Andrea L. Henning

Reduced inflammatory and muscle damage biomarkers following oral supplementation with bioavailable curcumin

Background Exercise-Induced Muscle Damage (EIMD) and delayed onset muscle soreness (DOMS) impact subsequent training sessions and activities of daily living (ADL) even in active individuals. In sedentary or diseased individuals, EIMD and DOMS may be even more pronounced and present even in the absence of structured exercise. Methods The purpose of this study was to determine the effects of oral curcumin supplementation (Longvida® 400 mg/days) on muscle & ADL soreness, creatine kinase (CK), and inflammatory cytokines (TNF-α, IL-6, IL-8, IL-10) following EMID (eccentric-only dual-leg press exercise). Subjects (N = 28) were randomly assigned to either curcumin (400 mg/day) or placebo (rice flour) and supplemented 2 days before to 4 days after EMID. Blood samples were collected prior to (PRE), and 1, 2, 3, and 4 days after EIMD to measure CK and inflammatory cytokines. Data were analyzed by ANOVA with P < 0.05. Results Curcumin supplementation resulted in significantly smaller increases in CK (− 48%), TNF-α (− 25%), and IL-8 (− 21%) following EIMD compared to placebo. We observed no significant differences in IL-6, IL-10, or quadriceps muscle soreness between conditions for this sample size. Conclusions Collectively, the findings demonstrated that consumption of curcumin reduced biological inflammation, but not quadriceps muscle soreness, during recovery after EIMD. The observed improvements in biological inflammation may translate to faster recovery and improved functional capacity during subsequent exercise sessions. General significance These findings support the use of oral curcumin supplementation to reduce the symptoms of EIMD. The next logical step is to evaluate further the efficacy of an inflammatory clinical disease model.

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.

A shirt containing multistage phase change material and active cooling components was associated with increased exercise capacity in a hot, humid environment

Abstract Recent advances in clothing design include the incorporation of phase change materials (PCM) and other active cooling components (ACC) to provide better body heat dissipation. The purpose of this study was to determine the effect of wearing a shirt containing multistage PCM/ACC on exercise capacity at low (5.0), moderate–high (7.5) and extreme (9.0) levels of the physiological strain index (PSI). Fourteen individuals tested two shirts (control vs. cooling) during 45-min of interval running in a hot, humid (35 ± 1 °C; 55 ± 6% RH) environment. The cooling shirt resulted in an 8% improvement in exercise capacity at a PSI of 7.5 (p < 0.05). The observed increase in exercise capacity would likely translate to a significant improvement in exercise performance. More research is needed to determine a best practice approach for the use of cooling clothing as a counter to exercise-induced heat exposure. Practitioner Summary: In this report, we demonstrate that when forced to exercise in a hot, humid environment, an individual’s exercise capacity may increase by as much as 8% when wearing a shirt composed of multistage phase change material and active cooling components.

Measurement of Low-Abundance Intracellular mRNA Using Amplified FISH Staining and Image-Based Flow Cytometry.

Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR‐gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR‐gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR‐gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low‐abundant intracellular mRNA expression. This protocol represents a continued effort by the authors’ laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease.

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).

Oral spore-based probiotic supplementation was associated with reduced incidence of post-prandial dietary endotoxin, triglycerides, and disease risk biomarkers

AIM To determine if 30-d of oral spore-based probiotic supplementation could reduce dietary endotoxemia. METHODS Apparently healthy men and women (n = 75) were screened for post-prandial dietary endotoxemia. Subjects whose serum endotoxin concentration increased by at least 5-fold from pre-meal levels at 5-h post-prandial were considered “responders” and were randomized to receive either placebo (rice flour) or a commercial spore-based probiotic supplement [Bacillus indicus (HU36), Bacillus subtilis (HU58), Bacillus coagulans, and Bacillus licheniformis, and Bacillus clausii] for 30-d. The dietary endotoxemia test was repeated at the conclusion of the supplementation period. Dietary endotoxin (LAL) and triglycerides (enzymatic) were measured using an automated chemistry analyzer. Serum disease risk biomarkers were measured using bead-based multiplex assays (Luminex and Milliplex) as secondary, exploratory measures. RESULTS Data were statistically analyzed using repeated measures ANOVA and a P < 0.05. We found that spore-based probiotic supplementation was associated with a 42% reduction in endotoxin (12.9 ± 3.5 vs 6.1 ± 2.6, P = 0.011) and 24% reduction in triglyceride (212 ± 28 vs 138 ± 12, P = 0.004) in the post-prandial period Placebo subjects presented with a 36% increase in endotoxin (10.3 ± 3.4 vs 15.4 ± 4.1, P = 0.011) and 5% decrease in triglycerides (191 ± 24 vs 186 ± 28, P = 0.004) over the same post-prandial period. We also found that spore-based probiotic supplementation was associated with significant post-prandial reductions in IL-12p70 (24.3 ± 2.2 vs 21.5 ± 1.7, P = 0.017) and IL-1β (1.9 ± 0.2 vs 1.6 ± 0.1, P = 0.020). Compared to placebo post supplementation, probiotic subject had less ghrelin (6.8 ± 0.4 vs 8.3 ± 1.1, P = 0.017) compared to placebo subjects. CONCLUSION The key findings of the present study is that oral spore-based probiotic supplementation reduced symptoms indicative of “leaky gut syndrome”.

