Taxifolin/Ergothioneine and Immune Biomarkers in Healthy Volunteers (TaxEr)

Study Overview
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Brief Summary
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The complexities of the immune system make measuring the impact of dietary interventions upon its function challenging. The immune system is highly responsive to environmental influences, including the diet. An individual's diet provides the energy required to mount a strong and protective immune response, the building blocks required for synthesis of immune mediators such as antibodies and cytokines, and can also indirectly affect immune function via changes in the gut microbiome. Immune function varies across the lifecourse, with a well understood decline in immune function with age, resulting in impaired vaccination responses and an increased risk of infections and of severe complications and mortality arising from common communicable diseases such as influenza. This impaired immunity with ageing is known as immunosenescence and this affects both innate and acquired arms of the immune system.

Detailed Description
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Expert guidance is available to inform the design of human nutrition trials to ensure they include the most relevant immunological outcomes (Albers, 2013). In this study, ex vivo phagocytosis and oxidative burst of immune cells will be the primary outcome, supported by other ex vivo immune measures of high clinical relevance including functional assessment of cytokine production and expression of activation markers. Human nutritional trials frequently omit to monitor the degree of immunosenescence in participants, even amongst studies conducted amongst older adults. For example, a recent review of pre- and probiotic trials which assessed immune responses in older adults identified that only two of thirty-six studies assessed any marker of immunosenescence (Childs & Calder, 2017). Taxifolin/DHQ is a naturally occurring polyphenol found in apples, onions and other fruits and bark extracts. Ergothioneine is an amino acid found in mushrooms, oats and some bean varieties. We hypothesise that Taxifolin/DHQ and/or Ergothioneine will alter immune function via their established antioxidant effects, and that the effects observed will vary between older adults relative to their degree of immunosenescence. Though current dietary guidelines advise consumption of 5 portions of fruits and vegetables per day, recent surveys reveal that fewer than 30% of adults achieve this. Antioxidants found within fruits and vegetables are understood to be one of the important aspects by which our diet can influence health. It is important to investigate the effects of such antioxidants through well designed and conducted human trials.

Official Title
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A Pilot Study of Dietary Taxifolin/Dihydroquercetin and Ergothioneine and Immune Biomarkers in Healthy Volunteers

Conditions
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Antioxidative Stress, Cold, Influenza, Aging, Inflammation

Intervention / Treatment
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* Dietary Supplement: Taxifolin
* Dietary Supplement: Ergothioneine
* Dietary Supplement: Control


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: age 50-65yr BMI 18.5-30kg/m2 Willing to avoid consumption of foods rich in Taxifolin/DHQ and Ergothioneine during the study period Willing to avoid taking any other food supplements or high doses of vitamins during the study period Able to provide written informed consent. Exclusion Criteria: Use of prescription medication which may influence immune function, such as anti-inflammatory or immunosuppressant medication Diabetes requiring any medication Liver cirrhosis A history of drug or alcohol misuse Asplenia or other acquired or congenital immunodeficiencies Any autoimmune disease including connective tissue diseases Malignancy Laboratory confirmed SARS-CoV-2 infection within last 3 months self-reported symptoms of acute or recent infection (including use of antibiotics within the last 3 months)

