Health and gut function in older adults

To my mother who inspired this career path

Örebro Studies in Medicine 140

LINA ÖSTLUND-LAGERSTRÖM

"The gut matters" - an interdisciplinary approach to health and gut function in older adults

© Lina Östlund-Lagerström, 2016 Title: Health and gut function in older adults Publisher: Örebro University 2016 www.oru.se/publikationer-avhandlingar Print: Örebro University, Repro, April 2016 ISSN 1652-4063 ISBN 978-91-7529-130-7

Abstract Lina Östlund-Lagerström (2016): “The gut matters” - an interdisciplinary approach to health and gut function in older adults. Örebro Studies in Medicine 140. Improved life expectancy is a triumph of modern medicine. However, today’s senior citizens are predicted to soon consume 75% of the available health-care resources. Identifying new strategies to promote a healthy ageing process has thus become a priority. In contribution to the research field of healthy ageing this thesis is focused on the health and gut function of older adults. Paper I explored ‘optimal functionality’; a new approach to put the older adult’s own perspectives on health in focus. According to the results a plethora of factors related to the body, the self and the external environment needs to be considered in order to create a comprehensive understanding of the health experience in old age. Paper II characterised senior orienteering athletes as a new model of healthy ageing, due to their significantly better percived health as compared to other free-living older adults; in particular they report better gut health. As the gut is important to health maintenance and immune function paper III explored inflammation and oxidative stress in senior orienteering athletes, and older adults with gut problems, generally finding low levels in both groups. Subsequently, Paper IV investigated the health status of free-living older adults in Örebro County and also reports the results from a randomised controlled trial evaluating the effect of a probiotic supplement on self-reported health and gut symptoms. Two-thirds of the included older adults reported gut problems, however, the probiotic intervention failed to show any effects. This thesis provides additional perspectives on older adults health and gut function, by concluding that 1) optimal functionality may be a useful concept to map areas of importance to the older adult’s health experience, 2) senior orienteers may be regarded as a suitable model to study healthy ageing, 3) the prevalence of gut problems among the general population of Swedish older adults is high, but was not improved by probiotic supplementation with Lactobacillus reuteri. Keywords: healthy ageing, gut health, old age, senior orienteering athletes Lina Östlund-Lagerström, School of Medical Sciences, Örebro University, SE-701 82 Örebro, Sweden, [email protected]

Svensk sammanfattning Vår stigande medellivslängd är en sann framgångssaga som kan tillskrivas den moderna hälso- och sjukvårdens framväxt. Dock har alla långlivade äldre blivit en utmaning för samhället, då de kräver ökade vårdinsatser under en längre period av sin ålderdom. Att identifiera nya strategier för att främja ett hälsosamt åldrande bör därmed prioriteras. Den här avhandlingen är ett bidrag till forskningsfältet ’det hälsosamma åldrandet’ och fokuserar på bibehållen hälsa och mag-tarm funktion hos äldre. Delarbete I undersökte begreppet ’optimal funktionalitet’ som en ny strategi för att sätta den äldre människans egna perspektiv på hälsa i fokus. Dess resultat visar att det finns en mängd faktorer i relation till jaget, kroppen och yttre omgivning som behöver övervägas för att skapa en fullständig förståelse för den äldres upplevelse av hälsa. I Delarbete II definierades seniora orienterare som en ny modell för att studera det hälsosamma åldrandet på grund av deras höga självupplevda hälsa och deras goda maghälsa. Eftersom magen är ett viktigt organ för vår hälsa och immunfunktion undersökte delarbete III nivåer av inflammation och oxidativ stress hos seniora orienterare. Resultaten jämfördes med äldre människor som har magproblem. Båda grupperna uppvisade dock låga värden på de undersökta parametrarna. I delarbete IV studerades hälsostatus hos äldre innevånare i Örebro kommun, och arbetet rapporterar även resultaten från en randomiserad kontrollerad studie vilken utvärderar effekten av ett probiotiskt kosttillskott på äldres självrapporterade hälsa och mag-tarm problem. Två tredjedelar av den studerade populationen rapporterade magproblem, men kosttillskottet visade inga effekter på vare sig den upplevda hälsan eller magproblemen bland de äldre. Avhandlingen konkluderade följande: 1) optimal funktionalitet kan vara ett användbart koncept för att skaffa översikt av de faktorer som är särskilt viktiga för äldres upplevelse av hälsa, 2) seniora orienterare kan ses som en lämplig modell för att studera det hälsosamma åldrandet, och 3) prevalensen av magproblem bland äldre är hög, men symptomen förbättrades inte av ett kosttillskott innehållande den probiotiska bakterien Lactobacillus reuteri. Ytterligare forskning behövs för att vidareutveckla optimal funktionalitet som koncept samt för att mer utförligt karaktärisera seniora orienterare som en modell av ett hälsosamt åldrande. Vidare behövs mer kunskap om de magproblem som drabbar äldre och nya behandlingsalternativ bör studeras ytterligare, förslagsvis genom fler randomiserade kontrollerade studier av pro- och prebiotiska kosttillskott.

List of papers PAPER I. Exploring the concept of optimal functionality in old age Samal Algilani*, Lina Östlund-Lagerström*, Annica Kihlgren, Karin Blomberg, Robert J. Brummer, Ida Schoultz. Journal of Multidisciplinary Healthcare (2014) 7:69–79 *Both authors contributed equally to this work

PAPER II. Senior orienteering athletes as a model of healthy ageing: a mixed methods approach Lina Östlund-Lagerström, Karin Blomberg, Samal Algilani, Magnus Schoultz, Annica Kihlgren, Robert J. Brummer Ida Schoultz. BMC Geriatrics (2015) 15:76

PAPER III. Low levels of inflammation and oxidative stress in senior orienteering athletes Lina Östlund-Lagerström, John-Peter Ganda Mall, Samal Algilani, Dara Rasoal, Robert J. Brummer, Ida Schoultz. Submitted manuscript, 2016.

PAPER IV. Probiotic administration among free-living older adults: a double blinded, randomised, placebo-controlled clinical trial Lina Östlund-Lagerström, Annica Kihlgren, Dirk Repsilber, Bengt Björkstén, Robert J. Brummer, Ida Schoultz. Submitted manuscript, 2016. Published papers have been reprinted with permission from the publisher.