Consumption of a high-fat, high-calorie meal is associated with an increase in intracellular co-localization of PPAR-γ mRNA and protein in monocytes

Acute and habitual dietary habits contribute to the onset and progression of many forms of cardiovascular disease. Circulating peripheral blood monocytes have been a target of pre-clinical research related to the risk of atherosclerosis. Specifically, when monocytes migrate into the subendothelial space and endocytosize modified LDL (i.e. acLDL or oxLDL) they phenotypically transform into foam cells. The endocytosis of modified LDL is mediated by the scavenger receptor CD36, whose expression is in tern regulated by the transcription factor PPAR-γ. In this report, we describe a novel technique for the simultaneous measurement of intracellular PPAR-γ mRNA and protein in peripheral blood monocytes collected from human subjects in fasted state or 3 and 5-h after consuming a high-calorie (65% of daily calorie needs), high-fat meal. Intracellular detection and co-localization of PPAR-γ was made possible using a combination of image-based flow cytometry (MilliporeSigma FlowSight) and an amplified mRNA FISH staining technique (Affymetrix/eBioscience PrimeFlow). Consumption of a high-calorie, high-fat meal increased the percentage of co-localization at both 3 and 5-h post prandial compared to pre-meal. No obvious difference in co-localization was observed when cells were treated by acLDL in vitro. More research is needed to determine how to best use this method to study pre-clinical risk of atherosclerosis.

Oral Supplementation with Baker's Yeast Beta Glucan Is Associated with Altered Monocytes, T Cells and Cytokines following a Bout of Strenuous Exercise

Exercise and physical labor in extreme environmental conditions causes transient decreases in immune cell and cytokine concentrations, likely increasing the susceptibility to opportunistic infection. Bakers yeast beta glucan (BYBG) has been previously demonstrated to be an effective countermeasure in athletes, but its effectiveness in individuals of average fitness under similar physical stress is unknown. The purpose of this study was to determine if 10 days of oral supplementation with BYBG could modify previously observed suppression of monocytes, T cells, circulating and whole blood LPS-stimulated cytokines due to strenuous exercise. Venous blood samples were collected from 109 healthy volunteers prior to, immediately after, 2 and 4 h post-exercise. Monocyte and T cell concentration, cell-surface receptor expression and serum and LPS-stimulated cytokines were assessed. BYBG significantly (P < 0.05) altered total and classic monocyte concentration and expression of CD38, CD80, CD86, TLR2, and TLR4 on monocyte subsets. BYBG also significantly increased CD4+ and CD8+ T cell concentration and the exercise response of CCR7+/CD45RA- central memory (TCM) cells. Likewise, BYBG significantly (P < 0.05) altered serum IFN-γ and IL-2, and LPS-stimulated IFN-γ, IL-2, IL-4, and IL-7. Taken together these data support the hypothesis that oral BYBG supplementation modulates the expected exercise response for individuals of average fitness. This may result in a decrease in susceptibility to opportunistic infections after strenuous exercise.

Measurement of T‐Cell Telomere Length Using Amplified‐Signal FISH Staining and Flow Cytometry

Exposure to pathogen‐associated molecular patterns (PAMPS), damage‐associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral infection and/or tissue injury. Thus, the ability to accurately assess cellular maturity and health is important to fully understand immune status and function. The most common technique for measuring cellular maturity is to measure telomere length; however, existing techniques are not optimized for single‐cell measurements using flow cytometry. Specifically, existing methods used to measure telomere length are PCR‐based, making it difficult for a researcher to measure maturity within specific leukocyte subsets (e.g., T cells). In this report, we describe a new approach for the measurement of telomere length within individual T cells using an amplified fluorescence in situ hybridization (FISH) technique (PrimeFlow RNA Assay). The unique aspect of this technique is that it amplifies the fluorescent signal rather than the target up to 3000‐fold, resulting in the detection of as few as 1 copy of the target nucleic acid. While the current technique focuses on human T cells, this method can be broadly applied to a variety of cell types and disease models.