Figure 1.
Schematic of the closed laminar flow chamber setup used to test attachment of Giardia under varying media conditions.
Giardia, test medium, and control medium were flowed in separately at controlled flow rates. The micrograph shows a 20X phase image of a representative population of Giardia attached to glass. Attachment levels are quantified by counting the attached cells in the population during media changes. Scale bar = 50 µm.
Figure 2.
Detachment of Giardia from glass.
(A) Detachment from glass during three-minute exposure to growth media of varying osmolality. Traces are normalized counts of attached cells averaged over 3−4 independent experiments for each osmolality. Red arrow marks the time where test media reached cells. Shifting the medium osmolality either progressively higher (from dark blue to light blue traces) or progressively lower (from dark green to light green traces) caused cells to detach in correspondingly greater numbers. (B) Cell attachment levels after three-minute exposure to growth media of varying osmolality for all osmolalities tested. Data fit a Gaussian profile (dashed line). For comparison, the red line indicates the full range of osmolality measured in the human duodenum of fed and fasted subjects and the red box indicates range of mean values [11]. (C) Mean normalized attachment levels in control experiments. Giardia did not show significant short-timescale detachment in the presence of Giardia growth medium, C2BBe-1 growth medium (DMEM), growth media with cytochalasin D (0.1 mM) or metronidazole (30 µg/mL), or in HEPES-based media with elevated pH (pH = 8.03) or low pH (pH = 4.94). Asterisks indicate significant difference from attachment levels in unmodified growth medium (p<0.000005 for both). Error bars indicate standard deviation.
Figure 3.
Detachment under changes in solution composition, osmolality, and tonicity.
(A) Cells initially incubated in growth media of a slightly higher (green) or lower (blue) osmolality adjusted to the shifted osmolality of the incubation medium. Subsequent exposure to test media of lower and higher osmolality produced detachment in response to the magnitude of the osmotic shift from the new osmolality set point, not the original osmolality. Arrows indicate shift between the observed detachment behavior (circles) and the detachment behavior that would be expected if cells did not adjust (crosses). (B) Cell attachment versus osmotic change in all media used. Dashed line is the fit for attachment results in growth media of varying osmolality (Figure 2B). Similar osmolality-dependent results were found for exposure to DMEM growth medium (squares) and for brief exposure to pure NaCl (triangles) or sucrose (diamonds) solutions. (C) Isotonicity experiments. Bars represent the mean normalized cell attachment at the end of the three-minute experiment. In the presence of growth medium alone, cells did not detach. In medium diluted by half with water, 29% of cells detached (p = 0.00001), while all cells exposed to pure water detached (p<0.000005). This effect can be prevented by restoring the diluted medium to its original tonicity with sucrose, or by substituting a pure, isotonic sucrose or NaCl solution. The effect is not prevented by restoring medium osmolality with cell-permeant mono(ethylene glycol) (MEG), which is iso-osmotic but not isotonic (p = 0.0095). Error bars indicate standard deviation.
Figure 4.
C2BBe-1 monolayers as a model for studying Giardia attachment to the intestinal epithelium.
(A) Fluorescent image of Giardia (red) attached to a C2BBe-1 cell monolayer, overlaid on a 20X phase image. Scale bar = 50 µm. (B) Scanning electron micrograph of brush border-expressing C2BBe-1 monolayer cells grown on glass showing dense microvilli. Scale bar = 1 µm. (C) Scanning electron micrograph of Giardia attached to a C2BBe-1 cell monolayer. Scale bar = 2 µm.
Figure 5.
Detachment of Giardia from intestinal epithelial monolayers.
(A) Giardia detachment from C2BBe-1 monolayers in hypo- and hyperosmotic growth media (purple triangles) and a Gaussian fit (thin dotted line). Data from experiments on glass (Fig. 2B) are included for reference (black circles, thick dotted line). (B) Isotonicity experiments on monolayers. Results are similar to those for Giardia attached to glass (Fig. 3C), with MEG-supplemented medium failing to prevent detachment (p<0.000005), as did simple diluted medium (p = 0.0007). Error bars indicate standard deviation.
Figure 6.
Enhanced detachment with controlled tonicity swings.
Giardia attached to C2BBe-1 monolayers were exposed to hypertonic and hypotonic growth media (blue and light green panels, respectively) in a timed pattern selected to exploit cells' adjustment to tonicity changes. Nearly all cells exposed to swings of ±150 mOsm/kg followed by adjustment periods detached over the course of the experiments (light gray trace), and over half (red dashed line) of cells exposed to swings of ±100 mOsm/kg detached (gray trace). Cells cultured for 72 hours instead of 48 hours prior to experiments appeared to detach more readily (dotted line). Traces are the averaged attachment index for multiple experiments (n = 2 for ±100 mOsm/kg, n = 5 for 48-hour ±150 mOsm/kg, n = 3 for 72-hour ±150 mOsm/kg).