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In vitro omeprazole inhibits growth of gram positive and gram negative bacteria including Helicobacter pylori
A~44
AUA AU~IHAUI~
• BACTERIAL PRODUCTS AND, CYTOKINES UPREGULATE 11.-8 GENE EXPRESSION IN HUMAN COLONIC EPITHELIALCELL LINES. C. Jobin. H.H. Herfarth, R.B. Sartor. Center for GI Biology and Disease, Univ. of North Carolina, Chapel Hill The chemokine Interleukin-8 (IL-8) initiates proinflammatory responses: through its chemoattractant activity for neutrophils, lymphocytes and monocytes and its capacity to stimulate neutrophil activity. Epithelial cell lines secrete Ib8 protein after bacterial invasion or cytokine Stimulation, however, the impact of purified bacterial products on IL-8 gene : expression in epithelial cells is not yet completely understood. Therefore we analyzed the induction of IL-8 gene expression in HT-29 and Caco-2 cells after stimulation with cytokines or bacterial components which are in constant contact with colonic epithelial cells. Methods: HT-29 cells were cultured in DMEM and Caco-2 in EMEM supplemented with serum and additives. The cells were stimulated with either IL-113or TNF-¢ (2ng/ml) and by either LPS (endotoxin, tpg/ml) or peptidoglycan-polysaccharide polymers (PG-PS, 50pg/ml)' for 1-24 hrs. IL'8 gene expression was analyzed at the protein level by ELISA and at the RNA level by : quantitative RT -PCR: Results: IL-8 mRNA accumulation increased in HT-29 cells upon cytokine- (peak: between l h and 2h) or bacterial products stimulation (peak at 12h). This mRNA induction was followed by secretion of IL-8 protein. IL-8 induction was higher in cytokine-stimulated HT-29 cells compared to LPS or PG,PS stimulation (25 fold versus 7 fold, respectively). This induction of IL-8 did not require IL-1, since IL-1 mRNA remained undetectable by RT-PCR. In Caco-2 cells, IL-8 gone expression could be induced by IL-113 but not by TNF-a or bacterial products. Conclusions: These results show that cytokines and bacterial products present in the nflamed intestine can upregulate 11.,8 gone expression in epithelial eel! lines. IL-i[3 and TNF-c~ appear to be more potent stimuli for the IL-8 gene. The differential response to cytokine and bacterial product stimulation between enterocyte-like cells (Caco-2) and undifferentiated enterocytes (HT-29) may be representative of polarity of IL-8 responsiveness within the intestinal crypt. The high abundance of LPS or PG-PS in the intestinal lumen raises the possibility that these ubiquitous bacteria poymers are mportant p h y s o o g c and pathophysiologic stimulants of IL-8 in the gut epithelium.
• IN VITRO OMEPRAZOLE INtllBITS GROWTH OF GRAM r o s m VE AND GRAM NEGATIVE BACTERIA INCLUDING HELICOBACTER PYLORI D. Jonkers ~, E. Stobberingh~, and R. Stockbrfigger~ Depts. of Microbiology~ and Gastroanterology~, Academical Hospital Maastricht, The Netherlands. Omeprazolo, a substituted banzimidazole, is used for the Eeatment of peptic diseases and in combination with antibiotics for the eradication of Helicobacter ~oylori. Moreover, the inhibition of gastric acid secretion by omeprazole can result in bacterial overgrowth in stomach and duodenum. We wanted to study potential direct effects of omeprazole on bacteria commonly found in the stomach. Methods: The in vitro antibacterial effect of omeprazol¢ (100, 200, and 300/zg/ml) was assessed using bacterial growth curves. To study a possible influence of omeprazole' on the availability of cysteinc similar experiments were performed after supplementation of the medium With cysteine (0.01 and 0 . 1 % ) . In addition, possible interaction with amoxycilline and doxycycline (1-64 #g/ml) was studied using checkerboard titrations. Results: A dose-related and permanent ( > 24 hours) growth inhibition was seen on actively growing Gram positive cocci, i.e. Enterococcus faeealis and rStaohvlococcus aureu#. A smaller effect for 8 hours only, was found on Gram negative cocci, i.e. Escherichia coli and Klebsiella oneumoniae. After 24 hours incubation of H.Dvlori in the presence of omeprazole 200 /~g/ml bacterial count was reduced to zero compared to 10" cfu/ml in the control without omeprazole. Supplementation of the medium with cysteine reduced the inhibitory effect of omeprazole, suggesting that the inhibitory effect might partly be due to binding of omeprazole to cysteine present either in the bacteria or as a nutrient. No synergistic or antagonistic interaction of omeprazole with amoxycilline or doxycycline was observed for E.coli and Enterococcus faeealis.
