Identification and characterisation of a novel Interleukin-4 receptor antagonist for allergy treatment

 The main focus of the PhD studies was the identification and characterization of a peptide antagonist which inhibited and/or down-regulated the binding of Interleukin-4 receptor to its cytokines IL-4 and IL-13 for allergy treatment. In addition, dietary components were tested for their ability to reduce inflammatory pathways of allergy, including fatty acids, resolvins and coenzyme Q10. Lastly, the Deakin AIRwatch project was undertaken which included the collection of ryegrass pollen data from 2012-2014 and its correlation with meteorological variables in regional Victoria.

Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling

BACKGROUND/AIMS: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. METHODS: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. RESULTS: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. CONCLUSION: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10-9 M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit C AT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat . Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease ( p 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven C AT expression, while TNF- alpha -enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat /TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

A novel model of sensitization and oral tolerance to peanut protein

The prevalence of food allergic diseases is rising and poses an increasing clinical problem. Peanut allergy affects around 1% of the population and is a common food allergy associated with severe clinical manifestations. The exact route of primary sensitization is unknown although the gastrointestinal immune system is likely to play an important role. Exposure of the gastrointestinal tract to soluble antigens normally leads to a state of antigen-specific systemic hyporesponsiveness (oral tolerance). A deviation from this process is thought to be responsible for food-allergic diseases. In this study, we have developed a murine model to investigate immunoregulatory processes after ingestion of peanut protein and compared this to a model of oral tolerance to chicken egg ovalbumin (OVA). We demonstrate that oral tolerance induction is highly dose dependent and differs for the allergenic proteins peanut and OVA. Tolerance to peanut requires a significantly higher oral dose than tolerance to OVA. Low doses of peanut are more likely to induce oral sensitization and increased production of interleukin-4 and specific immunoglobulin E upon challenge. When tolerance is induced both T helper 1 and 2 responses are suppressed. These results show that oral tolerance to peanut can be induced experimentally but that peanut proteins have a potent sensitizing effect. This model can now be used to define regulatory mechanisms following oral exposure to allergenic proteins on local, mucosal and systemic immunity and to investigate the immunomodulating effects of non-oral routes of allergen exposure on the development of allergic sensitization to peanut and other food allergens.

Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

Cord blood monocyte-derived inflammatory cytokines suppress IL-2 and induce nonclassic "T(H)2-type" immunity associated with development of food allergy

Food allergy is a major health burden in early childhood. Infants who develop food allergy display a proinflammatory immune profile in cord blood, but how this is related to interleukin-4 (IL-4)/T helper 2 (T(H)2)-type immunity characteristic of allergy is unknown. In a general population-derived birth cohort, we found that in infants who developed food allergy, cord blood displayed a higher monocyte to CD4(+) T cell ratio and a lower proportion of natural regulatory T cell (nT(reg)) in relation to duration of labor. CD14(+) monocytes of food-allergic infants secreted higher amounts of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) in response to lipopolysaccharide. In the presence of the mucosal cytokine transforming growth factor-β, these inflammatory cytokines suppressed IL-2 expression by CD4(+) T cells. In the absence of IL-2, inflammatory cytokines decreased the number of activated nT(reg) and diverted the differentiation of both nT(reg) and naïve CD4(+) T cells toward an IL-4-expressing nonclassical TH2 phenotype. These findings provide a mechanistic explanation for susceptibility to food allergy in infants and suggest anti-inflammatory approaches to its prevention.

Impact of resistance exercise training on interleukin-6 and JAK/STAT in young men

The JAK/STAT signaling pathway is essential for myogenic regeneration and is regulated by a diverse range of ligands, including interleukin-6 (IL-6) and platelet-derived growth factor-BB (PDGF-BB). Our aim was to evaluate the responsiveness of IL-6 and PDGF-BB to intense exercise, along with STAT3 activation, before and after 12 weeks of resistance training. In young men, IL-6 and PDGF-BB protein concentrations were quantified in biopsied muscle and increased at 3 h post-exercise (17.5-fold and 3-fold, respectively). The response was unaltered by 12 weeks of training. Similarly, STAT3 phosphorylation was elevated post-exercise (12.5-fold), irrespective of training status, as was the expression of downstream targets c-MYC (8-fold), c-FOS (4.5-fold), and SOCS3 (2.3-fold). Thus, intense exercise transiently increases IL-6 and PDGF-BB proteins, and STAT3 phosphorylation is increased. These responses are preserved after intense exercise. This suggests they are not modified by training and may be an essential component of the adaptive responses to intense exercise.

Effects of resistance or aerobic exercise training on interleukin-6, C-reactive protein, and body composition

PURPOSE: To determine the effects of 10 wk of resistance or aerobic exercise training on interleukin-6 (IL-6) and C-reactive protein (CRP). Further, to determine pretraining and posttraining associations between alterations of IL-6 and CRP and alterations of total body fat mass (TB-FM), intra-abdominal fat mass (IA-FM), and total body lean mass (TB-LM). METHODS: A sample of 102 sedentary subjects were assigned to a resistance group (n = 35), an aerobic group (n = 41), or a control group (n = 26). Before and after intervention, subjects were involved in dual-energy x-ray absorptiometry, muscular strength and aerobic fitness, measurements and further provided a resting fasted venous blood sample for measures of IL-6, CRP, cholesterol profile, triglycerides, glucose, insulin, and glycosylated hemoglobin. The resistance and the aerobic groups completed a respective 10-wk supervised and periodized training program, whereas the control group maintained sedentary lifestyle and dietary patterns. RESULTS: Both exercise training programs did not reduce IL-6; however, the resistance and the aerobic groups reduced CRP by 32.8% (P < 0.05) and 16.1% (P = 0.06), respectively. At baseline, CRP was positively correlated with IL-6 (r = 0.35), (TB-FM) (r = 0.36), and IA-FM (r = 0.31) and was inversely correlated with aerobic fitness measures (all r values > or = -0.24). Compared with the resistance and the control groups, the aerobic group exhibited significant (P < 0.05) improvements in all aerobic fitness measures and significant reductions in IA-FM (7.4%) and body mass (1.1%). Compared with the aerobic and the control groups, the resistance group significantly (P < 0.05) improved TB-FM (3.7%) and upper (46.3%) and lower (56.6%) body strength. CONCLUSION: Despite no alteration in baseline IL-6 and significantly smaller reductions in measures of adipose tissue as compared with the aerobic training group, only resistance exercise training resulted in significant attenuation of CRP concentration.