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The objective of this pilot study was to investigate the acute effects on circulating lymphocyte subsets, antioxidant status, and cytokine profile after consumption of EpiCor® (EP) (Embria Health Sciences, Ankeny, IA, USA), a dried fermentate produced from Saccharomyces cerevisiae, using a placebo-controlled randomized crossover study design with 12 healthy adult human subjects. EP contains high levels of bioavailable antioxidants and strongly activates natural killer (NK) cells in vitro. EP consumption has been shown to increase erythrocyte hematocrit levels, boost mucosal immune protection, reduce cold/flu symptoms, reduce seasonal allergy symptoms and the need for rescue medication, and increase salivary secretory immunoglobulin A levels. This warranted further study on immune effects in humans. A within-subject analysis of data collected before and at 1 and 2 hours after consumption of a single dose of 500mg of EP versus placebo was performed. A transient reduction in circulating T and NK cell numbers was observed 2 hours post-consumption, suggesting that homing and recirculation of these cells, as part of healthy immune surveillance, were supported by EP. The increased expression of activation markers on the CD3− CD56+ NK cell population was significant for CD69 at 1 hour post-consumption (CD25, P<.07; CD69, P<.05), whereas for CD25 it was significant at 2 hours after consumption (CD25, P<.03; CD69, P<.15). A rapid increase in serum interferon-γ was observed at 1 hour post-consumption (P<.07; after removal of two outlying data sets, P<.05) and may have contributed to the effects seen on NK and T cell subsets. Significant increase in serum antioxidant protection was seen 2 hours after consumption (P<.04). Thus consumption of a single 500mg dose of EP provides a rapid and transient effect on the trafficking and activation status of specific lymphocyte subsets, as well as increased antioxidant protection.
The multifaceted health implications of viral diseases span a vast spectrum throughout poverty-stricken as well as industrialized countries. Acute viral diseases include those due to rhinovirus and rapidly mutating influenza virus strains. Chronic viral infections include those due to hepatitis virus, human immune deficiency virus, and many omnipresent viruses such as the herpes family of viruses. The viral load, along with confounding factors, may lead to increased risks of development of autoimmune disease and other chronic illness or cancer in some individuals. There is also a growing suspicion that chronic obscure viruses may be involved, directly or indirectly, in chronic pain and fatigue syndromes.1–3
The role of natural killer (NK) cells in combating viral disease is known to involve many aspects of NK cell biology and interaction with other cell types such as macrophages and dendritic cells. The production of interferons (IFNs), combined with NK cell trafficking, is an important aspect of immune surveillance against viral infections. NK cells are also an important part of eliminating malignant cells,4,5 and reduced NK cell numbers and function are reported in many cancers.6
The immune surveillance activities of many lymphocyte subsets, including NK and T lymphocytes, are closely regulated by the central nervous system. There is a distinct circadian rhythm in levels and activity of T and NK cells in the blood circulation,7,8 including opposing effects on naive and memory T cell subsets, where naive cells appear in the blood circulation at low levels during sleep and a gradual increase in circulating cells during waking hours, and the opposite effect is seen for memory T cells.9 This suggests that immune surveillance, involving exit from blood circulation and migration through tissue, is regulated, and individual fluctuations must be taken into account when analyzing a nutritional product's effect on numbers of leukocyte subsets in blood circulation.
Furthermore, NK cell biology is directly affected by psychological stressors and sleep disturbances. Acute stress results in reduced lymphocyte trafficking and surveillance, whereas relaxation supports immune surveillance, seen as reduced levels of circulating NK cells. Sleep disturbances are involved in reduced NK cell function in healthy volunteers10 and accumulation of NK cells in the blood circulation in diabetics.11 There is a documented link among reduced NK cell function, low melatonin levels,12 and high cortisol levels.13 Therefore, studies on acute changes to NK activation status and trafficking need to be carefully controlled for environmental stressors.
