Stains

Example 1

The ability of eyedrops to deliver Penl-XBIR3 in mice and rats was tested. Results are presented in FIG. 2 and FIG. 3.

In mice, Penl-XBIR3 (10 μg) eyedrops were applied, then the animals were sacrificed at the indicated times. In rabbits, 200 μg Penl-XBIR3 eyedrops or a saline vehicle were administered BID for 4.5 days. The final dose given 5 h prior to harvest of retinas. Plasma from rabbits obtained at baseline and harvest.

Retinal lysates were immunoprecipitated with XIAP, followed by western blotting for anti-His. XBIR3 contains a His tag, so uptake of XBIR3 is detectable using anti-His. Blots for the mouse and rabbit samples, along with graphs quantifying the results, are presented in FIG. 2. XBIT3 uptake was observed in both mouse and rabbit samples. Uptake in the mouse samples was detected by 1 h and maintained through 24 h. In rabbit there was significant XBIR3 in retina at 5d.

Baseline and post-treatment plasma from rabbits was analyzed by immunoprecipitation with XIAP followed by western blot with anti-His. A Ponceau protein stain was used to show input protein amounts. XBIR3 was not detected in rabbit plasma (FIG. 3), indicating that it remains localized in the eye.

Example 2

Antimicrobial activity of the compositions according to the invention has been compared with compositions comprising either only the modified clay particle comprising an antimicrobial compound (‘CPC’, prepared as in Ex. 1), or only a nonionic triblock copolymer (‘pluronic’). Salivary flora and actives (according to Table 1 below) were co-incubated overnight and at the end of incubation biofilm was stained with crystal violet. Detailed protocol as mentioned below:

Treatment and Biofilm Formation

Early morning saliva samples before brushing was collected from 4-5 people, pooled together and washed twice in saline. Absorbance was set to 0.2 OD620 nm in ultra-filtered tryptone yeast extract broth (2% sucrose) and used for experiments as mentioned in further steps. 2 ml of set culture was added into 24/12 well plate to which test actives at varying concentrations were added into each of the wells. The plate was incubated anaerobically overnight at 37° C.

Staining Protocol

At the end overnight incubation, decant the plate out over a biohazard bag to remove all the planktonic bacteria. Rinse the plate in a tray of water and decant the water out over the tray. This step was done once to remove the loosely adhered biofilm. Place the plate on a blotting paper/paper towel over the bench top. Stain all the test wells with 1 ml of 1% Crystal violet stain (CV) for 10 min. This step was done using a pipette. Decant the plate out over the biohazard discard bag to remove all the stain. Rinse the plate in a tray of water and pour the water out over the tray. This step was done thrice consecutively, in three separate trays of water. (Each tray procedure was repeated thrice-total 9 rinse). Cover the bench top with more blotting paper/paper towel and hit the plate against the bench top until all the wells are free of any liquid. This step was done to ensure that only CV remaining is bound to a biofilm at the bottom of a well. Leave the plate face up on the bench top at room temperature (23+2° C.) until it dries completely. Add 1 ml of 33% glacial acetic acid to the test wells to solubilize the biofilm bound CV stain. Allow the acetic acid to sit for 10 mins. Pipette up and down the mix of acetic acid and CV in the wells.

Transfer 10 μl of above solution mix to 90 ul of 33% acetic acid in a well of flat bottom 96 well plate. Mix the solution well and absorbance is taken at 540 nm. All the test actives were done in duplicates.

Example 4

HEK293-hAQP4-GFP and LPS-stimulated RAW264.7 were co-cultured with CD4 antibody, commercial AQP4 antibody, A002 antibody (ten-fold serial dilution) or culture medium only at 37° C. and 5% CO 2 for 6 hours. Cells were stain with Fixable Far Red-labeled anti-amine, PE-labeled anti-mouse CD11 b then analyzed the % of amine in CD11b-/GFP+ cells. Cell death(%) increased in ADCC=(% cell death in presence of IgG-% cell death in absence of IgG)/(% Cell death in maximum lysis-% cell death in absence of IgG)×100. Antibody-dependent cell-mediated cytotoxicity Assay (ADCC) of A002 antibody by immunofluorescent stain. HEK293-hAQP4-GFP and LPS-stimulated RAW264.7 were co-cultured with CD4 antibody, commercial AQP4 antibody, A002 antibody (ten-fold serial dilution) or culture medium only at 37° C. and 5% CO₂ for 6 hours. Then cells were stain with Propidium Iodide (PI). Histograms show quantification of Propidium Iodide (PI) of cells co-cultured with CD4 antibody, commercial AQP4 antibody or A002 antibody (ten-fold serial dilution) (FIG. 9). PI intensity was adjusted by Subtracting PI intensity of cells co-cultured with culture medium only.