Ages Eligible for Study
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Minimum Age: 50 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: A n=90 study (3 treatment arms, each n=30) providing participants with either 250mg/day Taxifolin/DHQ, 80mg/day Ergothioneine, or control.
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Taxifolin/Dihydroquercetin<br>250mg/day Taxifolin (also known as Dihydroquercetin). One capsule in the morning for 8 weeks. | Dietary Supplement: Taxifolin<br>* A naturally occurring polyphenol found in apples, onions and other fruits and bark extracts.<br>* Other names: Dihydroquercetin;|
| Experimental: Ergothioneine<br>80mg/day Ergothioneine. One capsule in the morning for 8 weeks. | Dietary Supplement: Ergothioneine<br>* An amino acid found in mushrooms, oats and some bean varieties.<br>|
| Placebo Comparator: Control<br>One capsule in the morning for 8 weeks. | Dietary Supplement: Control<br>* Microcrystalline cellulose.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Phagocytosis activity by granulocytes ex vivo | Mean fluorescence intensity per cell will be assessed by flow cytometry. | 8 weeks post intervention | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Percentage phagocytosis by monocytes ex vivo | Percentage of cells undergoing phagocytosis will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Phagocytosis activity by monocytes ex vivo | Mean fluorescence intensity per cell will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Percentage phagocytosis by granulocytes ex vivo | Percentage of cells undergoing phagocytosis will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Phagocytosis activity by granulocytes ex vivo | Mean fluorescence intensity per cell will be assessed by flow cytometry. | 4 weeks, 3 months post intervention | 
| Percentage oxidative burst by monocytes ex vivo | Percentage of cells undergoing oxidative burst will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Oxidative burst activity by monocytes ex vivo | Mean fluorescence intensity per cell will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Percentage oxidative burst by granulocytes ex vivo | Percentage of cells undergoing oxidative burst will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Oxidative burst activity by granulocytes ex vivo | Mean fluorescence intensity per cell will be assessed by flow cytometry. | 4 weeks, 8 weeks, 3 months post intervention | 
| Frequencies of naive T cells | The proportion of naive T cells will be assessed by flow cytometry. | 8 weeks | 
| Frequencies of memory T cells | The proportion of memory T cells will be assessed by flow cytometry. | 8 weeks | 
| CD57 expression upon T cells. | The proportion of T cells expressing CD57 (a marker associated with chronic immune activation) and the mean fluorescence intensity per cell will be assessed by flow cytometry. | 8 weeks | 
| CD28 expression upon T cells. | The proportion of T cells expressing CD28 (a cell surface marker required for T cell activation and survival) and the mean fluorescence intensity per cell will be assessed by flow cytometry. | 8 weeks | 
| Plasma lipid peroxides | Participant plasma lipid peroxides will be measured by colorimetric analysis. | 8 weeks | 
| Urinary isoprostanes | Participant urinary isoprostanes will be measured by commercially available ELISA. | 4 weeks, 8 weeks, 3 months post intervention | 
| Plasma isoprostanes | Participant plasma isoprostanes will be measured by commercially available ELISA. | 4 weeks, 8 weeks, 3 months post intervention | 
| Cytokine production by cryopreserved peripheral blood mononuclear cells in response to lipopolyssaccharide | A panel of pro- and anti-inflammatory cytokines secreted by immune cells ex vivo will be assessed by Luminex array. | 4 weeks, 8 weeks | 
| Cytokine production by cryopreserved peripheral blood mononuclear cells in response to influenza or coronavirus vaccine products | A panel of pro- and anti-inflammatory cytokines secreted by immune cells ex vivo will be assessed by Luminex array. | 4 weeks, 8 weeks | 
| Metabolomic analysis of urine samples | Full metabolic profiling of first-morning urine samples will be used to assess changes to metabolic activity of participants and their microbiome. | 4 weeks, 8 weeks, 3 months post intervention | 
| Metabolomic analysis of serum samples | Full metabolic profiling of serum samples will be used to assess changes to metabolic activity of participants. | 4 weeks, 8 weeks, 3 months post intervention | 
| Faecal microbiome analysis | Sequences of ribosomal RNA (rRNA) in participant faecal samples will be measured to assess changes in the numbers or proportions of bacterial genera and species/strains. | 4 weeks, 8 weeks, 3 months post intervention | 
| Incidence of self-reported seasonal cold, coronavirus and influenza-like illness. | A daily online form will be completed by participants to log any seasonal cold, coronavirus and influenza-like illness. | 4 weeks, 8 weeks, 3 months post intervention | 
| Duration of self-reported illness. | A daily online form will be completed by participants to log any self-reported illness. | 4 weeks, 8 weeks, 3 months post intervention | 
| Severity of self-reported illness. | A daily online form will be completed by participants to log any self-reported illness. | 4 weeks, 8 weeks, 3 months post intervention | 
| Self-reported medication use. | A daily online form will be completed by participants to log any medication use. | 4 weeks, 8 weeks, 3 months post intervention | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Phagocytosis, Immunosenecence, Cytokine, Antioxidant, Metabolome, Microbiome