Aging Resilience Through Microbiota Optimization and Regulation

Skeletal muscle loss in old age is a critical point for the development of very serioushealth conditions affecting the quality of life of older adults, such as loss ofautonomy, loss of healthy cognition, and the development of complex metabolic diseases.Additionally, intestinal microbiota can be modulated through fecal microbiotatransplantation (FMT), and possibly this modulation could have direct effects thatpromote muscle health. Therefore, this project aims to investigate whether modifying theintestinal microbiota in older adults through FMT using young and trained donors canenhance skeletal muscle health and function, leading to greater resilience, reducingcognitive decline, and metabolic dysfunction associated with aging, especially instressful contexts.The donors would be young and trained individuals. Why trained donors? There is growingevidence that the composition of the intestinal microbiota is regulated by physicalexercise. In animal studies, chronic exercise has been shown to increase the biodiversityof the intestinal microbiota and reduce systemic and intestinal inflammation. Thebacterial profile associated with exercise is specific, for example, it does notcorrespond to that associated with a healthy diet, suggesting that physical activityallows for the development of a specific microbial profile in intestinal bacterialcommunities. Several studies have observed that changes in the intestinal microbiotaassociated with exercise are beneficial for promoting intestinal mucosal integrity andoverall metabolic function in the individual.In humans, intestinal bacterial biodiversity has been observed to be higher in athletescompared to sedentary subjects (normalized by body weight, age, and gender). In fact,there is evidence that a chronic exercise routine can restore the intestinal microbiotato a healthy profile in individuals with intestinal dysbiosis. In rats and mice, chronicexercise increases the biodiversity of Firmicutes, Bifidobacteria, and Actinobacteria andreduces the proportion of Bacteroides. Some data show how the presence of intestinalmicrobiota affects muscle performance, for example, it was found that germ‐free mice hadgreater swimming endurance compared to germ‐free mice, and when the latter were colonizedwith bacteria in the gut, the mice increased their physical endurance and swam forlonger.The main objective of this project is to carry out fecal microbiota transplants (FMT)from physically active young donors to a cohort of older individuals to evaluate theireffect on muscle, cognitive, and metabolic function. FMT in older adults is planned to beperformed by administering lyophilized microbiota capsules. By restoring microbialdiversity, it is expected to improve the quality and function of skeletal muscles,leading to greater cognitive and metabolic resilience. It is important to mention thatmodifications of the intestinal microbiota have been associated with direct and specificeffects on brain and metabolic functions, so the FMT from young and trained donorsproposed in this project could have. This project has great potential to develop aninnovative approach to treating highly debilitating diseases affecting older adults,based on the lyophilization and encapsulation of intestinal microbiota from young andtrained donors, which can be easily preserved in a conventional freezer. This approachhas been previously implemented for the treatment of C. difficile infection, withoutstanding results. This idea will have a profound impact on the quality of life ofadult and elderly patients, as it can be an alternative or complementary option tophysical exercise therapy that older adults must undergo to prevent or treat sarcopenia,but often cannot be adequately achieved due to physical limitations associated with oldage. Due to the high percentage of older adults worldwide and the high prevalence ofsarcopenia among older adults, the researchers aim to address a significant public healthissue with a large target population eager for options to promote muscle health,functional autonomy, as well as cognitive and metabolic well‐being.RESEARCH QUESTION. The hypothesis is as follows: Intestinal microbiota transplantationfrom young, trained donors promotes resilience through the enhancement of musclefunction, thereby preserving cognitive and metabolic performance in older individualsexposed to stressful situations.GENERAL AND SPECIFIC OBJECTIVES. General Objective To demonstrate that fecal microbiotatransplantation from young and physically trained donors promotes muscle performance,preventing cognitive and metabolic decline in older individuals in a stressful context.Specific Objectives To evaluate the effect of fecal microbiota transplantation (FMT) fromtrained young donors on the muscular health of aged individuals, correlating the resultswith the