Related publications Increasing the qualitative understanding of optimal functionality in older adults: a focus group based study. Samal Algilani, Lina Östlund-Lagerström, Ida Schoultz, Robert J. Brummer, Annica Kihlgren. Accepted for publication, BMC Geriatrics, 2016.

Table of Contents ABBREVIATIONS ...................................................................................13! PREFACE ................................................................................................14! INTRODUCTION ...................................................................................16! Healthy ageing ......................................................................................... 16! Understanding health from the older adults perspective ....................... 17! The importance of the gut in healthy ageing............................................. 18! Probiotics as a non-invasive strategy to improve health ....................... 21! Rationale.................................................................................................. 22! AIM .........................................................................................................23! METHODS ..............................................................................................24! Sample ..................................................................................................... 24! Scoping study (Paper I)............................................................................. 25! Considerations of the scoping study ..................................................... 27! Mixed-method (Paper II) .......................................................................... 27! Content analysis ................................................................................... 28! Considerations of the mixed-methods design ....................................... 30! Assessment of inflammation and oxidative stress (Paper III) .................... 31! Considerations of the biological parameters assessed ........................... 32! Randomised controlled trial (Paper IV) .................................................... 33! RCT considerations ............................................................................. 35! Statistics ................................................................................................... 37! Ethical considerations .............................................................................. 37! Public outreach ........................................................................................ 38! RESULTS .................................................................................................39! PAPER I. Optimal functionality in old age is a multifaceted concept ........ 40! PAPER II. Senior orienteering athletes may be considered a new model of healthy ageing ...................................................................................... 41! PAPER III. The levels of inflammation and oxidative stress are low in senior orienteering athletes ....................................................................... 44! PAPER IV. Probiotic supplementation shows no significant effects on gut complaints or self-reported health in free-living older adults .............. 45! DISCUSSION ...........................................................................................48! Health in relation to old age..................................................................... 48!

Interpreting health from an individual perspective ............................... 49! A new model of healthy ageing ................................................................ 50! The gut as an important denominator of health ....................................... 53! Methodological considerations and limitations ........................................ 54! The RCT outcome in paper IV ............................................................. 54! Validity and reliability of questionnaire data in paper II-IV ................. 55! Comparing senior athletes to older adults with gut problems in paper III ........................................................................................... 57! Trustworthiness of the focus group discussions in paper II................... 58! Considerations of the database search in paper I .................................. 58! Originality and representativeness of paper I-IV................................... 59! Gender perspective ............................................................................... 60! Future perspectives ................................................................................... 60! CONCLUSIONS ......................................................................................63! ACKNOWLEDGEMENTS ......................................................................65! REFERENCES .........................................................................................68!

Abbreviations AAD CRP EQ-5D-5L FGAS FGD FORT GMMS GSRS HADS HI IFN IL IPAQ IQR ITT L MeSH MMSE MoCA PPI PSS RCT ROS SD SF-36 TNF WHO

Antibiotic associated diarrhoea C-reactive protein EuroQol Frändin-Grimby Activity Scale Focus group discussion Free Oxygen Radicals Test Gastrointestinal motility modulating substances Gastrointestinal Symptoms Rating Scale Hospital Anxiety and Depression Scale Health Index Interferon Interleukin International Physical Activity Questionnaire Inter-quartile range Intention-to-treat Lactobacillus Medical Subject Heading Mini Mental State Exam Montreal Cognitive Assessment Proton pump inhibitors Perceived Stress Scale Randomised controlled trial Reactive oxygen species Standard deviation Short Form-36 Tumour necrosis factor World Health Organization

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Preface This thesis is focused on health maintenance and gut function among older adults, and has primarily been motivated by the societal concerns arising from the steadily growing population of older adults. Even tough a true success story, the increasing life expectancy have paradoxically resulted in more years with late-life health complications leading to individual suffering and increased pressure on the health-care system. Hence, there is a pressing need to facilitate perceived health among older adults, with the ultimate goal to increase individual wellbeing and delaying the first contact with health-care. Numerous factors are likely to reduce the perceived health of older adults and thus needs recognition in order to reach this goal. Which these factors are the older adults themselves have the best knowledge about and characterising groups of successful agers will be an important step towards their identification. Furthermore, discovering noninvasive and cost-effective strategies to increase the health and optimal functionality of older adults will be key in managing the societal consequences of their constant increase. This thesis has been produced within an interdisciplinary context. In early 2012 I got the opportunity to start my PhD studies at Örebro University and during the following four years I have been part of two research centres, i.e. the Nutrition and Physical Activity Research Centre (NUPARC) and the Nutrition-Gut-Brain Interaction Research Centre (NGBI). Currently the studies performed in these two research centres are mainly focused on two agendas, 1) optimal functionality in older adults and 2) intestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and the decreased gut function associated with ageing. The research activities in the centres are performed in an interdisciplinary fashion, bringing together researchers with many different competences to create a common basis for knowledge production. Hence, the researchers within NUPARC and NGBI come from a variety of research fields and professional background. For example, the co-authors of my four papers consist of individuals with expertise in biomedicine, gastroenterology, paediatrics, elderly care, emergency care, psychiatry and bioinformatics. Adding my own background within sport sciences this creates a great diversity of competences and previous experiences. Interdisciplinary research may be defined as joint, coordinated, and continuously integrated efforts by individuals with different disciplinary backgrounds working together and producing joint scientific papers. Together the researcher of 14

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NGBI and NUPARC have set the common goal for the research presented in this thesis and worked jointly on the design and execution of the studies as well as the presentation in the four included papers. This has produced a thesis that has a broad focus on the health of older adults and combines a number of different methods to explore this matter; generating new common knowledge on this particular subject.

Single discipline

Multidisciplinary

Interdisciplinary

A schematic overview of multi- versus interdisciplinary research In contrast to multidisciplinary teams, working in parallel on basis of their different disciplines, interdisciplinary teams work jointly from a discipline specific base to address a common problem (1). An interdisciplinary team agree on a common goal and then coordinates their input, while a multidisciplinary team set individual discipline dependent goals and then regularly meet to evaluate their work towards these goals (2).

