Juice Plus Inflammaging and Cardiovascular Disease Prevention Study
Juice Plus Inflammaging and Cardiovascular Disease Prevention Study
Low- grade inflammation is a pathological feature of a wide range of chronic conditions,
 including the metabolic syndrome, cardiovascular diseases (CVD) and the accelerated reduction
 in bone density. Previous research shows that diets rich in fruits and vegetables can reduce
 chronic inflammation. To date there is no data on multiyear clinical interventions assessing
 the effect of plant-based dietary supplements on low-grade inflammation, cardiovascular
 disease prevention and indicators of biological aging, including individuals' cognitive
 function. In this study, the investigators are thus exploring whether separate ingestions of
 two plant-based nutritional products over 2 years, are able to modulate low-grade
 inflammation, parameters of CVD prevention, circulating micronutrients, upper respiratory
 tract- and gastro-intestinal symptoms, quality of life, indicators of biological aging, and
 cognitive function in overweight seniors.
Inflammation is a central component of innate (non-specific) immunity. The termination of
 inflammation is an active process involving cytokines and anti-inflammatory mediators like
 lipids and micronutrients. Inflammation acts as both, 'friend and foe'; it is an essential
 component of immunosurveillance and host defense, but a chronic low-grade inflammatory state
 is a pathological feature of a wide range of chronic conditions, such as the metabolic
 syndrome or cardiovascular diseases (CVD). In addition, low-grade inflammation and oxidative
 stress are often associated with increased body fat mass, obesity but also accelerated
 reduction in bone density.

 There is a substantial amount of evidence to suggest that many foods, nutrients and
 non-nutrient food components modulate inflammation both acutely and chronically. Nutritional
 regimens with adequate intake of micronutrients, vegetables, and fruits, low in sugar and
 saturated fats, like the Mediterranean diet or a vegetarian dietary regimen, can reduce
 chronic inflammation and oxidative stress.

 To date there is no data on multiyear clinical interventions assessing the effect of
 plant-based dietary supplements on low-grade inflammation, cardiovascular disease prevention
 and indicators of biological aging, including individuals' cognitive function. In this study,
 the investigators are thus exploring whether separate ingestions of two plant-based
 nutritional products over 2 years, are able to modulate biomarkers of low-grade inflammation
 and CVD prevention, plasma concentrations of micronutrients, upper respiratory tract- and
 gastro-intestinal symptoms, quality of life, indicators of biological aging, and cognitive
 function in an overweight/obese cohort of middle-aged, elderly people.

 Volunteers expressing interest to take part in the study, will need to attend a screening
 visit where their eligibility will be assessed. For participants with confirmed eligibility,
 they will need to attend a baseline visit and consecutive study visits at 6, 12, 18 and 24
 months. Markers of low-grade inflammation and CVD, micronutrients status, respiratory tract
 symptoms, gastrointestinal symptoms and quality of life will be assessed at baseline, 6, 12,
 18 and 24 months. Bone quality and telomere length will be assessed at baseline, 12 and 24
 months.

 Propensity score approach:

 Since this is a long-term trial and in order to maximize adherence to the ingestion of the
 products, volunteers will be allowed to choose their preferred nutritional product. Instead
 of using randomization, the investigators have chosen a propensity score (PS) approach which
 helps to reduce bias with regards to random significances. More specifically, for each
 participant an individual score based on certain parameters, likely to affect the primary
 outcomes, will be calculated and will be matched with another participant with the same PS
 across the three groups. PS matching will be implemented without replacement and setting the
 caliper equal to 0.025. Furthermore, to ensure an adequate number of matching PSs between
 groups, about a 3-fold number of subjects needed to enter the study, will be pre-screened.

 Sample size:

 With a sample size of 20 subjects per group (total sample size = 60), the disjunctive power
 for testing each primary endpoint (i.e., the probability of establishing a significant effect
 in supplementation-control or between-supplementation comparisons) is 78%, 51%, 97% for
 TNF-α, homocysteine and vitamin C (1st ranked parameter for each co-primary outcome),
 respectively.