Antioxidant uptake and protection in blood and tissue are important factors for immune health. Poor antioxidant status has been related to chronic inflammatory problems and cardiovascular disease.14 Bioavailability of antioxidants in vivo is the desired end point for antioxidant documentation for nutritional products. The Cell-based Antioxidant Protection in Erythrocytes (CAP-e) assay (patent pending, NIS Labs, Klamath Falls, OR, USA) has been shown to be useful for evaluating rapid changes in antioxidant status after consumption of a single dose of antioxidant-rich foods.15 The data reflect not only the uptake of antioxidants involved in blood circulation, but they also measure those antioxidants that are present in serum and able to enter into and protect cells from oxidative damage.16
The Saccharomyces cerevisiae–based dry fermentate nutritional product EpiCor® (EP) (Embria Health Sciences, Ankeny, IA, USA) has a high antioxidant content as evaluated by the chemical oxygen radical absorbance capacity assay17 and was shown to possess antioxidants capable of entering into and protecting live cells in vitro.16 Another yeast-based extract has similar immune-activating properties in vitro.18
EP has been used in previous studies relating to immune health and function. A 5-week placebo-controlled pilot study involved daily consumption of 500mg and showed trends of increased mucosal protection and reduced allergies.19 This was followed by three larger studies. Two 12-week, randomized, double-blinded, placebo-controlled clinical trials20,21 used 500mg/day and found that subjects had significantly fewer cold/flu symptoms than subjects taking placebo. Another 12-week, randomized, double-blinded, placebo-controlled study investigated the effects of EP on allergic rhinitis:22 it found EP to significantly reduce the mean severity of specific allergic rhinitis symptoms. Significant outcomes observed included reduced nasal congestion and rhinorrhea and self-reported parameters such as total number of days with nasal congestion and nasal secretions on a quality of life questionnaire, as well as reduced use of rescue medication.22
The purpose of the clinical study reported here was to expand on previous in vitro and clinical work examining the effects of EP on immune cells in vivo, with a special focus on NK cell activation, antioxidant status, and cytokine profile.
Phosphate-buffered saline (PBS) (pH 7.4), sodium azide, gallic acid, bovine serum albumin, Histopaque® 1077, and Histopaque 1119 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dichlorofluorescein diacetate was obtained from Molecular Probes (Eugene, OR, USA), a subdivision of Invitrogen (Carlsbad, CA, USA). 2,2′-Azobis-(2-amidinopropane) dihydrochloride (AAPH) was obtained from Wako Chemicals (Richmond, VA, USA). The S. cerevisiae–based fermentation product EP was obtained from Embria Health Sciences. The placebo was encapsulated white rice flour, shown to possess no measurable antioxidant protection of live cells in vitro. For the clinical study, both EP and placebo were encapsulated in non-transparent blue capsules in order to blind subjects and clinic staff.
The following monoclonal antibodies were purchased from Becton-Dickinson (San Jose, CA, USA): CD25–fluorescein isothiocyanate (FITC), CD34-FITC, CD45-FITC, CD69-FITC, CD56-phycoerythrin, CD3–peridinin-chlorophyll-protein complex (PerCP), CD45-PerCP, and isotype control antibodies.
Two different types of consumables were prepared: EP and placebo. EP is a proprietary high-metabolite S. cerevisiae–based dried nutritional fermentate. As a placebo, white rice flour was used. Because EP is dark brown and the rice flour is white, non-transparent, blue capsules were used for encapsulating. Thus, at each visit, each person would consume a single blue capsule in the presence of the study coordinator. Study participants, nurses, and laboratory assistants were blinded so nurses and staff interacting with the study participants, as well as the staff processing samples, conducting tests, and performing data entry and analysis, were unaware of the content of capsules.
A double-blinded randomized placebo-controlled crossover study design was used (Fig. 1). Twelve people were recruited for the study upon informed consent approved by the Sky Lakes Medical Center (Klamath Falls) Institutional Review Board (protocol number FWA 2603) (Table 1).
The following exclusion criteria were used: under 18 or over 55 years of age, pregnancy, severe asthma and allergies requiring daily medication, any known chronic illness, frequent recreational drug use, impaired digestive function (including previous major gastrointestinal surgery), and allergies to yeast.
The volunteers were scheduled on two study days at least 2 weeks apart. Testing was always performed at the same time of the day (8–11 a.m.) to minimize the effect of circadian fluctuations. Because of the interference from exercise23 and stress9,24–26 with the release versus homing of lymphocytes, effort was taken to minimize any physical and mental stress prior to and during testing. In addition, on each study day, volunteers were instructed to complete a questionnaire aimed at monitoring any exceptional stress-related circumstances that might affect the person on that particular study day. Predetermined criteria for exclusion from data analysis included sleep deprivation and acute anxiety. After completing the questionnaire, volunteers were instructed to remain quiescent for 3 hours, comfortably seated in a chair. After the first hour, the baseline blood sample was drawn. Immediately after the baseline sample was drawn, a consumable was provided with water. Blood samples were drawn at 1 and 2 hours after ingestion of the consumable. At each blood draw, 8mL of blood was drawn into serum separator tubes: 6mL of blood was drawn into heparin, and 2mL blood was drawn into EDTA. Serum was harvested and kept frozen at −80°C until testing for antioxidant status and cytokine profile. The blood drawn into EDTA was used for obtaining a complete blood count (CBC) with differential, using a Coulter counter (Micro Diff II, Beckman Coulter, Brea, CA, USA). All CBCs were performed in triplicate within an hour of drawing the sample.