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Introduction The development of the industrialised world has led to a better living standard, promoting greater life expectancy for the worldwide population. Although a great success, this progress has not come without complications. The proportion of citizens in the Western world1 reaching old age2 is increasing at an alarmingly high rate, and is estimated to soon outnumber young children. Unfortunately an increased lifespan is not always equal to more healthy years. On the contrary older adults have an increased risk of various disorders, commonly manifested as multi-morbidities (3), and this segment of the population is predicted to consume about 75% of the collective health-care resources (4). As a step towards managing the great societal challenge arising from a growing population of older adults research initiatives to promote healthy ageing has become a priority. Identifying factors that are of importance to support a healthy ageing process and maintained functional capacity are essential in order to increase the proportion of independent free-living older adults and thereby limit the use of the health-care resources.

Healthy ageing To further increase the knowledge on how to age healthy there is a need to identify groups of older adults that are successful in this sense. Groups of adults reaching an exceptionally high age, such as octo- and centenarians, are often used to study healthy ageing due to their success in reaching a high age. Yet, healthy ageing it is not solely about reaching a high age, but rather about experiencing wellbeing and maintained function. Successful ageing has previously been described as, avoiding disease and disability, maintaining high physical and cognitive function and continuously engaging in social and productive activities (5–7). Ergo, the suggested models are likely to fall short of addressing healthy ageing from a holistic perspective. Hence, it is essential to identify cohorts of older individuals experiencing wellbeing in combination with maintained physical and cognitive function at an advanced age; to allow the identification of important factors to promote health and independence through life.

The Western world here refers to Europe, America, Russia, Northern Asia, Australia and New Zealand.

1

2

In this thesis an older adult is defined as an individual aged ≥65 years.

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Master athletes, i.e. individuals that continue their participation in competitive sports at older ages (8), has been suggested to be an ideal model to study healthy ageing as the process of ageing may be an end-product of disuse and physical inactivity (9,10). In addition, high mid-life leisure-time physical activity is associated with increased survival (11). Hence, it seems logical to study groups of older adults who maintain a high level of physical activity throughout life. In support of master athletes as a model of healthy ageing elite athletes live longer than the general population (12). However, this fact does not stand without dispute, for example longer life span cannot be consistently proven among “power athletes”, e.g. baseball players, weightlifters and wrestlers, whom even tend to display a decreased life-expectancy as compared to the general population (13). Furthermore, excessive training may be associated with health risks (14,15), for example former elite athletes display increased risk of lower-limb osteoarthritis (16) and increased probability to develop atrial fibrillation, i.e. abnormal heart rhythm (17). In addition, elite athletes show a reduced thymic output and rearrangements in the T-cell compartment, resembling a state of premature ageing of the immune system (18). Previous studies have reported that highly active individuals have an increased susceptibility to upper respiratory tract infections (19,20) in combination with lowered levels of protective saliva Immunoglobulin A (19,21). Therefore, master athletes may be afflicted with health abnormalities that disaffirm their suitability as an ideal healthy ageing model. At present no gold standard model of a healthy ageing population exists (22) and new models to study the healthy ageing process is needed in order to circumvent the weaknesses of the current ones. Based on the findings outlined above, new models of healthy ageing would preferably consist of older individuals displaying a high health status, maintained physical and cognitive function, regular physical exercise - at a moderate intensity - and routine engagement in social activities.

Understanding health from the older adults perspective This thesis classifies individuals of ≥65 years of age as older adults. In most developed countries this age cut-off is commonly accepted for defining an older adult (23) and in Sweden it corresponds to the first opportunity to receive pension benefits. Although this age definition is often used it is, however, not entirely without argument. At any age there is a great variance in functional capacity and health status between individuals, and people over 65 years are certainly not a homogenous group. On the conLINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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trary, people seem to become even more different from one another as they grow old; some will continue to work while others will chose to retire early to enjoy the freedom of spending time as one prefers, and yet some will have to stop working at an early age due to ill-health and disability. Thus, interpreting older adults health from an individual perspective seems inevitable. In research healthy ageing is commonly addressed with a biostatistical approach (24,25), whereby health is described as identical to the absence of disease. The World Health Organization (WHO), however, defines health in a more holistic sense describing it as a state of complete physical, mental and social wellbeing and not merely as the absence of disease or infirmity (26). A definition that clearly emphasizes the importance of experiencing subjective health and wellbeing alongside objective health, i.e. absence of clinically diagnosed disease. According to Nordenfelts holistic theory of health (25) an individual can be ill not only if the person’s chance of survival has been lowered but also if she does not feel well or are hindered in pursuing a goal (separate from the organism’s survival). The holistic theory of health is thus more in agreement with WHO as compared to the biostatical view. Still, the importance of the biostatistical approach to define health is not negligible, as we also are in need of more objective ways to evaluate human health. Nevertheless, solely adopting a biostatistical approach is likely to result in a narrowed “disease-directed” thinking, emphasising the negative aspects of reaching a high age. This way of characterising ageing may incorporate negative beliefs into the older person’s own understanding of what constitutes ageing (27–29), hampering the possibility to focus on maintenance of mental and physical wellbeing through life (30). Hence, defining ageing merely by loss of function may be counterproductive in the quest to promote older adults sense of wellbeing and independence. Therefore, it is important to allocate focus to perceived health and functionality at the individual level. Ergo, there is a need of new approaches to characterise and monitor the health of older adults, based on their own perceptions of what constitutes an ideal health and optimal functionality.

The importance of the gut in healthy ageing There are supposedly a vast number of factors that may be addressed in order to increase the older individual’s perception of health and optimal functionality, and identifying appropriate targets for health interventions are important to enable preventive work. In suggestion such targets should 18