degree of stress exposure of the elderly recipients of FMT.To assess the effect of fecal microbiota transplantation (FMT) from trained young donorson cognitive parameters of elderly recipients, correlating the results with individualsstress levels and modifications of muscle function after FMT.To evaluate the effect of fecal microbiota transplantation (FMT) from trained youngdonors on metabolic parameters of elderly recipients, correlating the results withindivididuals stress levels and modifications of muscle function after FMT.In these objectives, researchers aim to investigate whether FMT from trained young donorscan improve muscle function in older individuals, with beneficial consequences oncognitive and metabolic functions, correlating the results with the degree of stressexposure of the participants.EXPERIMENTAL DESIGN. The research is structured as a randomized, placebo‐controlled,double‐blind clinical trial. Researchers will work with a group of 92 elderly individuals(aged 65‐84 years, both women and men). All the characteristics mentioned in theexperimental design, in addition to the fact that through its implementation researchersaim to answer the question: Does the treatment (FMT in this case) generate a benefit (inthis project, a muscular, cognitive, or metabolic benefit) in the context of a conditionor disease (aging and stressful environment)? and also to evaluate the safety of theintervention, classify this research as a Phase II clinical study. Participants will berecruited at Clínica Universidad de los Andes or the Institute of Nutrition and FoodTechnology (INTA) of the Universidad de Chile, after signing an informed consentdocument. FMT will be administered using capsules of lyophilized fecal microbiotamanufactured at the Center for Biomedical Research and Innovation (CIIB) of theUniversidad de los Andes.Fecal Microbiota Donors. The donors will be young individuals (aged 20 to 35 years, 2males and 2 females), physically active, who engage in at least 150 minutes ofhigh‐intensity aerobic physical activity per week, as this type of training improves thediversity and production of short‐chain fatty acids (SCFAs) of the human intestinalmicrobiota. The Global Physical Activity Questionnaire (GPAQ) and actigraphs will be usedto measure physical activity in donors 2 weeks before stool donation. For donorselection, individuals will be considered to have reached a metabolic equivalent (MET) of600 minutes or more per week.Donors will meet all criteria for donating fecal microbiota (criteria already establishedfor FMT treatment in patients with CDAD.During the initial stage of the selection process, the medical history and risk behaviorof potential donors will be assessed using a specialized questionnaire. The questionnairewill be administered by a medical professional. Objective evaluation includes theconsequences of a specific response for the selection process. The topics and elements tobe addressed in this questionnaire are listed in Table 1.TABLE 1. Elements to Evaluate During Donor Selection Prior to Approval for FecalMicrobiota Transplantation (FMT)Infectious Diseases ‐ History of or exposure to infectious diseases with chronic activity: HIV, hepatitisB virus (HBV), hepatitis C virus (HCV), unsuccessfully eradicated Helicobacterpylori, syphilis, malaria, trypanosomiasis, tuberculosis, Chagas disease,strongyloidiasis. ‐ Any currently active or relevant infection within the last 6 months. ‐ Live attenuated virus vaccination within the last 8 weeks. Country of birth. RiskBehaviors ‐ Current or previous intravenous drug use. ‐ High‐risk sexual behavior within the last 6 months. ‐ Travel to high‐risk foreign countries within the last 6 months. ‐ Current occupation in an environment facilitating the acquisition of potentialpathogens (e.g., veterinarian, animal caretaker, ranger, prison worker). ‐ Tattooing, piercing, or acupuncture within the last 6 months. ‐ Major surgery within the last 6 months. ‐ Contact with human blood (e.g., accident, needle stick injury) within the last 6months. ‐ History of incarceration. ‐ Prior tissue/organ transplant. ‐ Blood product transfusion (e.g., packed red blood cells, plasma, platelets,immunoglobulins) within the last 6 months of medical history.Medical History ‐ Chronic diseases. ‐ (Risk of) Creutzfeldt‐Jakob disease. ‐ Allergies or atopy (e.g., food or drug allergies, asthma). ‐ Hospitalization within the last 4 months. ‐ Ongoing pregnancy. ‐ Antibiotic treatments, scheduled or received within the last 3 months. ‐ Regular medications or nutritional supplements. ‐ BMI (accepted if ≥20 and ≤25 kg/m2). ‐ Age (accepted if ≥18 and ≤30 years).Intestinal Health ‐ Previous or scheduled gastrointestinal surgery, except for appendectomy. ‐ Gastrointestinal symptoms in the last 3 months (e.g., diarrhea, constipation,hematochezia, vomiting, abdominal pain), or adenomatous polyps