Objective: The immune response is linked to the progression of atherosclerotic cardiovascular disease (CVD). Free autoantibodies targeting ApoA-I (apolipoprotein A-I) have been identified as a component of the inflammatory milieu in patients and have a moderate association with CVD progression. Based on the presence of these antibodies and the high concentration of circulating ApoA-I, we hypothesized that antibodies bound to ApoA-I as an immune complex would be predictive of incident adverse CVD outcomes. Approach and Results: The presence of ApoA-I/IgG immune complexes (ICs) in plasma was confirmed by ELISA in 3 subject cohorts. Characterization of the protein components of ApoAI/IgG ICs indicate that ICs are not correlated with total ApoA-I concentration and are enriched in the anti-inflammatory subclass, IgG4, relative to total plasma IgG (>30% versus 6%). In 359 patients with coronary artery disease (CAD), there were 71 incident adverse CVD events (death, myocardial infarction, and stroke) during a median 4.1-year follow-up. In Cox proportional hazard regression analysis, low levels of ApoA-I/IgG ICs were independent predictors of adverse cardiovascular outcomes after adjustment for age, sex, diabetes mellitus, estimated glomerular filtration rate, presence of obstructive CAD, heart failure, total cholesterol, and HDL (high-density lipoprotein) cholesterol (adjusted hazard ratio of 1.90 [95% CI, 1.03–3.49; P =0.038] between the lowest and the highest tertiles). Conclusions: Low levels of ApoA-I/IgG ICs are associated with an increased risk of adverse events in patients with CAD, raising their potential to be used as a biomarker to predict CVD progression.

Cyanuric chloride has been utilized in the development of new synthetic lipid compounds using two differing schemes. The resulting lipids, presented in this manuscript, were characterized and evaluated for their ability to form nanoparticles and subsequently tested for their utility in various biological applications, including gene delivery and immunization. Of the 12 lipids synthesized, 8 formed nanoparticles that remained stable, based on dynamic light scattering, for at least one month. The compounds were then assessed for their toxicity, and subsequently tested for their ability to encapsulate drugs, genes and peptides. While the compounds did not seem to encapsulate carboxyfluorescein, we demonstrate that these lipids are capable of plasmid delivery in vitro, and inducing antibody profiles similar to other hydrophobic anchors in liposomal peptide vaccines. This strategy for accessing diverse lipid compounds offers a way to easily optimize lipid-based therapeutics for research in an expedited manner.

Non-viral vectors for in vivo delivery of plasmid DNA rely on optimized formulations to achieve robust transgene expression. Several cationic lipids have been developed to deliver nucleic acids, but most recent literature has focused on mRNA due to its increased expression profile and excluded plasmid DNA, which may have the advantage of being less immunogenic. In this study, we describe the in vivo evaluation of cationic triazine based lipids, previously prepared by our group. We identify one lipid with limited in vivo toxicity for studies to optimize the lipid formulations, which include an evaluation of the influence of PEG and helper lipids on transgene expression. We then demonstrate that lipoplexes, but not lipid nanoparticles, formed from triazine lipids achieve similar transgene expression levels as AAV vectors and offer enhanced expression as compared to a commercially available cationic lipid, DOTAP. Importantly, the lipid nanoparticles and lipoplexes induce minimal antibody profiles toward the expressed protein, while serving as a platform to induce robust antibody responses when directly delivering the protein. Collectively, these data demonstrate the potential for triazine based lipids as non-viral vectors for gene delivery, and highlights the need to optimize each formulation based on the exact contents to achieve enhanced transgene expression with plasmid DNA constructs.

Atherosclerosis is responsible for a large percentage of all-cause mortality worldwide, but it is only now beginning to be understood as a complex disease process involving metabolic insult, chronic inflammation, and multiple immune mechanisms. Abs targeting apolipoprotein A-I (ApoA-I) have been found in patients with cardiovascular disease, autoimmune conditions, as well as those with no documented history of either. However, relatively little is known about how these Abs are generated and their relationship to diet and sex. In the current study, we modeled this aspect of autoimmunity using anti–ApoA-I immunization of male and female C57BL/6 mice. Unexpectedly, we found that autoantibodies directed against a single, previously unknown, epitope within the ApoA-I protein developed irrespective of immunization status or dyslipidemia in mice. When total IgG subclasses were analyzed over the course of time, we observed that rather than driving an increase in inflammatory IgG subclasses, consumption of Western diet suppressed age-dependent increases in IgG2b and IgG2c in male mice only. The lack of change observed in female mice suggested that diet and sex might play a combined role in Th1/Th2 balance and, ultimately, in immunity to pathogen challenge. This report demonstrates the need for inclusion of both sexes in studies pertaining to diet and aging and suggests that further study of immunogenic epitopes present in ApoA-I is warranted.

Strategies that introduce students to unconscious bias and social determinants of health (SDOH) are critical to develop them as effective health care providers. We developed a semester-long activity that utilizes disease-relevant scientific literature to implement cultural humility training in a second-year rheumatology pharmacy course. Students were first re-introduced to implicit bias and then completed an anonymous survey at the beginning and conclusion of the course using a 5-point Likert scale to assess their perceptions of the role of biases and SDOH in patient care. Throughout the semester, five journal articles were assigned that relate to course material and focus on one characteristic (e.g., gout—gender). Students’ evolved perceptions of SDOH were compared to baseline data and characteristics of assigned articles indicate an improved understanding of SDOH including race/ethnicity (3.0 to 4.4, p < 0.0001); gender (2.8 to 4.0, p < 0.0001); and religion (2.3 to 2.9, p < 0.01). Among characteristics that were not directly discussed in the assignments, only education showed a significant increase (3.0 to 3.6, p < 0.01). Scientific articles that focus on health inequities relevant to course-specific diseases provide a strategy to integrate discussions that help students evaluate their biases and SDOH with the goal of improving patient care.