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preferably allow for non-invasive treatment that is easy to apply in the older population. Gastrointestinal conditions are a widespread phenomenon among older adults (31–33) and have been described to negatively affect both functional status and quality of life (34), as well as overall health (35,36) and immune function (37). Accordingly, gut-health may be a particularly important denominator of wellbeing and health among older adults. By becoming old the human body will experience a number of physiological alterations in the function of the gastrointestinal system resulting in an increased prevalence of gastrointestinal disorders with increasing age (38,39) . Age-related physiological changes occurs throughout the whole length of the gastrointestinal tract, for example causing difficulties with swallowing in the upper part and decreased colonic motility in its lower parts (39). Furthermore, ageing has a profound influence on the immune system (40–42), and it has been suggested that the age-associated alterations arise in the mucosal immune system of the gastrointestinal tract rather than in the systemic immune compartments (34,43). The gastrointestinal epithelial and mucosal layer provides an important barrier towards the outside world, highly selective in the process of allowing or rejecting molecules passage trough the intestinal wall. Despite the fundamental importance in maintaining the body's defence, changes in the barrier function of the intestinal epithelium have been little studied during ageing. There are indications from animal studies that the intestinal permeability increases with advancing age (44,45). Furthermore, there is evidence that some intestinal cells may develop an age-related senescence-associated phenotype, induced by DNA damage (46,47). The senescence-associated phenotype is likely to increases the production of inflammatory mediators and reactive oxygen species (ROS), for example myenteric neurons in older animals have been shown to secrete elevated ROS levels (46,48). Mitochondria are the main generator of ROS, i.e. superoxide, hydroxyl, peroxy radicals and hydrogen peroxide, as by-products from the electron transport chain (49). ROS have the potential to cause damage to all sorts of biological molecules, such as DNA, proteins and lipids (50) and may further damage the mitochondria causing a positive feedback loop of increased ROS production and additional mitochondrial damage (51). An increased ROS production in ageing has led researchers to believe that oxidative stress plays an important part in acceleration of the ageing process (49,52). In the gut, ROS-induced damage has been proposed as a mechanism contributing to the ageing of enteric neuronal cells (53), with the potential to effect both intestinal motility (54) and barrier function (55). In addition, elevated LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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levels of ROS has been shown to increase the passage of bacteria over the intestinal barrier (56). The physiological alterations in the gastrointestinal system also change the habitat for the vast number of microorganisms populating the intestinal tract. In resent time these microscopic bugs have received a tremendous attention due to their proposed impact on human health (35,36). More than a trillion commensal bacteria inhabit our intestine and they have coevolved with us since the beginning of mankind. This evolutionary companionship fuels anticipations about the bugs playing an important part in our health maintenance. Contemporary high-throughput methods, e.g. next generation sequencing of the bacterial 16S ribosomal RNA, have allowed for more extensive studies of the intestinal microbiome in health and disease. So far disturbances in the microbial ecology has been connected to a large number of disease states, such as inflammatory bowel disease, diabetes, gastric cancer and allergies (36). The microbiota has also been suggested to be associated with intestinal barrier and immune function in ageing (57). The gut microbiota starts to develop in conjunction with our birth and gradually matures to an adult-like form at during the first years of life (58). It remains rather stable throughout adulthood, in absence of disease, but several changes has been described to occur in the aged microbiota (59,60). The age-associated microbiota seems to be characterised by a reduced biodiversity, a compromised stability and an increased number of pathobionts3 (37). The age-associated microbiota has further been suggested to be a major driver of the persistent low-grade pro-inflammatory process that characterise older individuals (37). Increased inflammation in older adults is a common consequence of immunosenescence, i.e. the process of a declining immune function that occurs in ageing. This immune deterioration is characterised by a remodelling of the immune cell profile (40) and increased levels of systemically circulating pro-inflammatory factors (41), such as cytokines and C-reactive protein. Increased inflammation may be considered a predictor of morbidity and mortality among older adults (61). Except for the changes in systemic cellular immunity, alterations in the inflammatory cells of the gastrointestinal mucosa are also described to occur with age (57,62).

Pathobionts are bacteria that when present in too high numbers exerts negative effects on its host, such as driving persistent inflammation.

3

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Apart from having an impact on physiological health the microbiota has also been suggested to be important for maintenance of psychological health and cognition, as an important element in the gut-brain axis (35); facilitating the bidirectional signalling between the gut and the brain. Moreover, the gut microbiota has recently been identified as important in linking emotional and cognitive centres of the brain with peripheral intestinal functions (63). Dysbiosis in the intestinal microbial ecology has been proposed to influence a broad spectrum of diseases, including psychiatric disorders (64) and cognitive function (59), thus suggesting its importance also in promoting mental health in the older individual.

Probiotics as a non-invasive strategy to improve health According to the presumption that gut-health has a large impact on older adults health, subjective as well as objective, therapeutic strategies able to treat the age-associated gastrointestinal discomfort may have a great impact on perceived health and independence. Yet, the treatment options are rather scarce and only a few non-pharmaceutical treatments are available. Probiotic bacteria are defined as live bacterial organisms that provide beneficial effects on host health (65). Probiotic bacteria are able to modulate the ecological environment in the gastrointestinal tract by impacting the bacterial community already present. So far, clinical interventions investigating the effects of probiotic supplementation among older adults are rather scarce and show inconclusive results. To mention a few, a multicentre study investigating supplementation with Lactobacillus (L) acidophilus and Bifidobacterium bifidum found no effect on antibiotic associated diarrhoea (AAD) (66), while another trial investigating the effect of L. casei shirota found a decrease in occurrence of AAD (67). The same bacteria also produced a significant increase of natural killer cell activity and improved cytokine profile (68). Yet another study investigating the effect of L. rahmnosus on a number of different plasma cytokines only found changes in interleukin (IL) 8 levels (69). More studies are needed to further investigate the potential benefits of probiotic supplementation among older adults and it is likely that the bacterial strain of choice, as well as the administrated amount and duration, plays an important part for the treatment effect. The probiotic organism L. reuteri has been identified as a promising therapy in various gastrointestinal disorders (70,71) and patient groups. For example it has been shown to decrease infantile colic (72), prevent acute diarrhoea in children (73), increase bowel movements in constipated adults (74), and to reduce oral Candida growth in older adults (75). LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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Hence, making it a promising organism for probiotic interventions, with the potential to increase the gut-health of older adults.