or sessile serratedlesions (removed). ‐ Any other relevant clinical sign or symptom in the last 3 months (e.g., fever orrash).In addition to all the infectious agents described in Table 2, SARS‐CoV‐2 will also beruled out through RT‐PCR in the stool. The presence of E. coli ST131 will be studiedthrough classical culture and PCR typing, as well as the presence of antibioticresistance genes. If positive results are obtained for these markers, the individualcannot be a donor. For each dose of FMT, a minimum of 150 grams of stool will be used,transported to the laboratory under the most anaerobic conditions possible (GutAlivesystem https://www.microviable.com/en/gutalive‐2/) and in the shortest time possible fromcollection. Stool is a complex sample due to its variable water content, and the only wayto standardize the dose is by the weight of the stool, as the amount of final powder canvary greatly. Therefore, the number of resulting capsules may vary widely. However, themicrobial load will be equivalent as long as same logarithmic order is maintained. Stoolwill be lyophilized by mixing it with semi‐skimmed lactose‐free milk at a ratio of 1:2 asa cryoprotectant. The mixture will be homogenized in a Stomacher (230 rpm), solidresidues will be removed by gentle centrifugation (500 rpm 5 min), and then allmicroorganisms will be collected by centrifugation (10,000 rpm 15 min). The supernatantwill be removed, and the pellet will be frozen at ‐80°C for at least 1 hour, after whichit will be lyophilized at ‐50°C for 18‐20 hours. The obtained powder will be encapsulatedin enteric‐coated capsules in batches that will be stored in the absence of moisture at4°C with a shelf life of 6 months. An aliquot of all processes will be stored at ‐80°C,and sample and donor traceability will be carried out with RedCap software.Elderly Participants Number of participants: 92 Ethnicity : Chilean population. Chile hasa population with homogeneous ethnicity, with indigenous (40%) and European (60%)ancestries.Age: 65‐84 years Biological sex: 46 males and 46 femalesStudy's recruitment, randomization, intervention, and follow‐up process, organized in thefollowing steps: ‐ Recruitment: Participants are recruited and undergo an eligibility evaluation. Theyare assessed to determine if they meet the inclusion criteria and do not meet theexclusion criteria. ‐ Informed Consent: Eligible participants who meet the study criteria provide theirinformed consent to participate. ‐ Pre‐intervention Evaluations: Before the intervention, participants are assessed toclassify them based on their levels of stress and anxiety. ‐ Randomization: A total of 92 participants are randomized into 2 groups:Placebo group (n=46) IMT group (n=46)Intervention: Participants in the four groups undergo the assigned intervention:The first group receives a placebo capsule The second group receives IMT capsule(Intervention of Interest)Follow‐up: Follow‐up evaluations are conducted at weeks 1, 4, 8, and 20 post‐interventionfor all groups to assess the outcomes of the interventions.This research is structured as a randomized, placebo‐controlled, double‐blind clinicaltrial. This structure allows the study to compare the effects of the intervention (IMT)versus a placebo across different stress level classifications.Pre‐Intervention Assessments:The 14‐item Perceived Stress Scale (PSS‐14) will be used to assess the level of stress inparticipants. Depressive symptoms will be evaluated using the 15‐item GeriatricDepression Scale (GDS), which assesses mood disorder symptoms commonly associated withdepression experienced in older adults (both documents attached). Anxiety symptoms willbe measured using the Beck Anxiety Inventory (BAI), which is designed to obtain a measureof anxiety that is relatively independent of depression (attached). These instrumentshave high reliability and validity in community samples, which will allow us to classifyour volunteers into low stress‐anxiety and medium/high stress‐anxiety groups. A post hocanalysis will be conducted to evaluate the effects of IMT on participants with low andmedium/high stress levels.To favor the establishment of the new ecosystem, the intestinal microbiota density of theparticipants will be reduced before the FMT with rifaximin (a suitable non‐absorbableantibiotic for intestinal decontamination) for 3 days, 1,200 mg/day, administered in 2daily doses. The antibiotic and its administration schedule will be provided to theparticipants.The FMT will be conducted in the Clinical Trials Unit of the Universidad de los AndesClinic by administering 4 capsules (containing a compact lyophilized preparation)prepared from approximately 150 grams of feces. Control subjects will receive placebocapsules with identical appearance to the capsules containing the intestinal microbiotalyophilized material. Free transportation will be provided to the participants