Table 1.
Western blot results of different lectin reactions with different allergens.
Figure 1.
Comparative analysis of cysteine protease allergens and non-allergens in terms of mannosylation.
Allergens are strongly mannosylated and have stronger reaction with anti-mannose GNA compared to non-allergens. +++: strong reaction, ++: moderate reaction, +: mild reaction, −: no reaction.
Figure 2.
MFI ± SEM readings which represent the difference in uptake between natural and recombinant Der p 1 (1 µg/ml) by immature DCs.
There was a significant difference between natural and recombinant allergen uptake. The results suggest that the average mean of uptake for the recombinant preparation is higher than that for natural Der p 1. The results also show that the uptake of Der p 1 by immature DCs at 4°C is lower than the uptake at 37°C for both preparations. Both natural and recombinant Der p 1 were labelled with FITC. *P value<0.05.
Figure 3.
Confocal images of the difference between recombinant and natural Der p 1 (0.5 µg/ml) uptake by the same immature DC at 37°C.
The results suggest that the uptake of the recombinant preparation (A) is higher than that for natural Der p 1 (B) in the same DC. A. Green: rDer p 1 labelled with FITC, red: MR labelled with PE, blue: nucleus labelled with DAPI. B. Green: nDer p 1 labelled with Cy5, red: MR labelled with PE, blue: nucleus labelled with DAPI.
Figure 4.
A. Natural and recombinant Der p 1 uptake by immature DCs at 37°C compared to the non-allergen Staphopain B at the same conditions and concentrations. Results presented as MFI ± SDM and all preparations were labelled with FITC. B. Confocal images of the uptake of Staphopain B by immature DCs.
Figure 5.
The uptake of recombinant and natural preparations of Der p 1 (1 µg/ml) by immature DCs at 30 mins.
A. Green: rDer p1 stained with FITC, red: MR stained with PE, blue: nucleus stained with DAPI. B. Green: nDer p 1 stained with Cy5, red: MR stained with PE, blue: nucleus stained with DAPI.
Figure 6.
The co-localization of natural and recombinant Derp1 (0.5 µg/ml) with LAMP-2 detected at 10 mins.
A. Green: rDer p 1 stained with FITC, red: LAMP-2 stained with PE, blue: nucleus stained with DAPI. B. Green: nDer p 1 stained with Cy5, red: LAMP-2 stained with PE, blue: nucleus stained with DAPI.
Figure 7.
Western blot against GNA (anti-mannose) of natural and recombinant Der p 1 before and after periodate treatment.
The blot shows minimal reaction with GNA for both preparations after periodate treatment, indicating that periodate removed most of the mannan. The concentration of the protein loaded in each well was 2.0 µg/ml.
Figure 8.
ELISA experiments showing the binding of natural Der p 1 and recombinant Der p 1 to anti-Der p 1 5H8 antibody before and after deglycosylation with periodate.
Der p 1 was used at the same concentration (2.0 µg/ml) for all conditions. Data show the average of triplicate experiments.
Figure 9.
Commassie blue stained gel of natural Der p 1 before and after deglycosylation with periodate showing a slight decrease in the MW of deglycosylated Der p 1.
Figure 10.
A. The MFI ± SEM readings for the uptake of different concentrations of nDer p 1 and rDer p 1 by immature DCs compared to the periodate treated preparations. Both nDer p 1 and rDer p 1 were treated with periodate for 30 mins and 1 hr, then their uptake was measured. The results show a significant decrease in uptake of periodate treated preparations compared with the untreated ones.*** P value<0.001, all Der p 1 preparations were labelled with FITC. B. Confocal images showing the uptake of periodate treated Der p 1 by immature DCs.
Figure 11.
Binding of MR C-type lectin-like carbohydrate recognition domains 4–7 with different glycoforms of Der p 1, nDer p 1 and rDer p 1 (concentrations at 2 µg/ml).
***P value<0.001.
Figure 12.
Differences in TSLP secretion in human BEAS-2B epithelial cells after 24 hrs stimulation with different glycoforms of Der p 1.
Concentration of all Der p 1 preparations used was 1 µg/ml. ***P value<0.001.