Rationale As the population of older adults is growing there is a need for further strategies to maintain their health and independence. Current approaches commonly characterise older adults on the basis of disease and loss of function and may thus hamper the possibility to focus on maintenance of health and optimal functionality through life. Ergo, there is a need of new approaches to characterise and monitor the health of older adults, based on their own perceptions of what constitutes an ideal health and optimal functionality. To gain further knowledge of what constitutes healthy ageing there is a need to identify groups of older adults that, in fact, are ageing well. The present models suggested for this purpose, e.g. centenarians and master athletes, may suffer from shortcomings in terms of health deficiencies. Hence, new models to study healthy ageing are warranted. Based on previous knowledge such models would preferably consist of older individuals displaying a high health status, maintained physical and cognitive function, regular physical exercise and routine engagement in social activities. In addition, identifying appropriate targets for health interventions in older individuals are important to enable preventive work at an early stage. Gastrointestinal problems are a widespread phenomenon among older adults negatively affecting both health status and quality of life. Accordingly, gut-health may be a particularly important denominator of health among older adults, hopefully allowing for non-invasive easy-to-use interventions to increase health via the gut microbiota, for example by probiotic supplementation. Based on the rationale above, this thesis was produced with the purpose to give additional voice to the individual preferences of health as explained by the older adults themselves. Applying a “down-up” approach, rather than the more common “top-down” where persons other than the older adults’ define their health status.

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Aim The overall aim of this thesis was to further explore the health and gut function among older adults, applying a down-up perspective, by exploring a new concept to increase the understanding of optimal functionality in old age (paper I), characterising a new potential model of healthy ageing (paper II-III), investigating the health status of Swedish older adults, in particular their prevalence of gut symptoms, and evaluating the effect of a dietary supplement containing a well-characterised probiotic bacterial strain (paper IV). The specific aims for the included papers were to: Paper I.

Explore the core of the concept of optimal functionality in old age.

Paper II.

Explore and characterise a Swedish population of senior orienteering athletes as a potential model of healthy ageing.

Paper III. Explore calprotectin, a marker of intestinal inflammation, and systemic inflammatory status in senior orienteering athletes, and older adults suffering from gastrointestinal discomfort. Paper IV. a) Investigate the over-all health status and prevalence of gastrointestinal symptoms among a Swedish population of free-living older adults, and b) to conduct a double-blinded, randomised, placebo-controlled trial with the aim to investigate digestive health and overall health status after the intake of a dietary supplement, consisting of the probiotic strain L. reuteri, during a three-month period.

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Methods In this section the methods applied in this thesis are presented together with some methodological considerations. Table 1 gives a brief overview of the four included papers. For a more detailed description of study design and procedure please see the respective full-text paper (paper I-IV), included at the end of this thesis. Table 1 Overview of the four included studies, paper I-IV Questionnaires* Analysis

Study

Design/Approach

Study population

Data

I

Scoping study

15 older adults

Literature, (focus group discussions)

II

Cross-sectional, descriptive, mixed-methods

136 senior orienteering Questionnaires, athletes (122 for ques- focus group tionnaire data and 14 discussions for focus group discussions), 238 older adults

EQ-5D-5L, FGAS, GSRS, HADS, HI

Mann-Whitney u-test, inductive content analysis

III

Cross-sectional, explorative

30 senior orienteering athletes + 20 older adults with gut problems

Questionnaires, calprotectin (faeces), CRP and FORT (plasma)

FGAS, GSRS

Spearman correlation, Mann-Whitney u-test

IV

Cross-sectional, descriptive; longitudinal, randomised controlled trial

307 older adults

Questionnaires

EQ-5D-5L2, Mann-Whitney FGAS, GSRS1, u test, Students HADS2, HI, t-test MMSE, MoCA, PSS2

Scoping review

*: EQ-5D-5L = EuroQol; FGAS = Frändin-Grimby Activity Scale; GSRS = Gastrointestinal Symptoms Rating Scale; HADS = Hospital Anxiety and Depression Scale; HI = Health Index; MMSE = Mini Mental State Exam; MoCA = Montreal Cognitive Assessment; PSS = Perceived Stress Scale (further presented in Table 2) 1: Primary outcome parameter in the randomised controlled trial 2: Secondary outcome parameters in the randomised controlled trial

Sample This thesis classifies individuals’ of ≥65 years of age as older adults. Three populations of older adults have been included in the four papers: 1) senior orienteering athletes, i.e. older adults that are actively practicing

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orienteering4, 2) free-living older adults, i.e. older adults living in ordinary housing (as opposed to for example in nursing homes) and 3) older adults displaying gut problems, as estimated by self-reported data from the Gastrointestinal Symptoms Rating Scale. The study populations for each study are briefly presented in Table 1.

Scoping study (Paper I) Paper I presents a scoping study, executed as suggested by Arksey and O’Malley in 2005 (76). Performing a scoping study is a process whereby suitable literature is identified, examined, and conceptually reviewed. Arksey and O’Malley identified five stages to be followed when conducting a scoping study: 1) the identification of a research question, 2) finding the relevant studies, 3) the selection of studies to be included in the review, 4) data extraction from the included studies and 5) assembling, summarizing, and reporting the results of the review. The research question addressed in paper I was to identify the core of the concept of optimal functionality in old age, by exploring already known factors and functions of importance to the older adult’s health experience. The primary step was to identify a suitable search term to retrieve appropriate literature from scientific databases. Conducting an initial search using the term ‘optimal functionality’ only retrieved articles focusing on physiological functionality and did not generate relevant articles to define the concept of optimal functionality from a more holistic perspective. Hence, we choose to turn to our target group, the older adults themselves, to aid in the matter of how to define the search term. In this purpose, we conducted two focus group discussions (FGDs), addressing the topic of how older adults perceive and maintain health, including 15 older adults ≥65 years of age. From the FGDs it became clear that experiencing life satisfaction, on a personal level, serves as an important foundation for a positive health interpretation, which was further facilitated by an active life style both on the physical and social level. Ergo, personal satisfaction serves as a basis for the experience of optimal functionality, as you may not function optimally in a holistic perspective without experiOrienteering is an endurance running sport that is performed outdoors. The athletes navigates their way through the landscape by the help of a map and a magnetic compass, passing obligatory control points along the course. The individual executing the correct course in the shortest time stands as the winner of the race. 4