Summery/conclusion: The indicated antibacterial properties of omeprazole possibly counteract gastrointestinal bacterial overgrowth, a known result of profound gastric acid inhibition, and may also be of additional advantagein the eradication of H.evlori.
GASTROENTEROLOGY, VoI. 108, NO. 4
• NF-kB ACTIVATION AND IL-8 EXPRESSION IN STIMULATED COLONIC EPITHELIAL CELL LINES. C. Jobin, S. Haskilll and R. B. Sartor. Ctr for GI Biology and Disease, 1 Lineberger Comprehensive Cancer Ctr, Univ. of North Carolina, Chapel Hill The IL-8 chemokine possesses pro-inflammatory properties that may contribute to induction and perpetuation of inflanimation in a variety of diseases. The IL-8 gene promoter possesses a binding site for several transcriptional factors including NF-kB. The activity of NF-kB is in part regulated by IkB, which is a cytoplasmic protein that binds to the Re A subunit of NF-kB and sequesters this protein in the cytoplasm, thereby inhibiting NF-kB-dependant gene transcription. Therefore IkB is considered an anti-inflammatory molecule. We now characterize the signaling pathway associated with IL-8gene expression in IL-113and TNF-(x-stimulated epithelial cell lines. Methods: HT-29 and Caco-2 cells were stimulated with either IL-113 or TNF-o~ (2ng/ml) and IL-8 expression was analyzed at different intervals at the protein level by ELISA and at the RNA level by quantitative RT-PCR. The steadystate level of cytoplasmic IkB protein was evaluated by Western Blot and nuclear NF-kB activity was determined by an Elactrophoretic Mobility Shift Assay (EMSA) technique. Results: In Caco-2 cells, only IL-113 induced IL-8 gene expressmn and protein synthesis. The Western blot of IkB shows degradation of this protein between 10-30 min after IL-113 stimulation with a reappearance at 60 min. Cycloheximide prevented reaccumulation of IkB, confirming that the disappearance of IkB was due to proteo ys s Concurrent with IkB disappearance, NF-kB was translocated to the nucleus in stimulated Caco-2 cells as documented by EMSA. IL-8 gene expression could be induced in HT-29 cells by both IL-113and TNF-oc. EMSA revealed that after 30 min of stimulation, NF-kB protein was translocated to the nucleus, although no IkB degradation was detected, Conclusion: These results show that NF-kB is a part of the signaling pathway involved in IL-8 gene induction. In Caco,2 cells, NF-kB activity is regulated by IkB, whereas in HT-29 cells, NF-kB activity appears to be controlled by an inhibitory molecule other than IkB. Finally Caco-2 cells are an appropriate system to study the effect of IkB overexpression on IL-8 gone transcription.
The etiology of inflammatory bowel disease (IBD) is unknown. There is considerable progress in recent years in understanding the immunological aspects of the disease. Immunosuppressive drugsare used increasingly in recent years and it is not clear if such therapies have altered the clinical outcome and reduced the need for surgical procedures. Purpose: The purpose of our study was to assess the clinical outcome and number of surgical procedures done among IBD patients treated with and without 6Mercaptopur ne (6-MP) Method IBD registry of Rochester commun ty wh c h was formed from hospitalization data of patients admitted in 8 hospitals in the city since 1973. As of 12/98, 2713 patients were registered: Crohns disease (CD) = 1679, ulcerative colitis (UC) = 1034. Review of hospital records of IBD patients since 1980 in 5 hospitals identified 120 patients who received 6-MP treatment. Data of clinical outcome of these patients and duration of 6-MP treatment and other medication profile were obtained from physicians followup data. 6-MP treated patients were found to be either steroid-nonresponders or those treated long-term with steroids, flying to be weaned off. Results: One hundred twenty patients were treated with 6-MP of whom 44 discontinued therapy within 6 months due to reversible adverse effects of the drug or following surgery shortly after initiation of 6-MP treatment and before the favorable effect of therapy was apparent. Of the remaining 76 patients (CD=70, UC=6)who all had 6-MP therapy > 6 months (mean 28+4 months, range 6-132 months), 19 patients required surgery while on 6,MP therapy. The remaining 57 of 76 patients (75%)were successfully treated without the need forsurgery. Thiswas significant when compared to a similar group of~ patients treated with steroids and/or 5, aminosalicylate drugs, without 6-MP (368 of 696 patients during the same study period) (p
