Brian K. McFarlin

Baker's yeast beta glucan supplementation increases salivary IgA and decreases cold/flu symptomatic days after intense exercise.

ABSTRACT Strenuous exercise, such as running a marathon, is known to suppress mucosal immunity for up to 24 hr, which can increase the risk of developing an upper respiratory tract infection (URTI) and reduced performance capacity (Allgrove JE, Geneen L, Latif S, Gleeson M. Influence of a fed or fasted state on the s-IgA response to prolonged cycling in active men and women. Int J Sport Nutr Exerc Metab. 2009;19(3):209–221; Barrett B, Locken K, Maberry R, Schwamman J, Brown R, Bobula J, Stauffacher EA. The Wisconsin Upper Respiratory Symptom Survey (WURSS): a new research instrument for assessing the common cold. J Fam Pract. 2002;51(3):265; Carpenter KC, Breslin WL, Davidson T, Adams A, McFarlin BK. Bakers yeast beta glucan supplementation increases monocytes and cytokines post-exercise: implications for infection risk? Br J Nutr. 2012;1–9). While many dietary interventions have been used to combat postexercise immune suppression, most have been ineffective. The key purpose of this study was to determine if bakers yeast β-glucan (BG) could positively affect the immune system of individuals undergoing intense exercise stress using two experiments. In the first (E1; N = 182 men and women), BG was compared to placebo supplementation for the incidence of URTI symptoms for 28 days postmarathon. In the second (E2; N = 60 men and women) changes in salivary immunoglobulin A (IgA) were evaluated after 50-min of strenuous cycling when participants had been supplemented for 10 days with either BG (250 mg/day) or placebo (rice flour). For E1, subjects reported URTI symptoms using a daily health log. For E2, saliva was collected prior to, immediately, and 2-hr postexercise using a salivette. Data for E1 and E2 were analyzed using separate analyses of variance (ANOVAs) with repeated measures (p < .05). In E1, BG was associated with a 37% reduction in the number of cold/flu symptom days postmarathon compared to placebo (p = .026). In E2, BG was associated with a 32% increase in salivary IgA (p = .048) at 2 hr after exercise compared to placebo. In summary, the present study demonstrates that BG may reduce URTI symptomatic days and improve mucosal immunity (salivary IgA) postexercise.

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.

Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst

Granulocytes play a key role in innate immunity and the most common functional assays are phagocytosis and oxidative burst. The purpose of this technical note is to use image‐based flow cytometry to divide activated granulocytes into unique subsets based on their degree of phagocytosis and oxidative burst in response to different experimental incubations. Prior to the experiments, all reagents were titered to determine the lowest dose that resulted in an acceptable signal to noise ratio. Heparinized, whole blood (100 µl) was mixed with one of two bioparticles (E. coli and S. aureus) and DHE (10 µg/ml) and incubated for 5, 10, 20, 40, 60, 80, 100, 120, and 140 min in a 37°C water bath. An additional tube kept on ice was used as a negative control. All subsequent processing steps were completed on ice in the dark to minimize additional activation of cells. After the 37°C incubation, N‐ethylmaleimide (15 mM) was added to halt phagocytosis, preventing the uptake of additional microparticles. Suspensions were labeled with CD66b‐APC and CD45‐APCeFluor780 for 60 min and a fix/lyse solution was added. Prior to acquisition, 7AAD was added to stain nuclear DNA. A minimum of 5,000 granulocyte (CD66b+) events were acquired using a Millipore‐Amnis FlowSight equipped with blue (488 nm, 60 mW), red (642 nm, 100 mW), and side scatter (785 nm, 12 mW) lasers. Samples were compensated and analyzed using Amnis IDEAS software (v.5.0.983.0). Image‐based analysis allowed us to divide activated granulocytes into three distinct subsets, whose relative abundance changed as a function of both bioparticle type and incubation length. The method described in this technical note represents a potential novel adaptation to common methods of assessing granulocyte function. More research is needed to test and validate our image‐based method in clinical conditions that impair granulocyte function.

