approximately 10 6 caco - 2 cells were seeded per 1 cm 2 transwell filter . cells were incubated for 16 h at 37 ° c . in absence of antibiotic or fcs with different doses of bacteria , as indicated in the figure legend . fresh overnight cultures of lpr , bl818 and e . coli tg - 1 bacteria were used . cells were then washed prior to enumeration . bound bacteria were counted by plating on mrs or lb plates . for each experiment , triplicate tests were performed . data were expressed as means of bound bacteria per 100 caco - 2 cells ± sem . triplicates were performed for each experiment . in a subsequent experiment , cells were incubated with 2 × 10 7 bacteria for 16 hours at 37 ° c ., in the presence of increasing doses of either siga or sc as indicated in the legend to fig3 . cells were then washed prior to enumeration . bound bacteria were counted by plating on mrs or lb plates . for each experiment , triplicate tests were performed . data were expressed as means of bound bacteria per 100 caco - 2 cells ± sem . triplicates were performed for each experiment . a preferential binding to polarized caco - 2 cells of lpr or bl818 is observed in comparison to e . coli tg - 1 ( fig2 ). there is a dose - dependent binding capacity of probiotics to intestinal epithelial cells . it can be observed that binding properties could be differentiated between the two strains . for subsequent experiments , 2 × 10 7 cfu of probiotics were used , as this amount did not lead to any ph change in the medium on one hand , and showed an efficient binding ratio on the other hand . increasing the dose of monoclonal siga potentiated the capacity of both lpr and bl818 to bind to polarized caco - 2 cell monolayers . when associated with the 20 bacteria , secretory component did not exhibit such properties ( fig3 ). the 1 μg dose of siga that confers a significant improvement in probiotic binding capacity was selected for subsequent experiments . this dose leads to the formation of a final complex constituted of approximately 50 , 000 to 100 , 000 units of siga for 1 bacterium . approximately 10 6 caco - 2 cells were seeded per 1 cm 2 transwell filter . cells were incubated for 24 h at 37 ° c . with 2 × 10 7 cfu of bacteria in absence of antibiotic or fcs . bacteria were tested either alone or in combination with siga or sc at concentrations indicated in the legend to fig4 . transepithelial electrical resistance ( ter ) was measured at 3 , 6 , 9 , 15 and 24 h . controls include incubation with siga and sc alone . triplicates were performed for each experiment . a 20 - 25 % increase in transepithelial electrical resistance ( ter ) resulted from the incubation of polarized caco - 2 cell monolayer with lpr or bl818 alone , suggesting that probiotics potentiated epithelial barrier function . this remained true when the bacteria were combined with siga or sc ( fig4 ). siga or sc by themselves did not lead to any ter change . approximately 10 6 caco - 2 cells were seeded per 1 cm 2 transwell filter . cells were incubated for 16 h at 37 ° c . with 2 × 10 7 cfu of lpr in absence of antibiotic or fcs . bacteria were tested either alone or in combination with siga or sc at concentrations indicated in the legend to fig5 . s . flexneri , s . typhi and h . pylori ( 2 × 10 7 cfu ) were used as pro - inflammatory pathogens in control experiments . nuclear extracts were prepared and analysed by electrophoretic mobility shift assay ( emsa ) to examine the binding of nf - κb to a specific dna probe . cytoplasmic extracts were obtained in parallel and the presence of iκbα was analyzed by western blot using an anti - iκbα - specific monoclonal antibody to the protein . exposure of polarized caco - 2 cell monolayers to pathogenic bacteria led to a much more pronounced activation of nuclear nf - κb compared to non - pathogenic bacteria ( fig5 ). disappearance of iκbα ( lower panel ) reflects activation of the pathway leading to nuclear translocation of nf - κb . in that respect , while lpr alone has a mild effect on nf - κ b activation , combination of lpr with siga or sc reduced nf - κb activation in caco - 2 cells ( bl818 not tested ). as expected , incubation of epithelial cells with pathogenic s . flexneri led to a complete disappearance of iκbα expression . approximately 10 6 caco - 2 cells were seeded per 1 cm 2 transwell filter . cells were incubated for 16 h at 37 ° c . with 2 × 10 7 cfu of lpr in absence of antibiotic or fcs . lpr was tested alone or in combination with with either 0 . 2 μg of sc , 1 μg of polyclonal siga or 1 g of specific anti - s . flexneri lps sigac5 . after incubation with lpr cells were washed and then incubated with 10 7 s . flexneri for 6 hours , washed again and incubated with 50 g / ml gentamycin for 45 min . finally , cells were lysed and intracellular s . flexneri were enumerated on lb agar plates . triplicates were performed for each experiment . addition of lpr reduced infection of polarized caco - 2 cell monolayer by s . flexneri in a dose dependent manner . the effect was highly enhanced upon combination with siga . full prevention of infection was achieved when s . flexneri lps - specific sigac5 antibody was used ( fig6 ). approximately 10 6 caco - 2 cells were seeded per 1 cm 2 transwell filter . cells were incubated for 16 h at 37 ° c . with 2 × 10 7 cfu of lpr or bl818 in absence of antibiotic or fcs . probiotics were tested alone or in combination with with either 0 . 2 μg of sc or 1 μg of polyclonal siga . control s . flexneri was tested alone or in combination with 1 μg of specific anti - s . flexneri lps sigac5 . after washing , caco - 2 cells were directly recovered from the transwell filters and lysed . nuclei were removed and cell debris as well as cytoplasms were analyzed by western blot using anti - pigr antibody and antisera to human sc and β - actin as controls . triplicates were performed for each experiment . in a subsequent experiment cells were incubated following the same procedure and then recovered from the transwell filter after 8 , 16 and 24 h of incubation . quantitative analysis of pigr was performed by elisa on cell debris / cytoplasms fractions . total proteins were determined by the bca protein assay . values were normalized to protein content and data expressed as means of ng pigr / mg of total protein ± sem . pigr expression in epithelial cells was normalized to β - actin expression . as revealed by western blot ( upper panel ) and densitometric analysis of the respective signals ( lower panel ), there was an increase of pigr level following overnight exposure of polarized caco - 2 monolayers to combinations of lpr or bl818 with either siga or sc compared to probiotics alone ( fig7 a ). specific anti - s . flexneri lps sigac5 prevented interaction of the pathogen with the caco - 2 cell polarized monolayer , thus explaining the decrease in pigr expression when compared to s . flexneri treatment alone . the results further showed a time - dependent increase of polymeric ig receptor ( pigr ) level following exposure of polarized caco - 2 cell monolayers to probiotic combinations with siga or sc ( fig7 b ).
ways to increase the effectiveness of probiotics is disclosed . one embodiment relates to a combination of probiotics with secretory iga and possible uses of this combination . for example , a method of using a composition comprising secretory iga and at least one probiotic to prepare a product to treat or prevent infection is disclosed .