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Tagged random insertion mutagenesis of helicobacter pylori
GASTROENTEROLOGY Vol. 114, No. 4
A932 AGA ABSTRACTS •
G3818
EX-VIVO AUTORADIOGRAPHY AND F A C S - S C A N A N A L Y S I S DEMONSTRATION OF A SPECIFIC BINDING BETWEEN IL2-RECEPTOR POSITIVE CELLS IN CROHN'S DISEASE GUT A N D 123I-INTERLEUKIN-2 INJECTED IN VIVO. L. Bianc0ne, A. Signore, G. Ronga, P. Pozzilli, F. Pallone. Clinica Medica 2, Universifft La Sapienza, Roma; Dpt. di Medicina Sperimentale, Universit~ di R. Calabria, Catanzaro. Activated intestinal mononuclear cells (LPMC) expressing Interleukin-2 (IL-2) receptors (IL-2R) represent a major component of gut inflammation in crohn's Disease (CD). Recently, a higher uptake of 123I-IL2has been shown in CD gut. However, specific immunoreactivity of labelled IL2 to human IL2-R+ve LPMCs has not been demonstrated. Aims. To assess by ex-vivo micro-autoradiography whether 123I-IL2 injected "in vivo" in CD patients specifically binds to activated LPMCs infiltrating CD gut. We also evaluated by FACSscan analysis whether radioactivity of injected 123I-IL2 is associated with CD25+ve LPMCs. Methods. Ex-vivo micro-autoradiography and FACSscan analysis were performed in 3 CD patients undergoing ileo-cecal resection. During surgery patients were injected i.v. with 125-IL2 (50-150 pci) Autoradiography. Surgical samples were frozen in liquid N2. Criostatic sections were fixed and sequentially stained with a mouse anti-human CD25 (Beckton-Dickinson, CA), a biotin conjugated rat anti-mouse IgG and a peroxidase-conjugated streptavidin (Dako, Germany). Sections were deeped into K5 liquid emulsion (Ilford, UK), incubated 40 days at -70°C, fixed and stained with Haematoxilin-Eosin. FACSscan analysis. LPMC were isolated by DTl'-EDTA-Collagenase followed by Percoll and stained with a FITCconjugated anti-CD25 (Beckton Dickinson, CA). LPMC (180x106) were sorted by FACSscan (Beckton-Dickinson, CA). Sorted CD25+ve and CD25-ve cells were counted for 600 rain. in a high sensitive x-counter (LKB). Data were expressed as c.p.m./107 cells-c.p.m, background. Results. Autoradiography. Immunoperoxidase staining showed that 1231-IL2 specifically binds only to the surface of IL2-R+ve LPMCs. No significant 125I-IL2 binding was found to be associated to the IL2-R-ve LPMCs nor to other cell types. FACSscan analysis counting the radioactivity associated with the CD25+ve and CD25-ve LPMC. CD25+ve LPMC were 27% (Figure) and radioactivity was only associated to the CD25+ve cell population (200 vs 5 c.p.m/107 ceils). The number of IL2 molecules bound per CD25+ve cell was 480, assuming 1 125Imolecule/IL2 molecule. ImO-
&
!" o
orosomucoids (p=0.001 and p=0.002 vs negative groups). In the 1 year follow up, the mean otl-AT-C1 significantly increased at 12 months vs both baseline and 6 months values (p=0.005; p=0.009). At baseline, otl-AT-C1 was significantly higher in the subgroup of inactive patients developing clinical relapse (CDAI>200) in the next 6 months than in those still inactive at 6 months (p=0.03). Fecal ctl-AT-CI showed a 75% sensitivity, 90% specificity, 60% PPV and 94% NPV in predicting CD relapse in the next 6 months. A lower sensitivity and specificity was shown by ¢tl-AT-C1 in predicting CD relapse at subsequent observations (1-4 yrs). CONCLUSIONS. Fecal oil-AT clearance represents a sensitive and specific marker of early clinical relapse of CD in patients under regular surveillance during remission. • G3820