betweentheir homes and the Universidad de los Andes Clinic.Researchers will conduct this study using only non‐invasive tests and analyses. Allproposed assessments to measure muscle, cognitive, and metabolic functions will becarried out before and after the FMT (from 1 to 20 weeks after the FMT). Theseassessments will be conducted at the Ambulatory Care Health Center of the NationalInstitute of Agricultural Technology (CEDINTA). Free transportation will be provided tothe participants between their homes and the CEDINTA. All participants will undergomedical history before and after the intervention to obtain information about overallhealth status, medication use, antibiotics, and dietary supplements, history ofintestinal diseases, and any other intestinal disorder that may alter the intestinalmicrobiota.Researchers will calculate the Frailty Index (FI) in participants from all groups beforeand after the FMT. The FI will be assessed using a combined method of clinical andlaboratory parameters. Firstly, researchers will use a self‐reported FI created with 30variables to assess health status. Variables comprise different types of health problems,including diseases (n=9), symptoms (n=6), disabilities for living (n=6), psychologicalproblems (n=2), Romberg test, physical performance, Geriatric Depression Scale (GDS)score, and Mini‐Mental State Examination (MMSE) score. For binary variables, the presenceof an impairment will be coded as "1"; and its absence as "0". For each three‐levelvariable, 0.5 will be used to represent an intermediate response level. For the MMSE, itwill be coded as 0 if >23, 0.5 if MMSE=15‐23, and 1 if MMSE≤14, while GDS will be recodedas 0 if GDS<11) and 1 if GDS≥11. Secondly, a laboratory‐based FI (FI‐lab) will identify15 parameters based on 9 laboratory tests, in addition to pulse, systolic and diastolicblood pressure, pulse pressure, BMI, and waist circumference. A normal reference rangewill be used to compare each parameter, where "0" indicates values within the normalrange and "1" indicates values outside the reference range. Finally, researchers willcombine the two FIs to construct a 45‐item FI (FI‐combined). Each FI‐combined will bedefined as the score of each parameter of an individual divided by the total number ofparameters considered, with the FI ranging from a theoretical minimum of 0 (no impairedparameters present) to a maximum possible of 1.0 (all impaired parameters present).For the assessment of muscle sarcopenia, researchers will measure lean and fat mass usingdual‐energy X‐ray absorptiometry (DEXA) and creatinine excretion. A standard dynamometerwill be used to measure isometric strength. Additionally, the Short Physical PerformanceBattery and the 6‐minute walk test will be applied to estimate maximum oxygenconsumption. Physical activity will be evaluated using an actigraph for 7 days.Researchers will measure the thickness of the rectus femoris muscle and the musclepennation angle in the participants' thighs using ultrasound. As biomarkers forsarcopenia, creatine kinase (CK), skeletal muscle troponin T (sTnT), insulin‐like growthfactor‐1 (IGF‐1), C‐reactive protein, vitamin D, TNF‐α, IL‐6, IL‐1β will be measured inblood.For cognitive performance, participants will be assessed on a set of higher cognitiveabilities such as attention, executive functions, working memory, language, andprocessing speed, among others, before and after the FMT intervention. The MontrealCognitive Assessment (MoCA), Trail Making Test A and B, Digit Span Forward and Backward,Frontal Assessment Battery (FAB), and Verbal Fluency (FAS) will be administered(attachments). Participants' quality of life will be assessed using the SF‐36questionnaire, one of the most commonly used and evaluated instruments for Health‐RelatedQuality of Life (HRQoL).For metabolic function, a comprehensive analysis of metabolic markers in blood such asinsulin, glucose, total cholesterol, LDL‐cholesterol, HDL‐cholesterol, triglycerides, andtransaminases (ALT, AST, gGT) will be performed in fasting conditions. Researchers willstudy the presence of non‐alcoholic fatty liver disease (NAFLD) using ultrasound imaging.Additionally, concentrations of metalloproteinases‐9 and IL‐6 in serum will be measuredby ELISA, as these markers have been proposed to be correlated with the severity of NAFLDin humans. Non‐invasive scores of hepatic fibrosis will also be applied.With the bacterial genomic DNA isolated from the fecal samples of the participants,researchers will study the microbiota profile using shotgun metagenomics or 16S genesequencing. Samples will be collected from both young donors and older recipients beforeand after the FMT. Using bioinformatics tools, researchers will identify a bacterialsignature in older adults who received FMT from trained young donors. Researchers willdetermine if this microbiome signature correlates with muscular, cognitive, and