The EDTA samples were used to obtain triplicate CBCs with differential. These data were used to examine whether EP consumption affects the numbers of white blood cells, granulocytes, erythrocytes, monocytes, and lymphocytes. The data also served to calculate numbers of circulating T cells, NK cells, and stem cells, by merging flow cytometric data with the CBC data.
Freshly isolated peripheral blood mononuclear cells were placed in a V-bottom 96-well plate (NUNC, Copenhagen, Denmark) and washed in IF buffer (PBS containing 1% bovine serum albumin and 0.02% sodium azide). Cells were resuspended in 0.05mL of IF buffer, monoclonal antibodies were added, and plates were incubated in the dark at room temperature for 10 minutes. The cells were washed twice with an additional 0.15mL of PBS with 0.02% sodium azide. Following centrifugation and aspiration of supernatant, cells were resuspended in 0.05mL of PBS with 0.02% azide and transferred to 5-mL polystyrene tubes each containing 0.4mL of 1% formalin. Samples were stored in the dark and acquired by flow cytometry within 24 hours using a FACSCalibur™ flow cytometer (Becton-Dickinson). Files of 200,000 events were collected on each triplicate sample. The percentages of CD3+, CD3− CD56+, and CD34+ CD45dim subsets were analyzed separately and were analyzed again after multiplying with the lymphocyte cell counts, as obtained from the average of the triplicate lymphocyte counts obtained by the CBC with differential. For the CD3+ and CD3− CD56+, subsets, the expression of the two activation markers CD69 and CD25 was evaluated. Analysis was performed using FlowJo (Tree Star Inc., Ashland, OR, USA) software.
Gallic acid was prepared immediately prior to use by weighing 425mg into methanol (5mL). Gentle agitation was performed until all crystals were dissolved, and 0.4mL was transferred to PBS (49.6mL).
Red blood cells (RBCs) were obtained from a healthy human volunteer after informed consent, as approved by the Sky Lakes Medical Center Institutional Review Board. Peripheral venous blood was drawn into sodium heparin and layered onto a double-gradient of Histopaque 1119 and 1077. The vials were centrifuged and spun at 2,400rpm using a four-place swinging rotor for the IEC Centra MP4 centrifuge (Thermo Fisher Scientific, Waltham, MA) for 25 minutes. The peripheral blood mononuclear cell–rich upper interface and the polymorphonuclear neutrophil–rich lower interfaces were removed using sterile transfer pipettes. Packed RBCs were washed twice in PBS without calcium or magnesium and spun at 2,400rpm using a four-place swinging rotor for the IEC Centra MP4 centrifuge (Thermo Fisher Scientific) for 10 minutes. Then the core of the packed RBCs was transferred into new vials and again washed twice in PBS without calcium or magnesium and spun at 2,400rpm using a four-place swinging rotor for the IEC Centra MP4 centrifuge (Thermo Fisher Scientific) for 10 minutes. Aliquots were prepared by transferring packed RBCs (0.2mL) into 3mL of sterile PBS in sterile vials. Aliquots were stored at 4°C until use.
An aliquot of RBCs was washed twice in physiological saline. A cell suspension was then prepared for the CAP-e assay by adding packed RBCs (0.1mL) to PBS (10mL). The cell suspension was distributed in a V-bottom 96-well microtiter plate. Twelve wells were not treated with any source of antioxidants and served as negative controls (six wells) and positive controls (six wells) for minimum versus maximum oxidative damage. Twelve wells were treated with a standard source of a known antioxidant (gallic acid) across six different serial dilutions, where each dilution was tested in duplicate. The remaining wells were treated with serum, where each serum sample collected during the study was tested in quadruplicate. Cells were incubated with serum for 20 minutes in the accelerated CAP-e assay protocol. Antioxidants not absorbed by the cells were removed by washing twice in PBS by centrifuging at 2,400rpm for 2.5 minutes using a four-place swinging rotor for the IEC Centra MP4 centrifuge (Thermo Fischer Scientific). The cells were lysed, and the precursor dye was added to the wells. Incubation was performed at room temperature for 15 minutes. Oxidative damage was introduced using the peroxyl free radical generator AAPH for 1 hour. The green fluorescence intensity, as a measure of oxidative damage, was measured at 488nm using a Tecan (Zurich, Switzerland) Spectrafluor plate reader (40 flashes, optimal gain). Analysis was performed where the mean fluorescence intensity (MFI) of cells treated with serum was compared with that of cells treated with AAPH alone, to establish the cellular antioxidant protection provided by each serum sample.
Serum separator tubes were allowed to sit for 60 minutes at room temperature to facilitate proper coagulation; the vials were then centrifuged at 3,000rpm using a four-place swinging rotor for the IEC Centra MP4 centrifuge (Thermo Fisher Scientific) for 15 minutes. Serum was harvested using a sterile transfer pipette and aliquoted before freezing at −80°C. The serum was used for cytokine analysis where the relative levels of the six cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, and IFN-γ were measured using a flow cytometry-based bead array kit (human T helper [Th] 1/Th2 cytokine kit II, BD Biosciences, San Jose). Samples were tested in duplicate according to the manufacturer's specifications, and data were acquired immediately by flow cytometry, using a FACSCalibur flow cytometer. The analysis was performed using FlowJo software.
Statistical analysis involved simple comparisons between two mean values and was performed using Microsoft (Redmond, WA, USA) Excel. Statistical significance was tested using Student's t test with a P value of <0.5 indicating a significant difference between two data sets.
No statistically significant changes were seen in the absolute or relative changes in the number of white blood cells (leukocytes), RBCs (erythrocytes), or granulocytes, when comparing baseline data with 1-hour and 2-hour blood draws after consumption of EP versus placebo using a paired t test (Table 2).
Using the lymphocyte counts obtained by the CBC, combined with the evaluation of lymphocyte subsets from the flow cytometry analysis, specific changes were seen among the CD3+ T cell and the CD3− CD56+ NK cell subsets. The number of circulating CD3+ T lymphocytes decreased at 1 hour in the EP group when compared with placebo, and further decrease was seen at 2 hours, at which time the difference between changes after consumption of placebo and EP was highly significant (P<.01) (Table 2 and Fig. 2). The number of NK cells showed a trend toward reduction in the EP group when compared with the placebo group at 2 hours post-consumption. With the removal of two data sets from the analysis, the within-subject analysis revealed statistical significance (P<.05) (Table 2 and Fig. 3).
A mild reduction was seen for the numbers of CD34+ circulating stem cells at 2 hours; however, the observation did not reach statistical significance (P<.15) (Table 2).
The CD3+ T cell population and the CD3− CD56+ NK cell population were analyzed for expression of the two activation markers CD25 and CD69. No significant changes were seen on the T cells. In contrast, both markers were expressed at higher levels on NK cells after consumption of EP than after consumption of placebo. The relative increase in expression of the CD25 marker on NK cells is shown in Figure 4. This effect was already seen at 1 hour after consumption (P<.07) and reached statistical significance at 2 hours after consumption (P<.05). The relative increase in expression of the CD69 marker on NK cells is shown in Figure 5. This effect was already statistically significant at 1 hour after consumption (P<.05) but was returning to normal at 2 hours after consumption (P<.15).
We used the CAP-e assay to test in vivo antioxidant protection capacity of EP. The serum samples were tested in an accelerated CAP-e assay protocol using freshly isolated erythrocytes. Each serum sample was tested in quadruplicate. The data from each person's baseline serum sample were indicative of this person's antioxidant protection capacity before consumption of either EP or placebo. The level of oxidative damage in samples where cells were treated with post-consumption serum samples was compared with the oxidative damage in samples exposed to baseline serum. Each person's start level was set to “0,” and the relative change (increase, decrease) in serum antioxidant protection capacity was calculated. The average and SD values are shown in Figure 6. Consumption of 500mg of EP resulted in a statistically significant increase in antioxidant protection (P<.04).
Serum was analyzed for the panel of Th1/Th2 cytokines IL-2, IL-4, Il-6, IL-10, tumor necrosis factor-α, and IFN-γ, using a cytokine bead array for flow cytometry. Each person's changes after consumption of placebo versus EP were evaluated, and the normalized data for the study population are shown in Table 3. There was a trend to reduced levels of IL-6 and IL-10 at 2 hours, when comparing each person's response to EP versus placebo. The changes did not reach statistical significance for either IL-6 (P<.1) or for IL-10 (P<.2). A transient increase in IFN-γ was seen at 1 hour (P<.07), but levels returned to normal at 2 hours. The increase in IFN-γ was re-analyzed after removal of two outliers, and the re-analysis of the remaining 10 subjects reached statistical significance (P<.05).