THE SPECIFIC TYPE IV PHOSPHODIESTERASE INHIBITOR ROLIPRAM MITIGATES EXPERIMENTAL COLITIS IN MICE. C. Bidlin~,maier. G. Hartmann, MD, K. Tsch6p, MD, A. Eigler, MD, H. A. Lehr, MD* and S. Endres, MD. Division of Clinical Pharmacology, Medizinische Klinik, Klinikum Innenstadt, University of Munich, and *Department of Pathology, University of Mainz, Germany. Anti-tumor necrosis factor-a (TNF) antibody therapy has been successfully used in patients with Crohn's disease (Targan et al., 1997). The specific type IV phosphodiesterase inhibitor rolipram (Semmler et al., 1993) is a promising candidate among several agents known to suppress formation of TNF (Eigler et al., 1997). We examined the efficacy of rolipram both for prevention of ongoing colitis (study A) and for therapy of established colitis (study B). Colitis was induced in BALB/c mice by continuous administration of 5% dextrane sulfate sodium (DSS; 43 kilodaltons) dissolved in drinking water for 7 days. Treatment was performed with intraperitoneal injection of rolipram (2 x 5 mg/kg body weight/day). Therapy and scoring of colitis was performed in a blinded fashion~ In study A (therapy of ongoing colitis), the clinical colitis score, calculated by weight loss, stool consistency and bleeding (range 0, healthy, to 4, severely ill), was lower in the rolipram-treated mice (1.1 versus 2.6; p = 0.015; day 8; n = 13). Colon length as an indirect parameter for the degree of inflammation was less reduced in the rolipramtreated mice (length 15.4 cm versus 12.4 cm; p = 0.008; mice without DSS 17.5 cm). The histological colitis score (0, no changes, to 6, serious damage) was lower in rolipram-treated mice (1.4 versus 4.8; p = 0.022; n = 13). In study B (therapy of established colitis), rolipram-treated mice recovered more quickly (80 % versus 40 % recovery; p = 0.022; n =54; day 15). The colon of rolipram-treated mice was less inflamed (less reduced colon length) (length 14.4 cm versus 11.9 cm; p < 0.001; n = 54; mice without DSS 17.3 cm). We conclude that rolipram is effective in both prevention of ongoing colitis and therapy of established colitis. Specific type IV phospbodiesterase inhibition forms a pharmacological principle that should be further investigated for chronic inflammatory bowel disease in humans. • G3821 TAGGED RANDOM INSERTION MUTAGENESIS OF HELICOBACTER PYLORI, J.J.E Bi_ilsma, C.M.J.E. Vandenbroucke-Grauls, J.G.Kusters Dept. of Medical Microbiology, Vrije Universiteit Amsterdam. The Netherlands.
CD=S*
C025°
Conclusions. Ex vivo micro-authoradiography in resected CD patients demonstrated that 123I-IL2 injected "in vivo" specifically binds to IL2-R+ve LPMC infiltrating CD gut. FACSs scan analysis of LPMC also demonstrated a specific immunoreactivity of injected 123I-IL2against CD25+ve LPMCs. • G3819 FECAL al-ANTITRYPSIN AS EARLY INDICATOR OF C L I N I C A L RELAPSE IN CROHN'S DISEASE: RESULTS FROM A 4-YEAR FOLLOW-UP. L. Biancone, M. Silvestri, F. Pallone. Clinica Medica 2, Universit~ "La Sapienza", Roma; Dipartimento di Medicina Sperimentale, Universifft di Reggio Calabria, Catanzaro. BACKGROUND. Fecal ctl-Antitripsin clearance (txl-AT-C1) is a marker of protein loss, reflecting an increased intestinal permeability. ¢tl-AT-C1 is increased during the active phases and in the early post-operative recurrence of Crohn's Disease (CD). However, the usefulness of fecal ctl-AT-C1 in predicting the early clinical CD relapse is unknown. AIM. To investigate, in a 4 years prospective longitudinal study, the possible usefulness of fecal ct 1-AT-C 1 in predicting the early clinical relapse of CD in inactive patients under regular follow up during remission. METHODS. Twenty-six patients with inactive ileal CD (CDAI < 150) on oral rnesalazine (2.4 gr/day) were enrolled and prospectively followed up for 4 years. Fecal otl-AT-C1 and concentration, daily stool weight and serum c~I-AT were measured at baseline (visit 1), after 1 week (visit 2) and 3 weeks from baseline (visit 3) in 26 patients and also every 3 months for 1 year in 10 patients. All patients underwent clinical assessment every 6 months for 4 years. Ten healthy volunteers were used as controls. Serum and fecal oil-AT were quantitated by radial immunodiffusion. RESULTS. In the short term follow up, fecal et 1-AT-C 1 did not significantly modify at different observations. Fecal ul-AT-C1 values were significantly related to CD activity as assessed by the CDAI (r=0.30; p=0.029). Fecal ctl-AT-CI was higher in CD patients showing positivity for CRP and
Recombinant DNA-based genetic research in H.pylori is hindered by a lack of convenient tools. The purpose of this study is to develop a method for generating tagged random insertion mutants in H.pylori. Our method is based On chromosomal integration of suicide plasmids. Methods. We used the apha3 cassette which is known to confer kanamycin resistance to H.pylori. We ligated this cassette into the Smal site of pBC, a plasmid unable to replicate in H.pylori. The resulting vector was called pBCa3. Chromosomal DNA was randomly restricted with Sau3AI and size fractionated to 500 bp. These fragments were ligated into the unique BamHI site in pBCa3 which is in front of the kanamycin cassette. The only way in which these suicide vector constructs can confer kanamycin resistance to H.pylori is by integration into the H.pylori chromosome through a homologous a single cross over event with the chromosomal DNA fragment present in the vector Results. Natural transformation of H.pylori with the constructs yielded 250 kanamycin resistant transformants per pg DNA. A control with no fragments of H.pylori ligated into the vector, resulted in 3 kanamycin resistant transformants per pg DNA. A Southern blot with 16 random selected transformants, probed with the kanamycine dassette, showed that the constructs integrate randomly into the chromosome. The mutant library is now screened for known virulence factors. A first screen indicated that circa 1% of the mutants was disrupted in urease activity and 2% was dismpted in motility. Conclusion. This new method of random insertion mutagenesis allows the direct generation of tagged mutants in H.pylori and the rapid identification of the genes involved.
A932 AGA ABSTRACTS •
G3818
EX-VIVO AUTORADIOGRAPHY AND F A C S - S C A N A N A L Y S I S DEMONSTRATION OF A SPECIFIC BINDING BETWEEN IL2-RECEPTOR POSITIVE CELLS IN CROHN'S DISEASE GUT A N D 123I-INTERLEUKIN-2 INJECTED IN VIVO. L. Bianc0ne, A. Signore, G. Ronga, P. Pozzilli, F. Pallone. Clinica Medica 2, Universifft La Sapienza, Roma; Dpt. di Medicina Sperimentale, Universit~ di R. Calabria, Catanzaro. Activated intestinal mononuclear cells (LPMC) expressing Interleukin-2 (IL-2) receptors (IL-2R) represent a major component of gut inflammation in crohn's Disease (CD). Recently, a higher uptake of 123I-IL2has been shown in CD gut. However, specific immunoreactivity of labelled IL2 to human IL2-R+ve LPMCs has not been demonstrated. Aims. To assess by ex-vivo micro-autoradiography whether 123I-IL2 injected "in vivo" in CD patients specifically binds to activated LPMCs infiltrating CD gut. We also evaluated by FACSscan analysis whether radioactivity of injected 123I-IL2 is associated with CD25+ve LPMCs. Methods. Ex-vivo micro-autoradiography and FACSscan analysis were performed in 3 CD patients undergoing ileo-cecal resection. During surgery patients were injected i.v. with 125-IL2 (50-150 pci) Autoradiography. Surgical samples were frozen in liquid N2. Criostatic sections were fixed and sequentially stained with a mouse anti-human CD25 (Beckton-Dickinson, CA), a biotin conjugated rat anti-mouse IgG and a peroxidase-conjugated streptavidin (Dako, Germany). Sections were deeped into K5 liquid emulsion (Ilford, UK), incubated 40 days at -70°C, fixed and stained with Haematoxilin-Eosin. FACSscan analysis. LPMC were isolated by DTl'-EDTA-Collagenase followed by Percoll and stained with a FITCconjugated anti-CD25 (Beckton Dickinson, CA). LPMC (180x106) were sorted by FACSscan (Beckton-Dickinson, CA). Sorted CD25+ve and CD25-ve cells were counted for 600 rain. in a high sensitive x-counter (LKB). Data were expressed as c.p.m./107 cells-c.p.m, background. Results. Autoradiography. Immunoperoxidase staining showed that 1231-IL2 specifically binds only to the surface of IL2-R+ve LPMCs. No significant 125I-IL2 binding was found to be associated to the IL2-R-ve LPMCs nor to other cell types. FACSscan analysis counting the radioactivity associated with the CD25+ve and CD25-ve LPMC. CD25+ve LPMC were 27% (Figure) and radioactivity was only associated to the CD25+ve cell population (200 vs 5 c.p.m/107 ceils). The number of IL2 molecules bound per CD25+ve cell was 480, assuming 1 125Imolecule/IL2 molecule. ImO-