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encing personal satisfaction. In addition, personal satisfaction is listed as a Medical Subject Heading (MeSH)5 term. Conducting a database search using this MeSH term, combined with elderly OR older adults OR old age OR old persons, retrieved research articles on the subject of perceived health, wellbeing and satisfaction. Personal satisfaction was thus judged to target the core of optimal functionality and, hence, this MeSH term was selected for the current scoping study; in an attempt to initially define the concept. Two scientific databases were chosen for the literature search: PubMed (US National Library of Medicine, Bethesda, MD, USA) and Cumulative Index to Nursing and Allied Health Literature (CINAHL). Inclusion criteria were: 1) complete peer-reviewed full-text articles reported in English, 2) defining older adults as 65 years and older, and 3) conducted in Western countries (including Australia and New Zealand). The date range was set from January 2002–July 2013, to reflect the current research performed during a 10-year period. The search retrieved a total number of 2454 titles over the two databases combined, of which 23 were duplicates. After the selection process (as described in paper I) 25 English, peerreviewed, full-text articles remained to be included in the review. The 25 articles included in the final review were analysed in a step by step process whereby: 1) factors related to the key search term were identified and extracted from the result section of each article; 2) the identified factors were organised into nine categories, i.e. mental aspects, activity aspects, autonomy aspects, capability aspects, social aspects, adjustment aspects, demographic aspects, health aspects, and environmental aspects; 3) the nine aspects were collapsed into three major themes, i.e. self-related factors (including mental; capability; adjustment aspects), body-related factors (including autonomy; health; activity aspects) and external factors (including demographic; social; environmental aspects). Consequently, the theme self-related factors focuses on mental wellbeing; the theme bodyrelated factors emphasizes physical wellbeing; while the theme external factors address the importance of demographics and environmental factors.

MeSH is an abbreviation of Medical Subject Heading, which is a controlled vocabulary for indexing scientific articles developed by the U.S. National Library of Medicine.

5

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Considerations of the scoping study The scoping study methodology aims to map the key concepts underpinning a research area that has not been comprehensively reviewed before (77) . In contrast to a systematic literature review, which commonly aims to provide answer to a question based on a relatively narrow range of quality assessed studies, a scoping study is less likely to address a very specific research question but rather to address wider topics where papers with any kind of research design may be included. A scoping study is thus a preferable method to initially map relevant literature in a certain field of interest. Hence, we found this method suitable for addressing the aim of Paper I, i.e. to initially explore the core of the concept of optimal functionality in old age. An advantage of the scoping study is that it may provide a comprehensive and transparent way to map a research area in a relatively short time as compared to a full systematic review. However, it would be incorrect to mistake the scoping study for a “quick” method, as it demands a rigorous work and a thorough study of the literature in order to synthesise and interpret the collected data (76).

Mixed-method (Paper II) The study presented in Paper II was designed as a mixed-method study (78), in which quantitative questionnaire data was integrated with qualitative data from focus groups discussions (FGDs). The included questionnaires are presented in Table 1 and are briefly described in Table 2. In total 136 senior orienteering athletes were recruited (Figure 1). For the quantitative part of the study 122 senior orienteering athletes were recruited based on the start list of the 2013 years O-Ringen6 international orienteering event. For the qualitative part of the study 14 (7 females and 7 males) active senior orienteers were recruited at local orienteering events, in order to facilitate their participation in the FGDs held at Örebro University. The orienteers were divided into two groups based on sex, under the presumption that female and male orienteers may have different experiences of their sport to share. The two FGDs were based on a semi-structured interview guide, including broad questions on the subject of health and physi-

O-Ringen takes place annually at different locations in Sweden and is the world’s largest orienteering event, yearly attracting 15.000–20.000 participants. Senior orienteers from the whole country, south to north, participate in the event. 6

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cal activity. Each FGD was audio-recorded with the participants’ permission and lasted 60–90 min. 14 senior orienteering athletes recruited at local orienteering events

122 senior orienteering athletes competing in 2013 O-Ringen

A

B#

Quantitative data: Questionnaires

D

Qualitative data: Focus group discussions

Quantitative data: Multiple-choice question

C#

Figure 1 overview of the mixed-methods design A) 122 orienteers ≥65 years were recruited from O-Ringen for questionnaire data collection, B) 14 orienteers were recruited at local orienteering events for FGDs, C) a multiplechoice question was developed based on the FGD findings and D) sent out to the 122 O-Ringen orienteers.

Content analysis The FGDs in paper II were transcribed and subsequently analysed by inductive content analysis, using an open coding approach (79). The following questions were formulated to use as a basis for the analysis: 1) “What is perceived as health and how is health maintained?” and 2) “What contributes to continuous engagement in physical activity?”. All focus group transcripts were read carefully and notations about meaningful content were made in the margins of the transcript documents. Headings were then collected from the margins and transferred to a coding sheet. To group the headings “sub-categories” were freely originated. The generated list of sub-categories was then organized under higher order headings, creating so-called “generic categories”. At this step of the analysis the data were classified as belonging to a particular group by distinguishing between data bearing separate meanings. The generic categories were further sorted into main categories depending on their relation to the three areas of: self, body, and external factors. An example of the analysis structure is provided in Table 3. 28

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Table 2 Overview of the questionnaires, included in paper II-IV Score Included in range paper:

Questionnaire

Variable

Scoring

EuroQol (EQ-5D, EQ-VAS) (80)

Measure of health status.

Five dimensions rated on a 5-point 1-5a scale using fixed respond alterna- 0-100a tives. One question rated on a VAS scale ranging from 0-100.

II, IV1,2

Frändin-Grimby Activity Scale (FGAS) (81)

Measure of habitual physical activity over a whole year.

Estimates physical activity summer 1-6a and winter using 6 fixed respond alternatives describing different levels of physical activity.

II, III1, IV

Gastrointestinal Symptoms Rating Scale (GSRS) (82)

Measure of gastrointestinal health, constructed by five domains: diarrhoea, indigestion, constipation, abdominal pain and reflux.

Fifteen questions rated on a 7point scale using fixed respond alternatives.

1-7b

II, III1,2, IV

Health Index (HI)

Measure of general health, rated on nine questions about energy, temper, fatigue, loneliness, sleep, dizziness, bowel function, pain and mobility.

Nine questions rated on a 4-point scale using fixed respond alternatives.

9-36a

II, IV1

Measure of psychological distress divided into two subscales: depression and anxiety.

Fourteen questions (7 questions per subscale) rated on a 4-point scale using fixed respond alternatives.

0-42b

II, IV1,2

(83)

Hospital Anxiety and Depression Scale (HADS) (84)

Mini Mental State Cognitive screening test. Exam (MMSE) (85)

Eleven tasks evaluating orienta0-30 a tion, memory, attention, language and visouspatial abilities.

IV1

Montreal Cognitive Assessment (MoCA) (86)

Cognitive screening test.

