Phil M. Choi

Evaluation of Lipopeptides as Toll-Like Receptor 2 Ligands

BACKGROUNDnPeptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines.nnnOBJECTIVEnThe aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay.nnnMETHODnAll of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction.nnnRESULTSnPam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2.nnnCONCLUSIONnFour alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Population histamine burden assessed using wastewater-based epidemiology: The association of 1,4‑methylimidazole acetic acid and fexofenadine

Systematic sampling and analysis of wastewater has become an important tool for monitoring consumption of drugs and other substances, and has been proposed as a method to evaluate aspects of population health using endogenous biomarkers. 1,4‑methylimidazoleacetic acid (MIAA) is an endogenous biomarker and metabolite of histamine turnover. Its urinary excretion is elevated in conditions such as mastocytosis, hay fever, hives, food allergies and anaphylaxis. The aim of this study was to develop and apply methods for MIAA in wastewater and compare its occurrence with antihistamine use in wastewater. Consecutive daily samples were collected from seven catchments serving populations from 3000 to 2 million and covering rural and urban communities during the 2016 Census in Australia. MIAA and the antihistamines (ranitidine, fexofenadine, cetirizine) were quantified consistently. Per capita excretion of MIAA (mg/d/capita) estimated from the WW concentrations were consistent with findings from previous clinical studies. We found significant positive correlations between loads of MIAA and fexofenadine (R2u202f=u202f0.68, pu202f<u202f0.0001) and cetirizine (R2u202f=u202f0.25, pu202f=u202f0.03) across the various catchments. Sewer reactor experiments on the degradation of MIAA and the antihistamines found that fexofenadine is stable for at least 24u202fh while MIAA, ranitidine and cetirizine are subject to degradation, and this should be considered in interpretations. To the best of our knowledge, this study is the first wastewater study to introduce and monitor an endogenous metabolite of histamine, and the first study to monitor and relate proxies of disease and treatment of disease.