&
!" o
orosomucoids (p=0.001 and p=0.002 vs negative groups). In the 1 year follow up, the mean otl-AT-C1 significantly increased at 12 months vs both baseline and 6 months values (p=0.005; p=0.009). At baseline, otl-AT-C1 was significantly higher in the subgroup of inactive patients developing clinical relapse (CDAI>200) in the next 6 months than in those still inactive at 6 months (p=0.03). Fecal ctl-AT-CI showed a 75% sensitivity, 90% specificity, 60% PPV and 94% NPV in predicting CD relapse in the next 6 months. A lower sensitivity and specificity was shown by ¢tl-AT-C1 in predicting CD relapse at subsequent observations (1-4 yrs). CONCLUSIONS. Fecal oil-AT clearance represents a sensitive and specific marker of early clinical relapse of CD in patients under regular surveillance during remission. • G3820
THE SPECIFIC TYPE IV PHOSPHODIESTERASE INHIBITOR ROLIPRAM MITIGATES EXPERIMENTAL COLITIS IN MICE. C. Bidlin~,maier. G. Hartmann, MD, K. Tsch6p, MD, A. Eigler, MD, H. A. Lehr, MD* and S. Endres, MD. Division of Clinical Pharmacology, Medizinische Klinik, Klinikum Innenstadt, University of Munich, and *Department of Pathology, University of Mainz, Germany. Anti-tumor necrosis factor-a (TNF) antibody therapy has been successfully used in patients with Crohn's disease (Targan et al., 1997). The specific type IV phosphodiesterase inhibitor rolipram (Semmler et al., 1993) is a promising candidate among several agents known to suppress formation of TNF (Eigler et al., 1997). We examined the efficacy of rolipram both for prevention of ongoing colitis (study A) and for therapy of established colitis (study B). Colitis was induced in BALB/c mice by continuous administration of 5% dextrane sulfate sodium (DSS; 43 kilodaltons) dissolved in drinking water for 7 days. Treatment was performed with intraperitoneal injection of rolipram (2 x 5 mg/kg body weight/day). Therapy and scoring of colitis was performed in a blinded fashion~ In study A (therapy of ongoing colitis), the clinical colitis score, calculated by weight loss, stool consistency and bleeding (range 0, healthy, to 4, severely ill), was lower in the rolipram-treated mice (1.1 versus 2.6; p = 0.015; day 8; n = 13). Colon length as an indirect parameter for the degree of inflammation was less reduced in the rolipramtreated mice (length 15.4 cm versus 12.4 cm; p = 0.008; mice without DSS 17.5 cm). The histological colitis score (0, no changes, to 6, serious damage) was lower in rolipram-treated mice (1.4 versus 4.8; p = 0.022; n = 13). In study B (therapy of established colitis), rolipram-treated mice recovered more quickly (80 % versus 40 % recovery; p = 0.022; n =54; day 15). The colon of rolipram-treated mice was less inflamed (less reduced colon length) (length 14.4 cm versus 11.9 cm; p < 0.001; n = 54; mice without DSS 17.3 cm). We conclude that rolipram is effective in both prevention of ongoing colitis and therapy of established colitis. Specific type IV phospbodiesterase inhibition forms a pharmacological principle that should be further investigated for chronic inflammatory bowel disease in humans. • G3821 TAGGED RANDOM INSERTION MUTAGENESIS OF HELICOBACTER PYLORI, J.J.E Bi_ilsma, C.M.J.E. Vandenbroucke-Grauls, J.G.Kusters Dept. of Medical Microbiology, Vrije Universiteit Amsterdam. The Netherlands.