Eleven tasks evaluating visouspatial abilities, naming objects, attention, language, abstraction, delayed recall and orientation.

0-30 a

IV1

Perceived Stress Scale (PSS) (87)

Measure of perceived stress, providing a total score.

Ten questions rated on a 5-point scale using fixed respond alternatives.

0-40b

II, IV1,2

a: High scores are favourable b: Low scores are favourable

1: The questionnaire was included in the descriptive study part 2: The questionnaire was included as an outcome in the RCT

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Table 3 Example of the FGD analysis structure Question used as basis for the analysis: What is perceived as health and how is health maintained? Sub-category1 Basic hygiene

Generic category2 Basic hygiene

Getting along with your gut Good gut-health

Good gut-health

Everyday exercise Keep Moving Not being still (not sitting at home) Physical Activity To be physically strong Feeling mentally stronger than others

Physical activity

Having a good time

Having a good time

Feeling desire to move Feeling desire to eat Keep a positive attitude towards life Wanting to do things Being in the forest Being outdoors Enjoying nature Enjoying the fresh air Exercising outdoors Fresh air and sunshine Picking berries and mushrooms (in the forest)

Staying positive to life

Experiencing culture To be a part of the cultural community To go to the theatre and cinema To keep up with news To read books To read magazines

Cultural engagement

Feeling mentally strong

Being outdoors

Main category3 Body-related factors

Self-related factors

External factors

1) Sub-categories were constructed based on text extracts from the interview transcript. 2) The subcategories were subsequently collapsed into generic categories and 3) organised under the three main categories of body-related, self-related, and external factors.

Considerations of the mixed-methods design The core assumption of mixed-methods research is that the combination of quantitative and qualitative approaches provides a more comprehensive understanding than either one could achieve separately (78,88). Integrating quantitative results with qualitative results are thus supposed to add dif30

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ferent perspectives and strengths to the understanding of a phenomenon (89) . As such, mixed-methods designs may be used in order to expand the scope of a study. A mixed-method study can be designed and executed in a large variety of ways, but primarily have three purposes: 1) triangulation, to ensure validation of data, 2) development, to guide the use of additional data collection or suggest other techniques of analysis and 3) complementarity, to clarify, explain, or more fully elaborate the results of a study (90). Paper I presents quantitative data from questionnaires, together with qualitative data from FGDs; here the qualitative data was collected in order to enrich the interpretation of the questionnaire data. Hence, our mixed-method approach in the current study falls under the purpose of complementarity, as suggested above. Further, in the purpose of triangulation a multiplechoice question was developed based on the FGD results, as schematically outlined in Figure 1, in order to validate these in a larger setting.

Assessment of inflammation and oxidative stress (Paper III) Paper III is a cross-sectional study exploring the relationships between guthealth, physical-activity, inflammatory status and oxidative stress among senior orienteering athletes (n=30) and older adults suffering from gut discomforts (n=20). In this purpose the levels of C-reactive protein (i.e. systemic inflammation) and hydroperoxide concentration (i.e. oxidative stress) were measured in plasma, as well as calprotectin (i.e. local inflammation) in stool samples. The C-reactive protein (CRP) is a historically highly preserved acutephase plasma protein of hepatic origin (91). Since its discovery, CRP has been studied as a screening device for inflammation, a marker for disease activity, and as a diagnostic tool (92). CRP is reported to increase rapidly in response to bacterial and viral infection, tissue injury and trauma (93), but modest increases have also been described as a sensitive marker of lowgrade inflammatory states (94), such as in ageing. In this thesis CRP was analysed in blood plasma using the high-sensitivity immunoturbidimetric assay CardioPhaseTM using the ADVIA 1800 chemistry system (SIMENS Healthcare Diagnostics Inc., NY, USA) at the Clinical Chemistry laboratory, Örebro University Hospital, Sweden. Hydroperoxide concentration in plasma may be used as an estimator of oxidative stress load. Oxidative stress arises as an imbalance between systemically circulating reactive oxygen species and an individual’s detoxification capacity. Increased oxidative stress is thought to accelerate human LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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ageing (52) and particularly hydrogenperoxide, which is considered to be the major ROS within the cell, is a potent inducer of senescence7 in many cell types (95). Oxidative stress was evaluated by a colorimetric assay named the Free Oxygen Radicals Test (FORT) from Callegari (Parma, Italy) using blood plasma. Calprotectin is an intracellular calcium binding protein, constituting about 60% of the total cytosolic protein content in neutrophils. Due to its strong correlation with neutrophil infiltration of the intestinal mucosa, calprotectin has been suggested as a marker of intestinal inflammation (96,97) . Increased levels of calprotectin in faeces have been described in several gastrointestinal disorders e.g. irritable bowel syndrome and inflammatory bowel disease. In addition, there have been indications of elevated calprotectin levels in older adults (98). Faecal calprotectin was measured by the CALPRO® (CALPRO AS, Lysaker, Norway) at the Clinical Chemistry laboratory, Örebro University Hospital, Sweden. CALPRO® is an enzymelinked immunosorbent based assay developed for the measurement of calprotectin in stool samples.

Considerations of the biological parameters assessed All biological samples were collected and analysed according to standardised and established methods. However, the processes of inflammation and oxidative stress are complex and extensive networks practically impossible to grasp by analysing single molecules. Hence, when targeting these biological processes the methods of choice are likely to fall short. For example, a major characteristic of the immunosenescence in ageing is the up-regulation of a multitude of pro-inflammatory agents, for example there is an increase in type 1 and type 2 positive CD8+ T-cells secreting a large variety of inflammatory cytokines, such as IL-2, IFN-γ, TNF-α, IL-4, IL-6 and IL-10 (40). CRP, which was included as the measure of systemic inflammation in paper III, fails to capture the complexity of these phenomena. However, CRP has been found to correlate well to IL-6 (61), which has been described as the interleukin for gerontologists (99). Possibly, supporting CRPs suitability for estimating systemic inflammation in this particular age group. The FORT method, evaluating oxidative stress, suf7

The term ‘senescence’ originates from Latin, meaning ”to grow old”. In the context of biological ageing it refers to the gradual deterioration of function that eventually leads to the death of an organism or a cell.

