Limits...
In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus

Paracellular permeability in Caco2 cell monolayers after infection with wild-type S. Typhi and its mutant strains (MOI 40:1). (A) FITC-BSA (40 kDa) net transport after infection with wild-type S. Typhi and attenuated strains. (B) FITC-Dextran (4 kDa) net transport after infection with wild-type S. Typhi or the attenuated mutants. Calcium-free medium supplemented with EGTA to disrupt TJs served as positive control. Results are expressed as mean ± SEM of triplicate samples for each condition and are representative of three experiments with similar results. p = ns compared to uninfected controls (ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569575&req=5

Figure 5: Paracellular permeability in Caco2 cell monolayers after infection with wild-type S. Typhi and its mutant strains (MOI 40:1). (A) FITC-BSA (40 kDa) net transport after infection with wild-type S. Typhi and attenuated strains. (B) FITC-Dextran (4 kDa) net transport after infection with wild-type S. Typhi or the attenuated mutants. Calcium-free medium supplemented with EGTA to disrupt TJs served as positive control. Results are expressed as mean ± SEM of triplicate samples for each condition and are representative of three experiments with similar results. p = ns compared to uninfected controls (ANOVA).

Mentions: We next sought to determine whether alterations induced by S. Typhi with regard to TEER correlated with changes in epithelial paracellular permeability. We evaluated alterations in barrier function in response to the pathogen, by measuring the trans-epithelial flux of fluorescently labeled Dextran (FITC-dextran, 4 kDa) and BSA (FITC-BSA, 40 kDa). As shown in Figure 4, Caco2 cells exposed to wild-type S. Typhi showed a significantly increased transport of both FITC-BSA (Figure 4A) and FITC-Dextran (Figures 4B,C) from the apical chamber to the basolateral side compared to uninfected monolayers. None of the mutant strains caused increased paracellular permeability to these markers. (Figures 4A–C). At a MOI 40:1, wild-type S. Typhi failed to increase the paracellular flux of labeled markers (Figure 5). EGTA was used as a maximum permeability positive control for its ability to completely open tight junctions. As expected, EGTA-treated monolayers showed a remarkably higher degree of transport of both tracers confirming the validity of the assays. Although we can’t exclude that to some extent the paracellular flux of labeled markers might be due to the minimal cell death we observed when infecting monolayers with wild-type S. Typhi, these results likely represent a S. Typhi-mediated increase in epithelial permeability resulting from modulation of intercellular tight junctions function. In light of this, the mutant vaccine strains appear to have either an attenuated or no effect on the tight junction barrier.


In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

Paracellular permeability in Caco2 cell monolayers after infection with wild-type S. Typhi and its mutant strains (MOI 40:1). (A) FITC-BSA (40 kDa) net transport after infection with wild-type S. Typhi and attenuated strains. (B) FITC-Dextran (4 kDa) net transport after infection with wild-type S. Typhi or the attenuated mutants. Calcium-free medium supplemented with EGTA to disrupt TJs served as positive control. Results are expressed as mean ± SEM of triplicate samples for each condition and are representative of three experiments with similar results. p = ns compared to uninfected controls (ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569575&req=5

Figure 5: Paracellular permeability in Caco2 cell monolayers after infection with wild-type S. Typhi and its mutant strains (MOI 40:1). (A) FITC-BSA (40 kDa) net transport after infection with wild-type S. Typhi and attenuated strains. (B) FITC-Dextran (4 kDa) net transport after infection with wild-type S. Typhi or the attenuated mutants. Calcium-free medium supplemented with EGTA to disrupt TJs served as positive control. Results are expressed as mean ± SEM of triplicate samples for each condition and are representative of three experiments with similar results. p = ns compared to uninfected controls (ANOVA).
Mentions: We next sought to determine whether alterations induced by S. Typhi with regard to TEER correlated with changes in epithelial paracellular permeability. We evaluated alterations in barrier function in response to the pathogen, by measuring the trans-epithelial flux of fluorescently labeled Dextran (FITC-dextran, 4 kDa) and BSA (FITC-BSA, 40 kDa). As shown in Figure 4, Caco2 cells exposed to wild-type S. Typhi showed a significantly increased transport of both FITC-BSA (Figure 4A) and FITC-Dextran (Figures 4B,C) from the apical chamber to the basolateral side compared to uninfected monolayers. None of the mutant strains caused increased paracellular permeability to these markers. (Figures 4A–C). At a MOI 40:1, wild-type S. Typhi failed to increase the paracellular flux of labeled markers (Figure 5). EGTA was used as a maximum permeability positive control for its ability to completely open tight junctions. As expected, EGTA-treated monolayers showed a remarkably higher degree of transport of both tracers confirming the validity of the assays. Although we can’t exclude that to some extent the paracellular flux of labeled markers might be due to the minimal cell death we observed when infecting monolayers with wild-type S. Typhi, these results likely represent a S. Typhi-mediated increase in epithelial permeability resulting from modulation of intercellular tight junctions function. In light of this, the mutant vaccine strains appear to have either an attenuated or no effect on the tight junction barrier.

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus