Purified Allergen Specific Immunotherapy

The research article deals with purified allergen specific immunotherapy (IT). The study investigates the efficacy of purified cockroach allergen immunotherapy with proteolytically active and inactive Per a 10 in allergic mouse model.. They carried out the study on Balb/c mice which were sensitized intraperitoneally with cockroach extract (CE) and purified allergen Per a 10 in separate groups. Mice were treated subcutaneously with phosphate-buffered saline (PBS), CE, active and inactive Per a 10 and challenged intranasally. Antigen specific IgE, IgG1 and IgG2a in serum and cytokines IL-4, IL-13, IFNγ, IL-10, TGF-β in bronchoalveolar lavage (BAL) fluid and spleen culture supernatant (CS) were estimated by enzyme-linked immunosorbent assay.

They determined specific immunoglobulin IgE, IgG1, and IgG2a levels in serum of different experimental groups by ELISA. Briefly, microtiter plates (Maxisorp; NuncTM Immunomodule, Roskilde, Denmark) were coated with 10 µg/ml of CE or 1 µg/ml of active Per a 10 in 0.1 M carbonate buffer (pH=9.6). Nonspecific sites were blocked with 3% defatted milk, washed, and incubated individually with mice sera for IgE (1:10), IgG1 (1:50), and IgG2a (1:50) estimation. After washing, the plates were incubated for 3 h with anti-mouse IgG1-peroxidase and anti-mouse IgG2a-peroxidase (1:1,000 PBS; BD Pharmingen, San Diego, CA, USA). For IgE estimation, the plate was incubated with biotinylated anti-mouse IgE (0.5 mg/ ml, BD Pharmingen, 1:1,000) at 25°C for 90 min.

Following washing, it was incubated with streptavidin– peroxidase (1:1,000; BD Pharmingen) for 30 min. Color was developed using o-phenylene di-amine hydrochloride (Sigma, USA), and the absorbance was read at 492 nm. Similar to antibody detection they utilized ELISA for determining cytokines levels in BAL fluid and splenocyte culture supernatant. IL-4 (detection limit; 7.8 pg/ml), IL-13 (detection limit; 62.5 pg/ml), IFN-γ (detection limit; 31.3 pg/ ml), IL-10 (detection limit; 31.3 pg/ml), and TGF-β (detection limit; 62.5 pg/ml) levels were determined in undiluted BAL fluid and culture supernatants by ELISA using paired antibodies according to manufacturer’s instruction (BD Pharmingen, USA and R & D systems Inc. Minneapolis, MN, USA for IL-13). Briefly precoated ELISA plates were utilized for cytokine detection, wherein antibodies to cytokines to be investigated were coated in the wells from the vendor. Also standards were provided for the quantification of the cytokines level, along with the detection antibody.

They showed that cockroach-specific immunoglobulin levels in serum mice sensitized with CE (0.325±0.071; P<0.01) and Per a 10 (0.356±0.075; P<0.01) showed significantly increased levels of specific IgE after sensitization in serum than normal PBS control (0.122±0.043). After completion of protocol, CE-treated mice showed non-significant reduction in specific IgE levels compared to PBS-treated mice (P> 0.01). Further, immunotherapy with Per a 10 (proteolytically active and inactive) reduced IgE levels effectively compared to PBS-treated mice (P<0.01). There was no significant difference in IgE levels between active Per a 10and CE-treated mice (P>0.01).

The enzymatically inactive Per a 10 showed significant reduction in specific IgE levels compared to active Per a 10- and CE-treated mice (P<0.01). Immunotherapy with Per a 10 (proteolytically active and inactive) in Per a 10-sensitized mice also showed significant reduction in specific IgE levels in comparison to PBS treated mice (P<0.01). Most importantly, IT with inactive Per a 10 showed significant reduction in specific IgE level compared to active Per a 10-treated mice in Per a 10-sensitized mice (P<0.01). Furthermore, IgE levels reduced significantly with active Per a 10 treatment in Per a 10-sensitized mice compared to CE-sensitized mice (#P< 0.01), whereas inactive Per a 10-treated mice showed remarkably significant reduction in IgE levels in active Per a 10-sensitized mice compared to CE-sensitized mice (P<0.001). IgG1 and IgG2a levels, however, did not change significantly in all the groups post-IT.

Cytokines in Bronchoalveolar Lavage Fluid and Splenocyte Culture Supernatant In CE-sensitized mice, IT with CE showed substantial reduction in IL-4 levels compared to PBS-treated mice (P>0.01). Per a 10 (proteolytically active and inactive) IT showed significant reduction in IL-4 levels than PBS-treated mice (P<0.01). The active Per a 10-treated mice, however, did not show significant reduction in IL-4 levels compared to CE-treated mice (P>0.01). But, IL-4 levels showed significant reduction in mice treated with proteolytically inactive Per a 10 compared to active Per a 10- and CE-treated group (P<0.01). Further, the IL-10 levels showed substantial increase in BAL fluid and culture supernatant post-IT with CE compared to PBS-treated mice (P>0.01).

Immunotherapy with Per a 10 (proteolytically active and inactive) showed significant increase in IL-10 levels compared to PBS-treated mice (P<0.01). However, Per a 10-treated mice did not show significant increase in IL-10 levels compared to CE-treated group (P>0.01), and inactive Per a 10 showed maximum increase in IL-10 levels post-IT followed by active Per a 10- and CE-treated mice (P<0.01) in CE-sensitized mice. Immunotherapy with Per a 10 (proteolytically active and inactive) in Per a 10-sensitized mice showed significant reduction in IL-4 levels compared to PBS-treated mice (P< 0.01). IL-10 levels showed significant increase in BAL fluid and culture supernatant post-IT with Per a 10 (proteolytically active and inactive) compared to PBS treated mice (P<0.01). Most importantly, IT with inactive Per a 10 showed significant reduction in IL-4 level accompanied by maximum increase in IL-10 level compared to active Per a 10-treated mice (P<0.001). IFN-γ, IL-13, and TGF-β levels, however, did not change significantly post-IT in all the groups. Additionally, IL-10 levels showed significant increase with active Per a 10 treatment.