Figure 1.
HCT-8 cells remain polarized after exposure to ricin for at least 16 hours.
A. Transepithelial cell resistance (TEER) as a marker for cell polarization in transwell-cultured HCT-8 monolayers. TEER of 2000 ohms/cm2 is considered indicative of polarization [11]. Resistance is shown at 0, 4, 8, 12, 16, 20, and 24 hours after administration of 3 µg/ml ricin (□), HRP (◊), or HRP + Ricin (Δ). B. Samples of media were collected from the basolateral chambers of the transwells at 4, 8, 12, 16, 20, and 24 hours and tested for cytotoxicity to Vero cells (absorbance is inversely proportional to cell death). Each point represents the average value from three wells, and the data shown are representative of three individual experiments. Statistical analysis was performed using two-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001.
Figure 2.
Labeled ricin bound to polarized HCT-8 cells on transwell membranes.
Polarized HCT-8 cell monolayers grown on transwell membranes were exposed to medium (A) or AlexaFluor® 488-labeled ricin (B, C, D). At 4 (A, B), 8 (C), and 16 (D) hours post-intoxication, transwell membranes were excised and cells were visualized with a confocal microscope. Three transwell membranes were removed at each time point. Magnification: 400X.
Figure 3.
Labeled ricin transits across HCT-8 cell organoids over time.
Organoids intoxicated with Alexa Fluor® 700-labeled ricin for 1 (A, B), 6 (C, D), and 24 (E, F) hours were removed from the vessels and processed. H&E staining was used to visualize the structure of the organoids (A, C, E; pink represents scaffold and purple represents HCT-8 cells). Confocal fluorescence microscopy was used to identify the labeled toxin (B, D, F); green indicates the presence of ricin and blue indicates DAPI-stained nuclei. White arrows show labeled ricin in the sections. For each time shown, tissues subjected to H&E or confocal microscopy are from the same organoid but are not serial sections. Magnification: 100x (A, C, E) and 100x (B, D, F).
Figure 4.
Visualization of labeled ricin in live mice.
Fluorescently-labeled ricin was administered intragastrically and mice were viewed after 3 (A), 16 (B), 24 (C), 48 (D), 72 (E), or 96 (F) hours post exposure. Ricin labeled with Alexa Fluor® 700 (A, C, D, E, F) was imaged at 690 nm and ricin labeled with Alexa Fluor® 633 (B) was imaged at 620 nm. X-rays were taken of the four sides of each mouse at each time to facilitate localization of the fluorescent signal. Each mouse is representative of three mice per time point, and the experiment was repeated three times.
Figure 5.
Labeled ricin disseminates to distal organs.
Organs were excised from mice at 16 hours (A, G), 24 hours (B, H), 48 hours (C), 72 hours (D) or 96 hours (E, F) following orogastric intoxication. Alexa Fluor® 633-labeled ricin (A), Alexa Fluor® 700-labeled ricin (B, C, D, E), PBS (F, G), or free Alexa Fluor® 700 label (H) was detected by fluorescence imaging as shown in; 1. stomach, 2. small intestine, 3. cecum, 4. large intestine, 5. kidneys, 6. liver, 7. heart, 8. spleen, 9. lung. *Mice that received PBS only were imaged at the same wavelength (690 nm) as mice that received AlexaFluor® 700-labeled ricin. ** Mice that received PBS only were imaged at the same wavelength (620 nm) as mice that received AlexaFluor® 633-labeled ricin. All PBS-treated mice had similar imaging profiles at all time points.
Figure 6.
Ricin binds to villi in the small intestine.
IF of small intestines from mice 24 hours after gavage with ricin (A) or PBS (B). Ricin was detected by IF after treatment with primary rabbit polyclonal anti-RTA and goat anti-rabbit Ig-AlexaFluor® 488 secondary antibody. Note that the small intestines from 3/3 mice euthanized at 24 hours post gavage were positive for ricin. Magnification: 400x. Normal human small intestinal tissue [A, (400x), B, (400x)] was overlaid with ricin (C), PBS (D), or ricin pre-incubated with lactose (E). White arrows indicate goblet cells and yellow arrows indicate the brush boarder. Ricin was detected in human tissues as described above. Yellow asterisks indicate ricin within epithelial cells and the lamina propria.
Figure 7.
Ricin binds to tubules of kidneys in ricin-intoxicated mice.
IF was used to detect ricin in the kidneys from a ricin-intoxicated mouse (A) and a PBS control mouse (B). Ricin was detected with a primary rabbit polyclonal anti-RTA and goat anti-rabbit Ig-AlexaFluor® 488 as the secondary antibody. Consistent with panel A, kidneys from 3/3 mice euthanized at 24 hours post gavage were positive for ricin. Magnification: 630x (A, B).
Figure 8.
Ricin causes mild damage to the lamina propria in the small intestine.
Small intestinal sections from mice orogastrically exposed to 10 mg/kg ricin (A, B, C) or PBS (D) were stained with H&E and assessed for damage at 16 (A), 24 (B, D), and 96 (C) hours. Magnification: 100X.
Figure 9.
Oral ricin intoxication causes vacuolated tubules in the kidneys of some mice.
Excised kidneys from mice orally exposed to ricin (A) or PBS (B) for 24 hours were sectioned and stained with H&E. Black arrows indicate vacuolated tubules. Vacuolation was noted in kidneys of 2/5 mice euthanized at 24 hours post exposure and in one additional mouse that died shortly before the designated time for euthanasia (48 hours). Magnification: 400X.