1. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: Role of iNOS and COX-2 
Silvia Resta–Lenert, Kim E. Barrett 
Gastroenterology 
Volume 122, Issue 4, Pages (April 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Effect of enteroinvasive bacteria on epithelial barrier function. Intestinal epithelial cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴), and TER was measured at the times indicated. Resistance values of cell-free inserts were subtracted from each measurement. (A and D) Time course of changes in TER determination in infected and in control monolayers. (B, C, E, F) Same as part A, except that cells were studied in the presence of L-NIL or NS398, respectively. The inhibitors were added 3 hours before TER measurement. Data in all panels are means ± SEM for 6 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (**P < 0.01; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Effect of conditioned medium from HT29/cl.19A on T84 cell TER. Uninfected T84 cells were exposed for 1 hour at 37°C to conditioned medium from controls (○), EIEC (●), SD (■), or ST/LA (▴)-infected HT29/cl.19A monolayers obtained after the times indicated in the presence or absence of inhibitors, and TER was measured. Resistance values of cell-free inserts were subtracted from each measurement. Data are means ± SEM for 4 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to cells treated with medium conditioned by uninfected control HT29/cl.19A cells are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Effect of enteroinvasive bacteria on baseline chloride secretion. Cell monolayers were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). At various times after infection, baseline chloride secretion was measured as Isc (A, D). The effect of pretreatment with L-NIL or NS398 is also shown for both cell lines tested (B, E, C, F). Data are means ± SEM for 6 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Effect of enteroinvasive bacteria on stimulated chloride secretory responses. Cell monolayers were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). At various times after infection, maximal chloride secretory responses to agonists were measured as changes in Isc (ΔIsc). Chloride secretory responses were induced in infected and control monolayers by (A, D) bilateral addition of Bt2-cAMP (3 mmol/L); (B, E) bilateral addition of 8Br-cGMP (300 μmol/L); or (C, F) serosal addition of galanin (100 nmol/L). Data are expressed as means ± SEM, n = 6. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls as assessed by ANOVA are indicated by asterisks (***P < 0.001).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Effect of enteroinvasive bacteria on stimulated chloride secretory responses in the presence of iNOS or COX-2 inhibitors. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). (A) Cell monolayers were treated for 3 hours before electrophysiological studies with iNOS inhibitors, L-NIL (50 μmol/L). Chloride secretory responses induced by Bt2-cAMP (3 mmol/L) were then measured at various time points in uninfected and infected cells. (B) Same as A, except that cells were pretreated with COX-2 inhibitor NS-398 (10 μmol/L). Data in both panels are means ± SEM for 6 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Effect of iNOS and COX-2 inhibitors on chloride secretory responses induced by galanin. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). Cell monolayers were treated for 3 hours before electrophysiological studies with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L), or the COX-2 inhibitor, NS-398 (10 μmol/L). Data are means ± SEM for 6 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Effect of iNOS and COX inhibitors on chloride secretory responses in T84 cells exposed to conditioned medium (CM). Uninfected T84 cell monolayers were exposed in Ussing chambers to filter-sterilized conditioned medium harvested in the (A) absence or (B, C) presence of inhibitors from infected and control HT29/cl.19A cells. Elicited chloride secretion was measured as changes in Isc. Symbols correspond to the treatments applied to HT29/cl.19A cells before harvest of conditioned medium: medium alone (○), EIEC (●), SD (■), or ST/LA (▴). Significant differences relative to results obtained using medium conditioned by uninfected control HT29/cl.19A cells are indicated by asterisks (*P < 0.05; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Enteroinvasive bacteria increase NO levels in HT29/cl.19A cells. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). Total nitrite production was assessed in the (A) absence or (B) presence of iNOS and COX-2 inhibitors. In B, each group of bars represents, from left to right, cells exposed to EIEC, SD, medium, or ST/LA for 12 hours, respectively. Data in all panels are means ± SEM for 4 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Effect of bacteria on NO production by T84 cell monolayers. T84 cells were exposed to (A) medium, EIEC, SD, or ST/LA or to (B) medium conditioned by these bacteria and harvested at various time points. Total nitrite production was assessed as indicated in the Materials and Methods section. No significant differences relative to uninfected controls were observed.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

