Design and conceptual framework

HEBE is a prospective cohort started in 2022, to evaluate the role of physical exercise in modulating the inflamm-aging by determining an impact on health and on the risk of developing NCD. The study has been registered in ClinicalTrials.gov (Identifier: NCT05815732; posted on April 18, 2023). The authors confirm that all ongoing and related trials for this intervention are registered.

The applied bottom-up approach involves the cooperation of 94 researchers in life sciences and medicine at the University of Milan, to create a synergy that holistically approaches the concept of inflamm-aging and evaluates its reduction by physical exercise, integrated by lifestyle changes.

Informed consent has been and will be obtained from all study subjects before their participation. This essential ethical principle was carefully observed throughout the recruitment and data collection phases of our study. Every participant was fully informed about the nature of the research, the procedures involved, and the potential risks and benefits. All methods employed in our study were conducted in strict accordance with the relevant guidelines and regulations. HEBE received Ethics Approval from the University of Milan Ethical Committee (approval 62/22, date 30/06/ 2022).

The overall project includes 10 Lines of Research (LR) as described:

LR1 ("proof of concept"), which is currently ongoing, proposes a lifestyle modification protocol to all University of Milan employees and will be used to evaluate the changes obtained in terms of quality of life and psychophysical well-being using questionnaires. LR1 is focused on physiological mechanisms and thus the recruitment involves only healthy subjects (accounting for cardiometabolic risk factors). We will broaden and deepen the study of biological processes at the basis of the senescence processes of the human organism, and in particular, we will define how these mechanisms change about the systemic and tissue / organ-specific inflammatory state, with physical exercise. The choice of measures that are carried out in the proof of concept, has been focused on the assumption that all levels of biological organization need to be investigated, from molecular, cellular, tissue, and organ studies, to studies that investigate model organisms as a whole (e.g., mouse model, zebrafish).

Concepts defined in LR1 will be then applied to 9 additional LRs, declining the common background of inflamm-aging and evaluating the effect of physical exercise on additional sub-cohorts according to the following lines of research (Fig 1).

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Fig 1. Conceptual framework for the HEBE research.

The concepts established in Line of Research (LR) 1 (“proof of concept”) serve as the foundation for investigating nine additional LRs. These LRs focus on distinct aspects of inflamm-aging and explore the impact of physical exercise on various sub-cohorts. The planned lines of research are as follows: LR2: Obesity and Comorbidity Development; LR3: Cancer and Inflamm-Aging; LR4: Endocrine Factors in Fragility; LR5: Cardiovascular Disease in Inflamm-Aging; LR6: Inflamm-Aging and Human Fertility; LR7: Psychological Stress and Inflamm-Aging; LR8: Respiratory Diseases and Inflamm-Aging; LR9: Neuroinflammation, Aging, and Frailty; LR10: Autoimmune Diseases and Inflamm-Aging. Each line of research extends from the foundational LR1, collectively contributing to a comprehensive understanding of inflamm-aging and its diverse implications.

https://doi.org/10.1371/journal.pone.0300011.g001

• LR2: Obesity and development of comorbidities;
• LR3: Cancer;
• LR4: Endocrine origin of fragility;
• LR5: Cardiovascular diseases;
• LR6: Human fertility;
• LR7: Psychological stress;
• LR8: Respiratory diseases;
• LR9: Neuroinflammation associated with aging and frailty;
• LR10: Autoimmune diseases.

Subjects enrolled in LR1—Phase 1

A lifestyle modification protocol has been proposed to all University of Milan employees, by sending an email from and to the institutional accounts. The University of Milan (“Università degli Studi di Milano”, UNIMI) is a founder and the only Italian member of the prestigious League of European Research Universities (LERU). UNIMI employees include 2605 teachers and 1872 technical and administrative staff. Those willing to adhere to the protocol have been asked to fill in at time zero (T0) a questionnaire that will investigate lifestyle habits (physical activity, sedentary lifestyle, nutrition, smoking, perception of stress, sleep, alcohol, etc.), and clinical individual characteristics (November 8, 2022, starting of the recruitment period, expected to end December 31, 2024). After answering the questionnaire questions, participants are asked to adhere as much as possible to specific recommendations in the following 6 months. Suggestions for a lifestyle change towards healthy habits are delivered to participants through texts published on the HEBE website (https://hebe.unimi.it), which has been specifically created and curated by researchers of the University of Milan with specific expertise in the topics of interest, to guarantee that all recommendations, indications, and messages delivered are based on scientific data and evidence-based medicine (EBM). Specifically, the HEBE website has been structured with a dedicated section named “live healthy”, including specific information related to physical activity, nutrition, psychophysical well-being (e.g., sleep habits, stress management, stopping smoking) and simple tools for the qualitative self-measure of health basic parameters, such as Body Mass Index, waist circumference, Healthy Diet Score, etc. This information is also provided weekly in the HEBE Social Media official Instagram profile (@hebe_unimi).