CD=S*
C025°
Conclusions. Ex vivo micro-authoradiography in resected CD patients demonstrated that 123I-IL2 injected "in vivo" specifically binds to IL2-R+ve LPMC infiltrating CD gut. FACSs scan analysis of LPMC also demonstrated a specific immunoreactivity of injected 123I-IL2against CD25+ve LPMCs. • G3819 FECAL al-ANTITRYPSIN AS EARLY INDICATOR OF C L I N I C A L RELAPSE IN CROHN'S DISEASE: RESULTS FROM A 4-YEAR FOLLOW-UP. L. Biancone, M. Silvestri, F. Pallone. Clinica Medica 2, Universit~ "La Sapienza", Roma; Dipartimento di Medicina Sperimentale, Universifft di Reggio Calabria, Catanzaro. BACKGROUND. Fecal ctl-Antitripsin clearance (txl-AT-C1) is a marker of protein loss, reflecting an increased intestinal permeability. ¢tl-AT-C1 is increased during the active phases and in the early post-operative recurrence of Crohn's Disease (CD). However, the usefulness of fecal ctl-AT-C1 in predicting the early clinical CD relapse is unknown. AIM. To investigate, in a 4 years prospective longitudinal study, the possible usefulness of fecal ct 1-AT-C 1 in predicting the early clinical relapse of CD in inactive patients under regular follow up during remission. METHODS. Twenty-six patients with inactive ileal CD (CDAI < 150) on oral rnesalazine (2.4 gr/day) were enrolled and prospectively followed up for 4 years. Fecal otl-AT-C1 and concentration, daily stool weight and serum c~I-AT were measured at baseline (visit 1), after 1 week (visit 2) and 3 weeks from baseline (visit 3) in 26 patients and also every 3 months for 1 year in 10 patients. All patients underwent clinical assessment every 6 months for 4 years. Ten healthy volunteers were used as controls. Serum and fecal oil-AT were quantitated by radial immunodiffusion. RESULTS. In the short term follow up, fecal et 1-AT-C 1 did not significantly modify at different observations. Fecal ul-AT-C1 values were significantly related to CD activity as assessed by the CDAI (r=0.30; p=0.029). Fecal ctl-AT-CI was higher in CD patients showing positivity for CRP and
Recombinant DNA-based genetic research in H.pylori is hindered by a lack of convenient tools. The purpose of this study is to develop a method for generating tagged random insertion mutants in H.pylori. Our method is based On chromosomal integration of suicide plasmids. Methods. We used the apha3 cassette which is known to confer kanamycin resistance to H.pylori. We ligated this cassette into the Smal site of pBC, a plasmid unable to replicate in H.pylori. The resulting vector was called pBCa3. Chromosomal DNA was randomly restricted with Sau3AI and size fractionated to 500 bp. These fragments were ligated into the unique BamHI site in pBCa3 which is in front of the kanamycin cassette. The only way in which these suicide vector constructs can confer kanamycin resistance to H.pylori is by integration into the H.pylori chromosome through a homologous a single cross over event with the chromosomal DNA fragment present in the vector Results. Natural transformation of H.pylori with the constructs yielded 250 kanamycin resistant transformants per pg DNA. A control with no fragments of H.pylori ligated into the vector, resulted in 3 kanamycin resistant transformants per pg DNA. A Southern blot with 16 random selected transformants, probed with the kanamycine dassette, showed that the constructs integrate randomly into the chromosome. The mutant library is now screened for known virulence factors. A first screen indicated that circa 1% of the mutants was disrupted in urease activity and 2% was dismpted in motility. Conclusion. This new method of random insertion mutagenesis allows the direct generation of tagged mutants in H.pylori and the rapid identification of the genes involved.