metabolicresilience, especially in the group experiencing higher stress. Researchers propose thatkey elements in this modified intestinal microbiota in older adults will serve asappropriate biomarkers for muscular, cognitive, and metabolic resilience in the contextof aging.SAFETY IMT performed by qualified personnel has not been associated with any seriousadverse events, but mild or common adverse reactions may occur. Marcella et al. (2021)analyzed the presence of adverse reactions to IMT in a systematic review, evaluating 129studies involving 4,241 patients. They found that the most common adverse effects weretransient diarrhea (10%), transient abdominal cramps/discomfort (7%), nausea, andvomiting (<5%) within 24 hours post‐IMT. Transient fever, abdominal distension, fatigue,and borborygmi have also been reported in less than 2% of cases. Constipation (2%) andexcess flatulence (3%) were reported during follow‐up.Severe adverse reactions, such as bacteremia and death as a direct consequence of IMT,are extremely rare (incidence <0.1%) and occurred only in patients with diseases causingdamage to the digestive mucosa. These diseases (inflammatory bowel disease, Crohn'sdisease, Clostridium difficile infection (CDI), and ulcerative colitis) are exclusioncriteria for this study. Another study in 2022 evaluated the safety associated with theuse of IMT in 5,344 patients affected by CDI. The researchers found that, overall, thesafety profile was favorable in a medically complex patient population that usually haspoor clinical outcomes. Among the cohort, 194 patients (3.6%) experienced one or moreserious adverse reactions (SARs). SARs were more common among patients with severe orsevere‐complicated CDI (n = 94 [48.5%]). Among the SARs, six events (0.1%) were possiblyrelated to IMT, all in patients who were severely immunocompromised. This included twopatients with fever, as well as one with abdominal pain in a patient withsevere‐complicated CDI and breast cancer. Additionally, two patients developed systemicinflammatory response syndrome‐one after a heart transplant and one after a kidneytransplant. There was one reported case of an inflammatory bowel disease (IBD) flare‐upin a patient with a history of ulcerative colitis not controlled with biological therapyand immunomodulators. None of the reported SARs were definitively related to IMT.Notably, no cases of sepsis or transmission of infectious diseases related to IMT,including infections with multi‐drug‐resistant organisms, were reported. This study willnot include patients with CDI or those who are severely immunocompromised.Finally, another systematic review and meta‐analysis in 2022 searched for IMT studies inpatients with CDI, using the rate of SARs related to IMT as the primary outcome.Secondary outcomes included SARs unrelated to IMT and minor adverse events associatedwith IMT. The combined analysis of 5,099 patients who received 5,551 IMTs showed thatSARs related to IMT developed in less than 1% of patients. The combined rate of SARsunrelated to IMT was higher, reaching 2.9%. The combined rate of minor adverse eventsalso showed infrequent self‐limited gastrointestinal and systemic discomfort. Thismeta‐analysis supports IMT as a safe option for treating recurrent CDI.For IMT performed exclusively in the elderly population, a study of 31 patients with anaverage age of 77 years with CDI reported that 87% of patients resolved their infection,and the most common adverse event reported in 4 (13%) of 31 respondents was thesubjective worsening of arthritis. Another study that performed IMT on 35 elderlypatients (average age 77 years) with CDI reported no serious adverse effects.In summary, the international experience using IMT reported in the literature shows thehigh safety of this procedure. However, the following risks should be considered: (1)Infection: Although IMT has been screened for pathogenic bacteria, viruses, fungi, andparasites, there is a risk of transmitting known and unknown infectious organismscontained in the donor's stool. There is also a theoretical risk of bacterial overgrowthin the small intestine. Rarely, bacteremia (e.g., E. coli, Klebsiella), sepsis, and fatalevents can occur after IMT; (2) Exacerbation of inflammatory bowel disease (IBD) inindividuals with underlying IBD; (3) Allergy/anaphylaxis to antigens present in thedonor's stool; (4) Transmission of non‐infectious diseases: There is a theoretical riskof developing diseases that may be related to the donor's gut microbiota. These includeobesity, metabolic syndrome, cardiovascular diseases, autoimmune conditions,allergic/atopic disorders, neurological disorders, psychiatric conditions, andmalignancies. This risk is very limited in the study, as individuals with these knownconditions are excluded from stool donation according to the donor screening proceduresexplained in this protocol.