After 6 months (T1), each subject is invited to fill in again the same questionnaire, integrated with additional questions to ascertain the subjects’ compliance. This second assessment is aimed at investigating the changes obtained in terms of lifestyle, quality of life, and psychophysical well-being. So far (September 20, 2023), 983 subjects filled out the questionnaire (21.9% of the involved UNIMI personnel).

At the end of this questionnaire (T0), the subjects also have the opportunity to express their willingness to be part of a personalized medical intervention (positive answer: n = 390, see Phase 2).

Sample size

For sample size assessment [19–22] we focused on the primary objective and the endpoint related to the variation of the measures of VO₂ max over the observation period. Using a Student’s t-test for paired data with a level of significance α = 0.050, a sample size of 100 pairs of pre- and post-intervention data reaches 80% power to reject the null hypothesis of no change in VO₂ max when the value of the effect size under alternative hypothesis is 0.28. The effect size is defined as d = (μ₁ –μ₂) / σ where σ the standard deviation of the population of pre vs post differences. According to Cohen (1988), the present effect size would be considered between low (0.2) and medium (0.5) reference values and therefore suitable for the needs of the study.

Subjects enrolled in Phase 2

Enrolment criteria of this subgroup were: i) absence of myocardial infarction, neurodegenerative and autoimmune diseases; ii) absence of cardiac arrhythmias; iii) absence of osteo-muscular problems that prevent the execution of physical exercise; iv) not pregnant / breastfeeding; v) absence of psychiatric pathologies; vi) absence of oncological pathologies in the last 3 years treated with chemotherapy and immunotherapy). The age range of the participants who have expressed their willingness to participate to the personalized physical exercise protocol spans from 30 to 70 years. All the subjects gave their written informed consent to the study.

Among all the subjects who meet the inclusion criteria, participants will be divided according to the following grouping criteria (strata):

1. Stratification by age (50% <50 years; 50% ≥50 years);
2. Stratification by sex (50% females; 50% males);
3. Stratification by Body mass index (50% with BMI <25; 50% with BMI≥25).

Within each stratum, the final selection of the 100 volunteers is carried out based on the order of joining the study (Fig 2). These 100 volunteers are invited to a preliminary online information meeting with the investigators of the study to verify their actual intention to actively participate in all phases of the project, as described below.

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Fig 2. Subgroup selection process for Phase 2 participants of the HEBE project.

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A similar approach will be defined also in the next phases of the Project (beginning in 2024), as we will recruit additional subgroups of subjects with the health conditions defined in the LR2-10.

During the first visit to set up the intervention program, all subjects enrolled in Phase 2 will be asked to fill in other questionnaires relevant to the aim of the study, to collect more detailed information, concerning specific aspects not investigated by the questionnaire administered in Phase 1.

In particular:

• Mental health continuum (MHC SF) questionnaire
• The hospital anxiety and depression scale (HADS) questionnaire
• Epworth Sleepiness Scale (ESS) questionnaire
• Pittsburgh Sleep Quality Index (PSQI) questionnaire
• Fertility questionnaire (developed ad hoc for the present study).

Biospecimen collection

The subjects who are enrolled in the personalized medical protocol are asked to donate 31 ml of blood (15 ml for biochemical analyses and 16 ml for circulating biomarker analyses), 15 ml of urine, a saliva sample, a nasal swab, and a blood card, a few days before (3–7 days) the first visit to set up the personalized protocol of physical exercise-based intervention. All samples are taken in close succession, after an overnight fasting period. Fasting blood samples are collected by a trained nurse by venipuncture in Ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson, Franklin Lakes, NJ, USA) between 9 and 10 a.m. and processed within 2 h from the phlebotomy. EDTA blood (16 ml) is centrifuged at 1200g for 15 min at room temperature to obtain plasma. Fig 3 shows how the sample is aliquoted and stored.

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Fig 3. Sample collection and processing workflow for participants enrolled in the personalized medical protocol.

https://doi.org/10.1371/journal.pone.0300011.g003

A 50 ml urine sample is also collected and frozen at -20°C within 2 hours. Saliva samples are self-collected by participants using Salivette® (Sarstedt S.R.L., Nümbrecht, Germany). Briefly, the subject removes the swab from the Salivette and places the swab in the mouth, and chews it for about 60 seconds to stimulate salivation. The swab with the absorbed saliva is then returned to the Salivette and the stopper is replaced immediately. A clear saliva sample in the conical tube is obtained by centrifugation for 2 minutes at 1,000 x g yields. Nasal microbiome collection is self-performed through nasal swabs by each subject. The swabs are then immediately placed in a tube of Universal Transport Media (D.I.D. Diagnostic International, Milan, Italy) and frozen at -20°C until DNA extraction. Finally, a drop of capillary blood is obtained from each subject by piercing the fingertip with a lancet device (Sterilance Safetylite III, VWR International S.r.l., Milan, Italy). A piece of filter paper (Whatman Protein Savercards, 903, Merck, Darmstadt, Germany) is then applied to the finger. The blood sample is dried at room temperature and then stored at -80°C until further analyses.

Variable collection

Clinical assessment.-Anthropometric parameter: Body weight is determined using a homologated electronic scale while height is measured with a statimeter, with the subject shoeless in the standing position. Body mass index (BMI) is then calculated as weight divided by the square of height (kg/m²). Waist circumference is measured on the upper border of the iliac crests using a flexible graduated measuring tape.

-Hemodynamic parameters: resting systolic/diastolic Arterial Pressure and resting Heart Rate are determined using an electronic UA-611 sphygmomanometer (A&D Medical, San Jose, CA) while the subject is supine (considering at least three repeated measures) or standing up.

Lifestyle assessment

• Physical activity is assessed by a modified version of the International Physical Activity Questionnaire (IPAQ) [25, 26], which focuses on intensity (nominally estimated in Metabolic Equivalents-MET- according to the type of activity) and duration (in minutes) of physical activity. We will consider the following levels: brisk walking (≈ 3.3 METs), other activities of moderate intensity (≈ 4.0 METs), and activities of vigorous intensity (≈ 8.0 METs) [27].
• Nutrition is assessed using the American Heart Association Healthy Diet Score [28, 29], taking into consideration fruit/vegetables, fish, sweetened beverages, whole grain, and sodium consumption.
• Sleep is assessed by inquiring about the number of hours on average slept every night and asking about the perception of sleep quality. Ad hoc questionnaires (Epworth Sleepiness Scale (ESS) questionnaire [30] and Pittsburgh Sleep Quality Index (PSQI) questionnaire [31]) are employed.
• Stress and somatic symptoms perception are assessed using a self-administered questionnaire [32, 33], providing nominal self-rated scales (higher values indicate higher degrees of symptoms) focusing on (i) the appraisal of the overall stress and fatigue perception by evaluation scales with integer scores from 0 (‘no perception’) to 10 (‘highest perception’) for each measure; (ii) the Short-Subjective Stress-related Somatic Symptoms Questionnaire (4S-Q), inquiring about four somatic symptoms accounting for the majority of somatic complaints. For scoring purposes, each response will be coded from 0 (‘no feeling’) to 10 (‘a strong feeling’); thus, the total score ranges from 0 to 40. Moreover, perceptions of quality of life, sleep, and overall health will be assessed using evaluation scales from 0 (‘worst quality’) to 10 (‘best quality’) for each measure.

Statistical analysis

The main outcome (variation of VO₂ max along the observation period) will be evaluated across the population strata and in association with the demographic, biological, and clinical covariates of interest. Descriptive analyses will be implemented to assess measured individual variations, in line with the repeated measures we collect. General linear analysis of variance/covariance models for repeated measures will then be used. Sensitivity analyses will be performed on the missing values and the robustness of the statistical models about extreme observations and assumptions on the distributions of the measures. The possibility of adopting non-parametric and/or robust regression methods of analysis will be evaluated. About the secondary/exploratory objectives regarding the biological variables considered in the project, analyses will be carried out taking into account the problem of the multiple statistical tests through the False Discovery Rate approach. Furthermore, projective techniques of multivariate analysis will be used such as Principal Component Analysis (PCA) and its non-linear and robust extensions according to inductive inference approaches (computational learning) to reduce the size of the problem and extract the most relevant information also by the structure of repeated measures of the study. A final statistical analysis plan (SAP) will be prepared during the conduct of the study before performing the final analyses.

Dissemination of results

A project’s website has been implemented and is updated regularly (https://hebe.unimi.it).

The main aim of the website is public engagement, for the promotion of a culture of health and the resulting prevention of disease, with a strong positive impact on the community.

The website is structured in sections aimed at informing citizens and patients in detail about the project and involved researchers, as well as providing recommendations related to physical activity, nutrition, and psychophysical well-being based on scientific evidence. It also strives to update the scientific most recent discoveries and Medical Society guidelines in the field of health and lifestyle and to advertise all the relevant events sponsored by HEBE.

Although the website is in Italian, a specific English section dedicated to national and international researchers is in progress, to provide useful material for scientists working in the Health field, such as the HEBE Study Protocol and scientific articles.

Moreover, information reported on the website is weekly published in the HEBE official Instagram profile (@hebe_unimi), through both posts and stories, creating a Social Community aimed at enhancing citizens’ engagement and awareness.

Both the website and the social media profile will support a comprehensive approach to health literacy among the aging population, promoting the idea that lifestyle changes can positively affect individual well-being and life quality in a personalized perspective.

Opportunities to collaborate

HEBE has been designed to be aware that much of its value would arise from involvement with other investigators, individually and within consortia. The HEBE project is open for collaboration with interested investigators. Proposals to test specific hypotheses on the HEBE cohort will be reviewed by our research team. Requests can be sent to the e-mail hebe@unimi.it.

Data protection and management

The data are collected in a database created using the REDCap (Research Electronic Data Capture) platform Harvard Catalyst, Boston, USA. REDCap is a secure non-profit web application, protected by personal login, and allows data traceability and the implementation of databases that comply with the current General Data Protection Regulation (GDPR) legislation. Data collection takes place by the documentation relating to the RDM UNIMI research data management policy.

Moreover, even if not legally necessary, the participating members of the project have signed a consortium agreement to define and regulate the management and use of all materials/data/results collected and produced by HEBE.