6:nap" 2.9* 0.07** 5.6 4.5
H202:
20mM 30m 3h 4.4 3.6
120mM 30m 3h
4.5 4.2 0.6*** 0.5**
3.4 0.3*
In N6:nap" acid and oxidative stress was also associated with reduced urease and catalase enzyme activities and changes in protein profiles. The ability of purified NapA to bind DNA was confirmed by gel shift assay. Conclusions: H.py/or/nap is a DNA binding (dps) homologue involved with acid and oxidative stress responses and is devoid of any neutrophil activating properties. Dr Logan and Dr Jenks are funded by the Wellcome Trust. G4197 HUMAN ANTI-THOMSEN-FRIEDENREICH (TF) ANTIBODIES, WHICH ARE UBIQUITOUS AND APPEAR ON WEANING, REACT WITH EPITOPES ON INTESTINAL BACTERIAL CELL WALLS. I London ], CA Hart2, Bo Jansson3, Jonathan M Rhodes 1. 1Department of Medicine, University of Liverpool, Liverpool, UK. 2Department of Medical Microbiology, Duncan Building, Royal Liverpool Hospital, Liverpool, UK. 3Biolnvent Therapeutic, Lund, Sweden. All human sera contain cold-reactive anti-TF antibodies, usually IgM or IgA subclasses, that appear in the serum on weaning. It has been suggested, but never proven, that they may reflect an immune response to "normal" bacterial antigens (Springer and Horton J. Clin. Invest. 1969; 48, 1280-1291). Interest in anti-TF antibodies has recently been heightened by the finding that some of these antibodies cause a dose-dependent proliferative effect on HT29 colorectal ceils that are known to express TF-antigen (Int. J. Cancer 1997; 73, 1-8 1997). Five human monoclonal anti-TF antibodies (TF-1 to 5) were supplied by Biolnvent (Lund, Sweden) for this study. These antibodies had been prepared following in-vitro stimulation by asialo-glycophorin of Leu-Leu-OMe4reated peripheral blood lymphocytes. Each antibody was screened against thirty-six diverse human bacterial isolates from a known library, including examples
from the Enterobacteriaceae, Bacteroidaceae, Micrococcaceae, Neisseriaceae and Bacilliaceae families. Antibody binding to bacteria was assessed using a haemagglutination inhibition technique. All red ceils were obtained from a single donor and pre-treated with sialidase to reveal surface TF-anfigen expression. Of the initial thirty-six bacterial isolates screened against each of the five antiTF antibodies, Enterobacter sakazakii was alone able to inhibit haemagglutination by an anti-TF antibody. This effect was consistently seen (n=6) with the TF-1 anti-TF monoclonal antibody. Enterobacter spp. inhabit soil and water. Enterobacter sakazakii has been associated with neonatal sepsis and is a rare cause of nosocomal infection. This study supports the hypothesis that anti-TF antibodies may be produced in response to exposure to environmental bacteria.
• G4198 HLA-DR4 SUBTYPES CARRYING VALINE AT RESIDUE 86 AS A SEVERITY MARKER OF ULCERATIVE COLITIS. G. L6pez-Nava*, M. Fermlndez-Arquero, J. Garcfa-Paredes*, F. Vidal, P. Vigil, J.B.A. Crusius**, A.S. Pefia***, M. Dfaz-Rubio*, E. G. de la Concha.
Depts. of Immunology and Gastroenterology, San Carlos University Hospital, Madrid and Depts. of Gastroenterology, Sant Pau Hospital, Barcelona, Spain and Vrije Universiteit**, Amsterdam, The Netherlands. Genetic factors are known to play a role in the predisposition to ulcerative colitis (UC) and several HLA-DRB1 alleles have been shown to be either positively or negatively associated with the disease. Notably, a negative association with DR4 has been reported in UC white patients, although no study of the different DR4 subtypes has been reported. The aim of the present study was to investigate further the association of the HLA-DR4 group and its allelic subspecificities with susceptibility and disease phenotype in UC. We have studied 135 unrelated Spanish Caucasian patients with UC and 275 ethnically matched controls. The disease was classified as extensive (inflammation proximal to the splenic flexure) in 74 patients or distal in 61 patients. No association was found between UC and HLA-DR4, but when the patients were classified according to disease extension, a statistically significant positive association was found between DR4 and extensive disease (27% versus 8% in distal disease; p=0.005, OR=4.15). DR4 subtypes were analysed and the association of the extensive disease phenotype was found with DRBI*0404 (9% vs 0% in distal disease, p=0.012), as well as with all subtypes carrying valine at position 86 (23% vs 5% in distal disease, p=0.003, 0R=5.77). HLA-DR4 subtypes carrying valine at position 86 were also significantly increased in patients with age of onset of UC under 25 years (27% vs. 11% in distal disease, p=0.04, OR=2.82) Conclusions: This study shows that HLA-DR4 subtypes differ in their association with UC disease phenotype. It suggests that Val86 HLA-DR4 subtypes are severity markers of UC in the Spanish population. They predispose to an early onset and to the development of extensive colitis. The association of the HLA-DR4 group of alleles with UC will depend in the different geographic areas on the frequency of the different DR4 subtypes in the normal population. G4199 IMPACT OF ULCERATIVE COLITIS VS CROHN'S DISEASE ON HEALTH RELATED QUALITY OF LIFE. J. L6pez Vivancos*, F. Casellas. X. Badfa +, J. Vilaseca, J-R. Malagelada. Digestive System Research Unit. Hospital General Vail d'Hebron. *Internal Medicine Service, Hospital General de Catalunya. +Institut de Salut P~blica de Catalunya. Barcelona, Spain. Ulcerative colitis (CU) and Crohn's disease (CD) share some clinical manifestations but have distinct physiopathological, epidemiological, morphological and clinical characteristics. Both conditions diminish health related quality of life (HRQOL), but whether impact on HRQOL and on different dimensions of life is different between UC or CD is unknown. Thus, we interviewed prospectively 116 patients with UC (clinically active in 29 and inactive in 87) and 95 with CD (active in 30 and inactive in 65). HRQOL was assessed using a validated spanish version of the Inflammatory Bowel Disease Questionnaire (IBDQ). Analysis included five dimensions: intestinal symptoms, general symptoms, emotional, social and functional. Two general HRQOL questionnaires (EUROOOL and Psychological General Well Being Index -PGWBI-) of HRQOL were also administered. RESULTS: Global IBDQ score was similar in UC and CD, for both inactive (median [95% CI]: 6.3 [5.9-6.2]) vs (6.2 [5.8-6.1]) and active (4.1 [3.6-4.4]) vs (4.1 [3.7-4.5]) disease, although the score was significantly lower in active disease (p<0.01). For each of the 5 dimensions assessed, scores were lower in active than in inactive disease, but again differences between UC and CD were not statistically significant. [ UC ACTIVE
UC INACTWE
CD ACTIVE [ CD INACTIVE
EUROQO[ dimensions visual scale
0.6 [0.6-0.7]* 50 [49-63]
1.0 [0.8-0.9] 80 [78-83]
0.6 [0.6-0.7]* 50 [45-62]*
0.9[0.8-0.9] 80 [76-83]
PGWBI
78 [70-82]*
107 [99-107]
74 [68-86]*
102 [92-103]
(* = p < 0.001, active vs inactive disease) In conclusion, the impact of the disease on global and each dimensional HRQOL score is similar in ulcerative colitis and Crohn's disease.