In this study we have shown evidence for rapid changes in serum antioxidant status, cytokine levels, and immune surveillance after consumption of a single dose of the immunogenic yeast product EP, compared with placebo. This is a direct extension of our previous publication showing direct activation of NK cells in vitro17 and in vivo.19 These combined data may help explain data from other clinical studies where reduced incidence and duration of cold/flu were observed.20,21
The acute effects of EP consumption included a highly significant decrease in numbers of circulating T lymphocytes, and a trend toward a reduced number of circulating NK cells suggests that ingestion of a single dose of EP led to increased T and NK cell trafficking and homing into tissue. The T and NK cells observed in the circulation at 1 and 2 hours after consumption had already increased expression of activation markers at 1 hour after consumption. The expression of the IL-2 receptor CD25 and of the marker CD69, associated with increased cytotoxic responsiveness, was increased on the surface of circulating NK cells. An increase in serum IFN-γ levels was seen at 1 hour, which did not reach statistical significance (P<.07). However, the trend to increased IFN-γ content may be causative for the observed effects on NK cell activation and trafficking (Fig. 7). It is important to note that the increases in IFN and cytokine levels are mild and do not reflect a pathological state of inflammation. Our previous placebo-controlled 5-week study of EP consumption in healthy people showed trends toward increased IL-4, IL-6, and IL-10.19 This is in contrast to the acute effects reported here, where a mild reduction is seen in serum levels of IL-6 and IL-10. It is possible that the data reflect differential acute versus cumulative effects of EP consumption.
The data presented here were generated by using a study protocol that was carefully planned to be able to control for normal circadian variations in levels of circulating leukocytes. The increase in total T cell numbers seen when placebo was consumed may reflect normal circadian rhythms, as it has been reported that T cell numbers are at a low level early in the morning and start to increase during the morning until noon.9,26 Our study protocol also aimed at a clinical study environment that controlled for psychological stress because acute stress negatively affects the biology of NK cells.
It is interesting that a trend toward decreased numbers of circulating CD34+ stem cells was observed (P<.12). This mild trend may signify a transient induction of stem cell homing into tissues. Previously, consumable plant- and microbial-based products and pure compounds such as fucoidan have led to changes in circulating stem cells, in part due to changes in chemokine expression.27,28 EP was shown by our team to compete with fucoidan-induced changes to expression levels of several chemokine receptors on leukocytes.17 The observation may warrant further research on the effects of EP on human stem cell biology, including trafficking, such as we documented for extracts from another microbial-based food, namely, the microalga Aphanizomenon flos-aquae.28 It is possible that the effects are direct and may be linked to the existence of a selectin ligand in EP.17 Alternatively, the effect may be indirect and associated with anti-inflammatory effects of EP, reducing the negative impact of inflammation on stem cell phenotype and function.
We found a statistically significant improvement in serum antioxidant protection capacity after ingestion of 500mg of EP. This may reflect the content of easily absorbed antioxidants existing in the native product, as well as antioxidant compounds released or generated as a result of normal digestive processes. Unknown metabolites may contribute significantly to the in vivo antioxidant protection of EP. By using the CAP-e assay to evaluate the serum capacity for antioxidant protection, such metabolites do not need to be chemically identified; as long as they are able to enter and protect living cells from oxidative damage they contribute to the CAP-e results. This is in contrast to other types of bioavailability studies, where chemical analysis is performed on serum to examine the sample for metabolites known to have come from the digestion of a natural product.
The data presented here involved a healthy study population. Future work should address lymphocyte subsets in a broader manner, by including staining for the major T cell subsets including T-regulatory cells, as well as staining for B cells and dendritic cells. Further clinical work should address whether EP consumption may support immune surveillance in populations experiencing stress and sleep disturbances. EP consumption may hold promise as a nutritional immune support in healthy subjects under acute stress, as well as for patients with specific chronic illnesses affecting innate and adaptive immune defense mechanisms.
The authors would like to thank Charlene Mogle, Wendy Zolczynski, Dana Honzel, Ariston Vallejos, and Carly Cox for their excellent technical assistance. The study was performed at NIS Labs, an independent contract research lab specializing in natural products research, and sponsored by Embria Health Sciences Inc.
G.S.J., K.A.R., K.F.B., S.G.C., and M.A.M. are employees of NIS Labs. S.R. and L.R. are employees of Embria Health Sciences Inc.