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Effects of apoB-derived peptide vaccination in a murine model of systemic lupus erythematosusSamuelsen, Brian 08 April 2016 (has links)
OBJECTIVE: Atherosclerotic disease progression is mediated in part, by immunological mechanisms. In recent years, interest has increased towards the prospect of modulating these immune mechanisms through vaccination to ameliorate the course of disease. Patients with lupus are at a significantly higher risk for accelerated atherosclerosis and related complications. The goal of this study was to assess the outcome of immunization in mouse models of lupus, and lupus with accelerated atherosclerosis.
MATERIALS/METHODS: Atherosclerosis-prone apoE^-/- mice and autoimmune gld mice were previously crossed to generate the gld.apoE^-/- mouse. Mice were treated with an apoB-100-derived vaccine, Alum (adjuvant control), or PBS control. The antibody response was determined by quantifying the amount of circulating anti-apoB100. Serum triglyceride and cholesterol levels were analyzed. Kidney tissue from gld and gld.apoE^-/- mice was processed and histologically analyzed, using glomerular tuft size as a measure of renal disease and by extension, autoimmune disease severity.
Results: Immunization led to a pronounced initial antibody response that was decreased by the endpoint of the study. No significant differences in serum triglyceride or cholesterol were observed regardless of treatment. Similarly, no significant differences were observed in glomerular tuft size.
Conclusion: The data suggests that immunization with an apoB-100- derived vaccine neither improves nor worsens autoimmune disease severity in the gld.apoE^-/- mouse model. It also appears that immunization is tolerated in the autoimmune background. While further study is necessary to determine the efficacy of immunization in reducing atherosclerotic disease in this model, this may be a possible therapy to lower incidence of atherosclerosis in lupus patients.
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Surface-Engineered Magnetic Nanoparticles for Sample Preparation and Analysis of Proteins and PeptidesPirani, Parisa 15 May 2015 (has links)
Sample preparation as an essential step in mass spectrometry-based analysis, plays a critical role in proteomics studies. Magnetic nanoparticles (MNPs) have been widely used in protein and peptide sample preparation due to their magnetic properties, biocompatibility, easy synthesis and surface functionalization. MNPs loaded with analyte or analyte modification reagent can be easily separated from the reaction medium by an externally applied magnetic field. The small size of MNPs provides high analyte loading and extraction capacity. Additionally, MNP can be decorated with different functional groups to achieve selective modification or extraction of analyte. In this study we have utilized silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) for protein and peptide sample preparation.
Fluorescence-based methods were utilized for quantitative and qualitative characterization of N-hydrosucccinimidyl (NHS) ester groups on the surface of Fe3O4@SiO2 MNPs. Fluorophore Dansylcadaverine was conjugated to NHS ester functional groups. Fluorometric measurement of cleaved dansylcadaveine was employed to determine the number of NHS ester groups per MNPs that was found to be 2.6 × 102 and 3.4 × 103for 20 nm and 100 nm Fe3O4@SiO2 MNPrespectively. The efficiency of labeling native bovine serum albumin (BSA) by NHS ester coated Fe3O4@SiO2 MNPs was also explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each MNP.
Lysine residues of apolipoprotein B-100 (apoB-100) on the surface of intact human low density lipoprotein (LDL) were labeled by NHS ester modified Fe3O4@SiO2 MNPs in aqueous solvents at room temperature. The MNP labeledapoB-100 was treated by SDS to remove lipids and then digested using trypsin. Tryptic peptides were eluted from MNPs by cleaving disulfide linkage between labeled peptides and MNPs. LC-MS/MS analysis found 28 peptides containing labeled lysine residues. These lysine residues should be on the solvent exposed surface of LDL since the large size of MNPs prevents contact of the labeling reagent to those lysines embedded inside the structure of LDL.
TCEP- immobilized Fe3O4@SiO2MNPs were fabricated and utilized for reduction of disulfide bonds in bovine pancreas insulin and two different cyclic peptides. Disulfide bonds were efficiently cleaved at room temperature in both organic and aqueous solvents confirmed by LC-MS/MS analysis of reduced/alkylated protein and peptides. Disulfide reduction and alkylation reactions was performed in one step and the reducing agent was simply separated from peptide and protein solution by magnetic separation.
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