AUA AU~IHAUI~
• BACTERIAL PRODUCTS AND, CYTOKINES UPREGULATE 11.-8 GENE EXPRESSION IN HUMAN COLONIC EPITHELIALCELL LINES. C. Jobin. H.H. Herfarth, R.B. Sartor. Center for GI Biology and Disease, Univ. of North Carolina, Chapel Hill The chemokine Interleukin-8 (IL-8) initiates proinflammatory responses: through its chemoattractant activity for neutrophils, lymphocytes and monocytes and its capacity to stimulate neutrophil activity. Epithelial cell lines secrete Ib8 protein after bacterial invasion or cytokine Stimulation, however, the impact of purified bacterial products on IL-8 gene : expression in epithelial cells is not yet completely understood. Therefore we analyzed the induction of IL-8 gene expression in HT-29 and Caco-2 cells after stimulation with cytokines or bacterial components which are in constant contact with colonic epithelial cells. Methods: HT-29 cells were cultured in DMEM and Caco-2 in EMEM supplemented with serum and additives. The cells were stimulated with either IL-113or TNF-¢ (2ng/ml) and by either LPS (endotoxin, tpg/ml) or peptidoglycan-polysaccharide polymers (PG-PS, 50pg/ml)' for 1-24 hrs. IL'8 gene expression was analyzed at the protein level by ELISA and at the RNA level by : quantitative RT -PCR: Results: IL-8 mRNA accumulation increased in HT-29 cells upon cytokine- (peak: between l h and 2h) or bacterial products stimulation (peak at 12h). This mRNA induction was followed by secretion of IL-8 protein. IL-8 induction was higher in cytokine-stimulated HT-29 cells compared to LPS or PG,PS stimulation (25 fold versus 7 fold, respectively). This induction of IL-8 did not require IL-1, since IL-1 mRNA remained undetectable by RT-PCR. In Caco-2 cells, IL-8 gone expression could be induced by IL-113 but not by TNF-a or bacterial products. Conclusions: These results show that cytokines and bacterial products present in the nflamed intestine can upregulate 11.,8 gone expression in epithelial eel! lines. IL-i[3 and TNF-c~ appear to be more potent stimuli for the IL-8 gene. The differential response to cytokine and bacterial product stimulation between enterocyte-like cells (Caco-2) and undifferentiated enterocytes (HT-29) may be representative of polarity of IL-8 responsiveness within the intestinal crypt. The high abundance of LPS or PG-PS in the intestinal lumen raises the possibility that these ubiquitous bacteria poymers are mportant p h y s o o g c and pathophysiologic stimulants of IL-8 in the gut epithelium.
• IN VITRO OMEPRAZOLE INtllBITS GROWTH OF GRAM r o s m VE AND GRAM NEGATIVE BACTERIA INCLUDING HELICOBACTER PYLORI D. Jonkers ~, E. Stobberingh~, and R. Stockbrfigger~ Depts. of Microbiology~ and Gastroanterology~, Academical Hospital Maastricht, The Netherlands. Omeprazolo, a substituted banzimidazole, is used for the Eeatment of peptic diseases and in combination with antibiotics for the eradication of Helicobacter ~oylori. Moreover, the inhibition of gastric acid secretion by omeprazole can result in bacterial overgrowth in stomach and duodenum. We wanted to study potential direct effects of omeprazole on bacteria commonly found in the stomach. Methods: The in vitro antibacterial effect of omeprazol¢ (100, 200, and 300/zg/ml) was assessed using bacterial growth curves. To study a possible influence of omeprazole' on the availability of cysteinc similar experiments were performed after supplementation of the medium With cysteine (0.01 and 0 . 1 % ) . In addition, possible interaction with amoxycilline and doxycycline (1-64 #g/ml) was studied using checkerboard titrations. Results: A dose-related and permanent ( > 24 hours) growth inhibition was seen on actively growing Gram positive cocci, i.e. Enterococcus faeealis and rStaohvlococcus aureu#. A smaller effect for 8 hours only, was found on Gram negative cocci, i.e. Escherichia coli and Klebsiella oneumoniae. After 24 hours incubation of H.Dvlori in the presence of omeprazole 200 /~g/ml bacterial count was reduced to zero compared to 10" cfu/ml in the control without omeprazole. Supplementation of the medium with cysteine reduced the inhibitory effect of omeprazole, suggesting that the inhibitory effect might partly be due to binding of omeprazole to cysteine present either in the bacteria or as a nutrient. No synergistic or antagonistic interaction of omeprazole with amoxycilline or doxycycline was observed for E.coli and Enterococcus faeealis.