11. Fig. 10
Enteroinvasive bacteria increase production of PGE2 by HT29/cl.19A cells. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). PGE2 production was assessed in the (A) absence or (B) presence of NOS and COX inhibitors. Monolayers were pretreated with L-NAME (100 μmol/L) or NS-398 (10 μmol/L) for 3 hours before the PG assay. In B, each group of bars represents, from left to right, cells exposed to EIEC, SD, medium, or ST/LA for 18 hours, respectively. Data in all panels are means ± SEM for 3 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (*P < 0.05; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

12. Fig. 11
Enteroinvasive bacteria increase cAMP and cGMP levels in HT29/cl.19A cells. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴), and (A) cGMP and (B) cAMP levels were measured. Where present, the NOS inhibitor, L-NAME (100 μmol/L), or the COX-2 inhibitor, NS-398 (10 μmol/L), were added 3 hours before the start of the experiment. Data in all panels are means ± SEM for 3 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

13. Fig. 12
Effect of guanylyl cyclase blockade on ion transport and barrier functions after infection with enteroinvasive bacteria. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). Cells were treated with ODQ (50 μmol/L) at the time of infection and for 3 hours before harvest. Baseline chloride secretion was measured as Isc (A), whereas barrier function integrity was assessed by measuring TER (B) at various time points. Data in both panels are means ± SEM for 3 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

14. Fig. 13
Effect of a peroxynitrite decomposition catalyst on altered chloride secretion and barrier function after enteroinvasive infection. HT29/cl.19A cells were exposed to medium (○), EIEC (●), SD (■), or ST/LA (▴). FeTPPS (50 μmol/L) was added to the cell cultures at the time of infection and 3 hours before harvest. TER and chloride secretion were assessed as described before at various times after infection. Data in both panels are means ± SEM for 3 experiments conducted in triplicate. Where error bars are not shown, they are contained within the symbol. Significant differences relative to uninfected controls are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

15. Fig. 14
Enteroinvasive bacteria induce the expression of iNOS in HT29/cl.19A cells. HT29/cl.19A cells were exposed to medium, EIEC or SD, and ST/LA for various times. Whole cell lysates were tested for iNOS (120 kd) by immunoblot after 3 hours pretreatment with medium or the NOS inhibitor, L-NAME (100 μmol/L), or the COX-2 inhibitor, NS-398 (10 μmol/L). Data in the lower panels are means ± SEM for densitometric analysis of 3–4 experiments with values expressed as arbitrary units. Each group of bars represent a time point for, from left to right, cells exposed to medium, ST/LA, EIEC, or SD, respectively. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

16. Fig. 15
Enteroinvasive bacteria induce the expression of COX-2 in HT29/cl.19A cells. HT29/cl.19A cells were exposed to medium, EIEC, SD, or ST/LA. Whole cell lysates were tested for COX-2 (70 kd) by immunoblot after 3 hours pretreatment with medium or the NOS inhibitor, L-NAME (100 μmol/L), or the COX-2 inhibitor, NS-398 (10 μmol/L). Data in the right-hand panels are means ± SEM for densitometric analysis of 3–4 experiments expressed as arbitrary units. Each group of bars represents a time point for, from left to right, cells exposed to medium, ST/LA, EIEC, or SD, respectively. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

17. Fig. 16
Enteroinvasive bacteria infection fails to induce iNOS and COX-2 in T84 cell monolayers. T84, HT29/cl.19A, and Caco-2 cells were exposed to medium or EIEC for up to 24 hours. Whole cell lysates were harvested at 24 hours and tested for the presence of (A) COX-2 (70 kd) and (B) iNOS (120 kd) by immunoprecipitation and Western blot. One of 3 representative blots is shown at the top of each panel in the Figure. Data presented in the bottom portion of each panel are means ± SEM for densitometric analysis of 3–4 experiments expressed as arbitrary units. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

18. Fig. 17
Enteroinvasive bacteria up-regulate the expression of CFTR and NKCC1. HT29/cl.19A cells were exposed to medium, EIEC, SD, or ST/LA. Membrane fractions from cell lysates were tested by immunoblot after 3 hours pretreatment with medium or the NOS inhibitor, L-NAME (100 μmol/L). (A) Immunoblots for CFTR. (B) Immunoblots for NKCC1. Data in the lower parts of both A and B are means ± SEM for densitometric analysis of 3–4 experiments expressed as arbitrary units. Each group of bars represent a time point for, from left to right, cells exposed to medium, ST/LA, EIEC, or SD, respectively. Significant differences relative to uninfected controls are indicated by asterisks (***P < by ANOVA).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions