School of Life and Environmental Sciences, Deakin University, Geelong VIC 3220, AUSTRALIA.
In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. Allergic reactions can result in life-threatening situations causing morbidity and high economic cost. Therefore, more effective reagents are needed for allergy treatment. In Chapter 2, IL-4Rα was targeted by employing a novel molecular method of phage display technology. A novel synthetic peptide (N1 biopep) was isolated using this technology, and demonstrated a strong affinity to the target IL-4R. This was followed by extensive immunoassays such as ELISA. Indeed, the N1 biopep has shown to be very promising in its capacity to significantly down-regulate the interactions between IL-4Rα and the cytokine IL-4.
In Chapter 3, peptides were commercially synthesized and used for in vitro assays and a HEK-BlueTM IL-4/IL-13 reporter cell line model, transfected with a gene producing an enzyme SEAP. SEAP acts as a substitute to IgE when cells are stimulated with cytokine IL-4 and IL-13. QUANTI-Blue was added as a substrate that breaks down in the presence of SEAP producing blue coloration. The blue color confirmed the activation of STAT6 pathway using a spectrophotometer. We have successfully used peptides N1 to IL-4Rα, K1 to IL-4 and P9 to IL-13 that demonstrated inhibition of SEAP production in HEK-Blue cells. A colorimetric analysis showed a >50% inhibition, resulting into less blue color with all three peptides. A statistical Student’s t-test revealed the significance of the results. Since IL4 and IL-13 interaction with IL-4R/IL-13R is a common pathway for many allergies, a prophylactic treatment can be devised by using such novel methods in the future.
In Chapter 4, it was hypothesized that a causal relationship exists between the intake of omega-3/6 fatty acids such as DHA, EPA, DPA and AA and atopic individuals suffering from allergies. In allergic cascades, cytokines IL-4 and IL-13 bind to IL-4 receptor and IL-13 receptor (IL-4R and IL-13R), respectively, which activate the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells. The overall aim here was to characterize omega-3/6 fatty acids and their effect on IL-4/IL-13 signaling. The fatty acids were tested in vitro with the HEK-Blue IL-4/IL-13 reporter cell line model. We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease of SEAP secretion as opposed to an increase in SEAP secretion with AA treatment. A statistical Student’s t-test revealed the significance of the results. We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model with varying potentials. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4Rα signalling, which regulates the STAT6 induced pathway in allergic cascades in vitro.
As literature suggests, resolvins and CoQ10 have shown promising results with down- regulation of allergy and inflammation. In Chapter 5, we assessed the effect of CoQ10 and resolvins on the HEK-Blue cell model of allergy. Through our series of experiments, we have shown the differential effects of resolvins and CoQ10 on SEAP secretion. A statistical Student’s t-test revealed the significance of the results. Resolvins, although anti-inflammatory by nature, showed an increase in SEAP secretion as opposed to CoQ10, which stood out as a promising candidate in repressing allergy. SEAP secretion was completely inhibited in the presence of CoQ10 even at lower concentrations, suggesting a novel therapeutic for allergy treatment.
Lastly, we assessed the distribution of ryegrass pollen in the regional city of Geelong (Victoria, Australia). The purpose of Chapter 6 was to establish a link between weather variables and the presence of ryegrass pollen (intact vs. ruptured) in the atmosphere. For this purpose, a Burkard volumetric air trap was installed at the Deakin University (Waurn Ponds, Geelong) for pollen collection (known as Deakin AIRwatch). Our results show that ryegrass pollen distribution is fairly correlated to weather variables such as rain, temperature, humidity and wind. Pollen counting from three years were incorporated as part of this study. We have successfully shown the presence of intact and ruptured pollen in air, which is important for future studies relevant to allergic individuals suffering from pollen allergy.
To sum up, chapters 2-5 have shown the use of different therapeutics to down-regulate allergic pathways which are common to all allergies. The isolated peptides along with n-3 fatty acids and CoQ10 can serve as novel candidates for allergy treatment. On the contrary, avoidance of pro-inflammatory products such as n-6 fatty acids and resolvins can prevent allergic symptoms. In chapter 6, we have conducted a thorough investigation of ryegrass pollen distribution in regional Victoria which can help alleviate allergy through awareness and avoidance of pollen allergy hence, improving quality of life. Our work paves a new and exciting path for the safe and effective treatment of allergies.