Human derived cell line
Caco-2 cells (ATCC Number: HTB-37) were used because they have the ability to differentiate into enterocytes of the upper villus forming monolayers. Cells were grown in high glucose (4.5 g/l) DMEM (Gibco, Life Technologies, UK) with 10% foetal calf serum (FBS) (Gibco, Life Technologies, UK), 1% non-essential amino acids, 50 mU/ml penicillin, and 50 mg/ml streptomycin. The Caco-2 cells were grown from 15 to 18 days after confluence on polycarbonate Transwell filters (pore size, 0.4 µm) (Costar Italia, Milan, Italy). MA104 cells (ATCC Number: CRL-2378) were used for viral titers and were grown in Medium 199 (Lonza, Belgium) with 5% FBS, 50 mU/ml penicillin, 50 mg/ml streptomycin, and 0.25 μg/ml amphotericin B.
Adsorption assays
For adsorption assays, 100 mg/ml DS was incubated with the medium alone or in the presence of RV (MOI 25) for 1 h at 37 °C. Then, the viral suspensions were probed with fluorescein isothiocyanate (FITC) conjugated anti-RV antibody (Abcam, ab31435) and examined using a Nikon Eclipse 80i epifluorescence microscope (FITC filter). The images were analysed using the NIS Elements D imaging software. As a negative control, a mixture of titanium dioxide, maltodextrin and glucose monohydrate was used (TMG).
The same assay protocol was used to evaluate the absorptive effect of DS on NSP4. Briefly, 100 mg/ml DS was incubated with medium alone or in the presence of NSP4 at different doses (50, 100 and 200 ng/ml) for 1 h at 37 °C. The slides were probed with an anti-NSP4 antibody and then with a fluorescein isothiocyanate (FITC) conjugated anti-rabbit antibody. Then, the slides were examined using a Nikon Eclipse 80i epifluorescence microscope (FITC filter). The images were analysed using the NIS Elements D imaging software.
RV positive cell staining
The Caco-2 cells were probed with FITC conjugated anti-RV antibody (Abcam, ab31435). Slides were mounted with Vectashield Mounting Medium with DAPI (Vector laboratories, Ltd, UK). The monolayers were examined using a Nikon Eclipse 80i epifluorescence microscope (FITC filter). The images were analysed using the NIS Elements D imaging software.
Viral load assay
A fluorescence focus assay in the MA104 cell monolayers was used to determine the viral titre, expressed as focus-forming units per millilitre of virus (FFU/ml). Briefly, MA104 cells were grown in 8-chamber slides (Lab-Tek chamber Slide, Nunc Inc, USA) and then infected with supernatants from the Caco-2 infection experiments. After viral absorption, the cells were fixed in methanol and probed with fluorescein isothiocyanate (FITC) conjugated anti-RV antibody (Abcam, ab31435). Fluorescence foci were counted individually on a Nikon Eclipse 80i epifluorescence microscope (FITC filter). The viral titre was calculated from the average number of foci per well adjusted for well volume and expressed as FFU/ml. As a negative control, a mixture of titanium dioxide, maltodextrin and glucose monohydrate was used (TMG).
Virus strain and Caco-2 cell infection protocol
The simian rotavirus strain SA11 (RV) (ATCC Number: VR-1565) was activated with 20 µg/ml trypsin for 1 h at 37 °C. The viral suspension was added to the apical side of the Caco-2 cell monolayer at a multiplicity of infection (MOI) of 25 to maximize the effect. After 1 h of incubation at 37 °C, the cells were rinsed three times and incubated in foetal calf serum–free medium for fixed times after infection. The time after infection was counted after the removal of excess viral particles. To test the effect of DS, RV was incubated with 100 mg/ml of DS for 1 h at 37 °C followed by centrifugation. The cells were then suspended and infected as previously described.
Ion transport studies
Infection with RV was performed with multiplicities of infection (MOI) of 25 for 2 h; next, the cell monolayers were mounted in Ussing chambers (Physiological Instruments, San Diego, CA). The following electrical parameters were measured at different time points after infection: transepithelial potential difference (PD), short-circuit current (Isc), and tissue ionic conductance (G). Isc is expressed as microamperes per square centimetre (μA/cm2), G is expressed as millisiemens per square centimetre (mS/cm2), and PD is expressed as millivolts (mV). An increase in PD indicates chloride secretion and provides a precise evaluation of enterotoxic effects, whereas an increase in G quantitatively correlates with epithelial damage.
Transepithelial electrical resistance measurements
The transepithelial electrical resistance (TEER) of cell monolayers grown on filters was measured using a Millicell-ERS resistance monitoring apparatus (Millipore). The net TEER (in Ohms/cm2) was calculated by subtracting the background from the actual value and multiplying the value obtained by the area of the filter (4.9 cm2).
Western blot analysis
After RV infection, cells were scraped into PBS buffer and lysed in HEPES-KCl buffer (KCl, 60 mM; β-mercaptoethanol, 14 mM; EDTA, 2 mM; HEPES pH 7.9, 15 mM; sucrose, 0.3 M; aprotinin, 5 μg/ml; leupeptin, 10 μg/ml; pepstatin, 2 μg/ml; phenylmethylsulfonyl fluoride, 0.1 mM) containing 1% Tergitol (Nonidet P-40). Total extracts were centrifuged at 1500g for 20 min at 4 °C. The protein content was determined by the Bradford method (Bio-Rad Laboratories, Munich, Germany). The supernatant containing the solubilized proteins was boiled for 5 min in Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue). Cell protein (50 μg/lane) was added to SDS-PAGE and transferred to a nitrocellulose membrane (BioBlot-NC-Costar; Corning Incorporated, Canada). Blots were probed for 1 h with specific NSP4 antibody. Bound antibody was detected using an anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody and developed by chemiluminescence reaction (Amersham Pharmacia Biotech, U.K.). All incubations and washes were carried out at room temperature with gentle shaking.
Reactive oxygen species (ROS) production
Reactive oxygen species production was measured using DCFH-DA spectrofluorometry. After stimulation, DCFH-DA (20 µM) was added for 30 min at 37 °C in the dark. Intracellular ROS production was measured in a fluorometer (SFM 25; Kontron Instruments, Japan). For DCF fluorescence imaging, Caco-2 cells were grown on the cover glass for 3 days, fixed and permeabilized with paraformaldehyde 4% and Triton 0.2% for 30 min at 4 °C. Cells were then incubated with DCF-HA 20 µM for 30 min at 37 °C in the dark. Fluorescence images from multiple fields were obtained using a Nikon Eclipse e 80i microscopy. The images were analysed using the NiS Elements D imaging software (Nikon Instruments, Inc., NY, USA).
Glutathione assay
The intracellular GSH/GSSG ratio was determined by a fluorimetric assay kit (Biovision, Milpitas, CA). The GSH content was normalized for protein content and expressed as % of control.
Statistical analysis
We used the GraphPad Prism Software (San Diego, CA) to evaluate the two-tailed unpaired student t test and a 2-tailed paired student t test to evaluate statistical significance. An alpha value of 0.05 was set for statistical significance; p values for each analysis are indicated in the figure legends. All experiments were repeated at least three times, and error bars indicate the standard deviation.