Eric A. Prado

Natural cocoa consumption: Potential to reduce atherogenic factors?

Short-term consumption of flavanol-rich cocoa has been demonstrated to improve various facets of vascular health. The purpose of the present study was to determine the effect of 4 weeks of natural cocoa consumption on selected cardiovascular disease (CVD) biomarkers in young (19-35 years) women of differing body mass indices (BMI; normal, overweight or obese). Subjects (n = 24) consumed a natural cocoa-containing product (12.7 g natural cocoa, 148 kcal/serving) or an isocaloric cocoa-free placebo daily for 4 weeks in a random, double-blind manner with a 2-week washout period between treatment arms. Fasted (>8-h) blood samples were collected before and after each 4-week period. Serum was analyzed to determine lipid profile (chemistry analyzer) and CVD biomarkers (26 biomarkers). EDTA-treated blood was used to assess monocytes (CD14, CD16, v11b and CD62L), while citrate-treated blood was used to measure changes in endothelial microparticles (EMPs; CD42a-/45-/144+) by flow cytometry. Natural cocoa consumption resulted in a significant decrease in haptoglobin (P = .034), EMP concentration (P = .017) and monocyte CD62L (P = .047) in obese compared to overweight and normal-weight subjects. Natural cocoa consumption regardless of BMI group was associated with an 18% increase in high-density lipoprotein (P = .020) and a 60% decrease in EMPs (P = .047). Also, obese subjects experienced a 21% decrease in haptoglobin (P = .034) and a 24% decrease in monocyte CD62L expression in (P = .047) following 4 weeks of natural cocoa consumption. Collectively, these findings indicate that acute natural cocoa consumption was associated with decreased obesity-related disease risk. More research is needed to assess the stability of the observed short-term changes.

Using image-based flow cytometry to measure monocyte oxidized LDL phagocytosis: A potential risk factor for CVD?

Obesity and cardiovascular disease is a worldwide health concern that has been a major focus in research for several decades. Among these diseases, atherosclerosis is one of the leading causes of death and disability nationwide. Circulating monocytes are believed to be primary cells responsible for foam cell formation. The present report describes a novel method for measuring monocyte oxLDL phagocytosis capacity using image-based flow cytometry. Human venous blood monocytes were incubated with different concentrations of oxLDL for different lengths of time to optimize the assay. High (post-meal) and low (pre-meal) responder samples were generated by feeding human subjects a high-fat (~85% of daily fat allowance), high-calorie (~65% of daily calorie needs) meal. This is a relevant model with respect to obesity and risk of developing atherogenesis. After the functional assay, classic (CD14+/CD16-) and pro-inflammatory (CD14+/CD16+) monocytes were assessed for oxLDL uptake, adhesion molecule expression (CD11b and CD18), and scavenger receptor expression (CD36) using an image-based flow cytometer (FlowSight). The present method represents a novel advance in methods available to detect the propensity of circulating monocytes to become intima foam cells. We found the assay to be most effective at separating high from low responder samples when using a fixed oxLDL concentration (120 μL/mL) and incubation length (1-h). In a clinical application, this method demonstrated that consuming a single high-fat meal causes an increase in the proportion of monocyte oxLDL phagocytosis and their adhesion capacity, suggesting a higher propensity to become foam cells.

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Using Image-Based Flow Cytometry to Assess Monocyte Oxidized LDL Phagocytosis Capacity

The examination of monocyte phagocytosis of modified lipoproteins is important to the understanding of plaque deposition and the development of atherosclerosis. Current methods lack the high-throughput image-based analysis capabilities, which may yield novel information concerning monocyte activity in disease processes. Specifically, this method identifies monocyte phagocytosis of oxidized LDL along with a change in adhesion molecules and scavenger receptors. Our laboratory is currently implementing this method as a means to study how acute dietary modifications alter risk of developing atherosclerosis.

Using Image-Based Flow Cytometry with a FISH-Based FlowRNA Assay to Simultaneously Detect Intracellular TNF-α Protein and mRNA in Monocytes Following LPS Stimulation

Existing methods of assessing monocyte inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) response to in vitro lipopolysaccharide (LPS) stimulation lack the ability to simultaneously detect intracellular mRNA and protein. This procedure takes advantage of new methodologies and instrumentation to simultaneously measure intracellular TNF-α mRNA and protein in CD14(+) monocytes after 1, 3, and 6 h of LPS stimulation. By assessing multiple timepoints, we are able to discern how LPS stimulation affects the temporal relationship between TNF-α mRNA and protein. By using image-based flow cytometry it is possible to co-localize mRNA and protein signals to identify the length of incubation that is needed to initiate protein translation.

Assessment of Granulocyte Subset Activation: New Information from Image-Based Flow Cytometry.

Analysis of granulocyte function can provide important information about the state of the bodys innate immune system. Existing flow cytometry methods that lack image-based analysis capabilities fail to fully evaluate granulocyte function. In the present method, we combine simultaneous detection of phagocytosis and oxidative burst in granulocytes to identify unique subsets of activated granulocytes. This analysis method provides novel information about granulocytes that allows our lab and others to evaluate the effectiveness of nutritional and lifestyle countermeasures, designed to improve immunity.