 In addition, it is estimated to have a maximum drop-out rate of 30% over 2 years. In order to
 ensure balanced distribution of subjects across the three different groups, the investigators
 will also stratify for gender and age. Based on this sample size calculation and in order to
 meet the stratification standards, 30 subjects will be recruited to be allocated to each
 group, corresponding to a total N of 90 subjects.

 Statistical analysis:

 Statistical analysis will be performed by using SPSS for Windows software, version 22.0.
 Metric data will be presented as mean ± SD. Statistical significance is set at P < 0.05. The
 Shapiro-Wilk test will be used to determine normal distribution. To check homogeneity of
 variances the Levene test will be used. Comparisons of mean values of metric baseline data
 between the 3 groups will be done by analysis of variance, ANOVA.

 If data are normally distributed and variance homogeneity is fulfilled, all metric analytes
 from blood (low-grade inflammation markers, CVD-prevention markers, micronutrients, telomer
 length, clinical chemistry etc) will be analyzed by one- and two-factorial (either 'time' or
 'time x treatment') repeated measures analysis of variance (ANOVA) and co-variance (ANCOVA,
 e.g. diet/dietary inflammatory index (DII), exercise), within each group and between groups.
 Student's t-test for paired samples will be used for within group analysis as soon as 6-month
 data are available. For post-hoc analyses the Bonferroni(-Holm) correction and/or Tukey´s
 post-hoc test will be used.

 If it is not possible to use metrical data, non-parametric tests will be used like the
 Friedman Test (within group) and the Kruskal Wallis test (between groups). If differences
 between groups reach significance the Tukey's post hoc test, the Bonferroni correction or the
 Bonferroni-Holm-method (for non-parametric data) will be used to determine the localization
 of the differences.

 A comprehensive correlation analyses to compute relations within each outcome-category and
 between the different outcome-categories will be conducted, also the PS-categories will be
 included into these correlation analyses.
Inclusion Criteria:

 -  90 male and female subjects

 -  age: 50 - 80 years

 -  post- or peri-menopausal

 -  Smokers and non-smokers

 -  BMI 25 to 40 kg/m2

 -  Dietary Inflammatory Index, DII: 0 to +10

 -  Fruit and vegetable intake <4 servings/d

 -  Adherence to a 6-week "wash-out" period

 -  Since the intervals between blood drawings are long (6 months), a temporary intake
 (e.g for a few days, one or two weeks) of some excluded drugs and food supplements
 does not necessarily mean exclusion from the study

 Exclusion Criteria:

 -  Age <50 and >79.9 years

 -  Dietary Inflammatory Index, DII: - 0.1 to -10

 -  Subjects with any kind of food allergy or histamine intolerance

 -  Aversion to stop the intake of nutritional supplements and food, that could interfere
 with the study outcome

 -  Food supplements, functional foods and dietetic products with anti-inflammatory or
 redox-biological relevance like omega-3 fatty acids, plant/herbal
 extracts/concentrates, vitamin- and mineral supplements

 -  Fruit and vegetable intake >3 servings per day

 -  Hypertension, starting with grade 2 according to the classification of the European
 Society of Hypertension: systolic blood pressure > 160 mmHg, diastolic blood pressure
 >100 mmHg

 -  Medication: any anti-inflammatory medication and medication with relevant antioxidant
 properties, blood pressure lowering medication, psychotropic drugs,
 immunosuppressives, cytostatics, anticoagulants, contraceptives, diuretics, pain
 medication

 -  Clinically relevant infectious disease

 -  Diabetes mellitus type I and type II

 -  Auto-immuno diseases

 -  Any stents and Coronary artery diseases (CAD)

 -  Cancer patients

 -  Pregnancy