Summery/conclusion: The indicated antibacterial properties of omeprazole possibly counteract gastrointestinal bacterial overgrowth, a known result of profound gastric acid inhibition, and may also be of additional advantagein the eradication of H.evlori.
GASTROENTEROLOGY, VoI. 108, NO. 4
• NF-kB ACTIVATION AND IL-8 EXPRESSION IN STIMULATED COLONIC EPITHELIAL CELL LINES. C. Jobin, S. Haskilll and R. B. Sartor. Ctr for GI Biology and Disease, 1 Lineberger Comprehensive Cancer Ctr, Univ. of North Carolina, Chapel Hill The IL-8 chemokine possesses pro-inflammatory properties that may contribute to induction and perpetuation of inflanimation in a variety of diseases. The IL-8 gene promoter possesses a binding site for several transcriptional factors including NF-kB. The activity of NF-kB is in part regulated by IkB, which is a cytoplasmic protein that binds to the Re A subunit of NF-kB and sequesters this protein in the cytoplasm, thereby inhibiting NF-kB-dependant gene transcription. Therefore IkB is considered an anti-inflammatory molecule. We now characterize the signaling pathway associated with IL-8gene expression in IL-113and TNF-(x-stimulated epithelial cell lines. Methods: HT-29 and Caco-2 cells were stimulated with either IL-113 or TNF-o~ (2ng/ml) and IL-8 expression was analyzed at different intervals at the protein level by ELISA and at the RNA level by quantitative RT-PCR. The steadystate level of cytoplasmic IkB protein was evaluated by Western Blot and nuclear NF-kB activity was determined by an Elactrophoretic Mobility Shift Assay (EMSA) technique. Results: In Caco-2 cells, only IL-113 induced IL-8 gene expressmn and protein synthesis. The Western blot of IkB shows degradation of this protein between 10-30 min after IL-113 stimulation with a reappearance at 60 min. Cycloheximide prevented reaccumulation of IkB, confirming that the disappearance of IkB was due to proteo ys s Concurrent with IkB disappearance, NF-kB was translocated to the nucleus in stimulated Caco-2 cells as documented by EMSA. IL-8 gene expression could be induced in HT-29 cells by both IL-113and TNF-oc. EMSA revealed that after 30 min of stimulation, NF-kB protein was translocated to the nucleus, although no IkB degradation was detected, Conclusion: These results show that NF-kB is a part of the signaling pathway involved in IL-8 gene induction. In Caco,2 cells, NF-kB activity is regulated by IkB, whereas in HT-29 cells, NF-kB activity appears to be controlled by an inhibitory molecule other than IkB. Finally Caco-2 cells are an appropriate system to study the effect of IkB overexpression on IL-8 gone transcription.
The etiology of inflammatory bowel disease (IBD) is unknown. There is considerable progress in recent years in understanding the immunological aspects of the disease. Immunosuppressive drugsare used increasingly in recent years and it is not clear if such therapies have altered the clinical outcome and reduced the need for surgical procedures. Purpose: The purpose of our study was to assess the clinical outcome and number of surgical procedures done among IBD patients treated with and without 6Mercaptopur ne (6-MP) Method IBD registry of Rochester commun ty wh c h was formed from hospitalization data of patients admitted in 8 hospitals in the city since 1973. As of 12/98, 2713 patients were registered: Crohns disease (CD) = 1679, ulcerative colitis (UC) = 1034. Review of hospital records of IBD patients since 1980 in 5 hospitals identified 120 patients who received 6-MP treatment. Data of clinical outcome of these patients and duration of 6-MP treatment and other medication profile were obtained from physicians followup data. 6-MP treated patients were found to be either steroid-nonresponders or those treated long-term with steroids, flying to be weaned off. Results: One hundred twenty patients were treated with 6-MP of whom 44 discontinued therapy within 6 months due to reversible adverse effects of the drug or following surgery shortly after initiation of 6-MP treatment and before the favorable effect of therapy was apparent. Of the remaining 76 patients (CD=70, UC=6)who all had 6-MP therapy > 6 months (mean 28+4 months, range 6-132 months), 19 patients required surgery while on 6,MP therapy. The remaining 57 of 76 patients (75%)were successfully treated without the need forsurgery. Thiswas significant when compared to a similar group of~ patients treated with steroids and/or 5, aminosalicylate drugs, without 6-MP (368 of 696 patients during the same study period) (p