Mexican American Children Have Differential Elevation of Metabolic Biomarkers Proportional to Obesity Status

Objectives: There is a health disparity for obesity among Mexican Americans compared with other racial/ethnic groups. In particular, Mexican American children who are obese are likely to become obese adults. The purpose of this study was to examine traditional and nontraditional risk factors in a subset of Mexican American children before their participation in a larger clinical weight loss study. Methods: Venous blood samples were collected from self-identified Mexican American children (12–14 years old) who were assigned to 1 of 3 weight groups based on their standardized body mass index; normal weight (N = 66), overweight (N = 23), or obese (N = 39). Serum was analyzed for interleukin-6, tumor necrosis factor-&agr;, C-peptide, ghrelin, glucagon-like protein, gastric inhibitory polypeptide-1, glucagon, insulin, leptin, macrophage chemoattractant protein 1, and pancreatic polypeptide using a Luminex MagPix-based assay. Total cholesterol, high-density lipoprotein-cholesterol, triglycerides, and glucose were analyzed using enzymatic assays. Data were analyzed for significance using separate analysis of variance tests, with significance set at P < 0.05. Results: Relative to normal weight and overweight children, obese children had significantly elevated C-peptide (P < 0.0001), insulin (P < 0.0001), leptin (P < 0.0001), macrophage chemoattractant protein 1 (P = 0.005), and tumor necrosis factor-&agr; (P = 0.006). Conclusions: We observed that Mexican American children as a function of body weight had elevated serum concentrations of several biomarkers that have been linked to chronic disease development in adults. More research is needed to understand how these differences affect disease risk in adulthood.

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.

An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques

Chronic vascular disease is partially characterized by the presence of lesions along the vascular endothelial wall. Current FDA-approved clinical techniques lack the ability to measure very early changes in endothelial cell health. When endothelial cells are damaged, they release endothelial microparticles (EMPs) into circulation. Thus, blood EMP concentration may represent a useful cardiovascular disease biomarker. Despite the potential value of EMPs, current flow cytometry techniques may not consistently distinguish EMPs from other small cell particles. The purpose of this study was to use imaging flow cytometry to modify existing methods of identifying EMPs based on cell-surface receptor expression and visual morphology. Platelet poor plasma (PPP) was isolated using four different techniques, each utilizing a two-step serial centrifugation process. The cell-surface markers used in this study were selected based on those that are commonly reported in the literature. PPP (100μL) was labeled with CD31, CD42a, CD45, CD51, CD66b, and CD144 for 30-min in dark on ice. Based on replicated experiments, EMPs were best identified by cell-surface CD144 expression relative to other commonly reported EMP markers (CD31 & CD51). It is important to note that contaminating LMPs, GMPs, and PMPs were thought to be removed in the preparation of PPP. However, upon analysis of prepared samples staining CD31 against CD51 revealed a double-positive population that was less than 1% EMPs. In contrast, when using CD144 to identify EMPs, ~87% of observed particles were free of contaminating microparticles. Using a counterstain of CD42a, this purity can be improved to over 99%. More research is needed to understand how our improved EMP measurement method can be used in experimental models measuring acute vascular responses or chronic vascular diseases.

Flow cytometry what you see matters: Enhanced clinical detection using image-based flow cytometry

Image-based flow cytometry combines the throughput of traditional flow cytometry with the ability to visually confirm findings and collect novel data that would not be possible otherwise. Since image-based flow cytometry borrows measurement parameters and analysis techniques from microscopy, it is possible to collect unique measures (i.e. nuclear translocation, co-localization, cellular synapse, cellular endocytosis, etc.) that would not be possible with traditional flow cytometry. The ability to collect unique outcomes has led many researchers to develop novel assays for the monitoring and detection of a variety of clinical conditions and diseases. In many cases, investigators have innovated and expanded classical assays to provide new insight regarding clinical conditions and chronic disease. Beyond human clinical applications, image-based flow cytometry has been used to monitor marine biology changes, nano-particles for solar cell production, and particle quality in pharmaceuticals. This review article summarizes work from the major scientists working in the field of image-based flow cytometry.

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.