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fers from similar shortcomings, particularly as oxidative stress occurs as a result of free-radical exposure and antioxidant depletion, i.e. an oxidantantioxidant imbalance (100). The FORT method merely measures the presence of hydroperoxide in plasma samples and thus fails to evaluate the antioxidant part of the system. Similarly, evaluating local inflammation by calprotectin gives a narrow picture of the inflammatory climate in the intestine, as it mainly correlates to neutrophil influx. In future studies these shortcomings may be addressed by including multiple measures of each phenomenon. However, the methods used in paper III are routinely applied in contemporary health-care and disease diagnosis and may be considered as robust measurements.

Randomised controlled trial (Paper IV) Paper IV presents a 12-week randomised controlled trial (RCT), investigating the effect of a probiotic dietary supplement on the gut-health and wellbeing of 307 older adults. A RCT is a type of study in which participants are randomly assigned to one of two (or more) clinical interventions to assess their effects on some selected parameters. The current RCT aimed to investigate the effect of the probiotic strain L. reuteri versus placebo on perceived health among older adults, as evaluated by self-reported questionnaires. See Table 4 for an overview of the study design and Table 2 for a brief presentation of the included questionnaires. As the health status of older adults may vary greatly the individuals enrolled in this RCT were recruited under the premise to execute the intervention and data collection in their home. Thus, a major challenge of this RCT was to organise home-visits to all 307 participants. To solve this logistic problem the study participants were divided among a suitable number of medical students (n=17) whom volunteered to aid in the data collection. The medical students, currently undergoing education at Örebro University, were introduced to the study and how to perform the data collection in a standardised manner. The medical students performed a home-visit to all their assigned study participants to collect the baseline data. Depending on how much assistance the older person needed to complete the questionnaires the medical student continued to visit the older adult’s home for each data collection time-point (i.e. time-point 0, 8 and 12 weeks of the trial) or made a contact over the telephone, at week 8 and 12, after the initial visit. At the end of the trial the medical students also transferred the questionnaire data, collected on paper, to digital form by entering the data in pre-prepared excel tables. The data could then be LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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linked together by using the participants individual study code, thus ensuring anonymity for each participant throughout the data analysis. Table 4 RCT overview Prior study start Time-point 0 weeks Month -1 Week -4-0

Study start Month 1 Week 1-4

Study on-going Time-point 8 weeks Month 2 Week 5-8

Study finish Time-point 12 weeks Month 3 Week 9-12

Baseline data: Demographic data, medications, physical activity, health status, diet

X

GSRS (primary outcome)

X

X

X

EQ-5D-5L (secondary outcome)

X

X

X

HADS (secondary outcome)

X

X

X

PSS (secondary outcome)

X

X

X

Stool frequency: daily diary (secondary outcome)

X

X

X

X

Before enrolment initial sample size estimation was made based on the distribution of gastrointestinal discomfort in a pilot population consisting of 40 older adults; 50% of the population was identified to suffer from gastrointestinal problems. However, to make a more specific power calculation, the 100 first participant enrolled in the RCT also completed the GSRS questionnaire, in order to identify a more reliable prevalence of gastrointestinal discomfort in the actual study population. According to this data 74.2% were identified to suffer from gastrointestinal discomfort. Thus we made the estimation that a sample size of 300 individuals would provided 80 % power to detect a minimum clinical improvement in gastrointestinal discomfort in 20% of the individuals in the intervention arm and in 5% of the placebo arm, allowing an estimated dropout rate of 17%. The power estimation was based on the presumption to remain within the 95% confidence interval. The primary outcome measure of the intervention was set to improvements of gut-health, as assessed by GSRS score. An improvement of 0.5 units was judged as clinically relevant. For secondary outcomes we chose improvements in HADS (psychological distress), PSS (perceived stress) and EQ-5D-5L (general health) score. All analyses were preformed blinded and

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followed the same analysis procedure as described below and briefly overviewed in Figure 2: As an initial step all data underwent an intention-to-treat8 (ITT) analysis. The ITT was followed by a per-protocol analysis; taking into account compliance and excluding dropouts. Subsequently we performed a responder-analysis, focusing on the subject whom had responded to the given treatment during the intervention. A responder was considered a subject compliant to the protocol and experiencing a relief in symptoms by a score of 0.5 (the minimal clinical relevant change) on the mean value of GSRS total score or on any of the GSRS domains. As a consequence of the responder analysis we found that the intake of gastrointestinal motility modulating substances (GMMS) and proton pump inhibitors (PPI) (as classified by the Swedish environmental classification of pharmaceuticals, available at www.fass.se) influenced the end point analysis. Hence, all study subjects whom had reported to take these medications were excluded from further analysis (n=71). Subsequently the median and mean score of the GSRS questionnaire for all subjects, excluding the ones taking GMMS and PPI but including non-compliant participants, were calculated for week 8 and 12 and tested against each other. The reasoning behind including the non-compliant subjects was to gain more power to the analysis. To stay true to the data the analyses were also performed excluding the non-compliant subjects, but since it only had minor effects on the results a decision was made to present the analysis including the noncompliant subjects. As a final step, all subjects reporting baseline problems at any of the outcome parameters were included in a mean value analysis to evaluate if the probiotic treatment would have a more pronounced effect in the ‘problematic cases’.

RCT considerations The RCT has been described as the most rigorous method of hypothesis testing available (101) and is thus regarded as the gold standard for evaluating intervention effectiveness. However, the quality of an RCT is highly dependent on the validity of the trial methodology (101). In order to assure

An ITT is based on the treatment assigned the study participants, rather than the treatment actually received. Hence, all subjects enrolled in a study have to be included in the analysis, regardless of drop-outs, lack of compliance or missing data, to be in accordance with the ITT principal. 8

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ITT analysis: including all subjects Per-protocol analysis: including compliant subjects, excluding drop-outs Responder analysis: including compliant subjects whom responded to treatment Mean/median value analysis: excluding PPI and GMMS using subjects but including non-compliant subjects

Excluding all PPI and GMMS using subjects

Mean/median value analysis: including subjects with baseline problems, while excluding PPI and GMMS using subjects but including non-compliant subjects

Figure 2 Overview of RCT analyses

quality the process of randomisation is of utter importance. Randomisation refers to the process of randomly assigning study participants to the treatment groups and should ensure equal probability of being assigned to any of the given treatments. Hence, randomisation is meant to balance potential confounding factors and to avoid selection bias; the main purpose being to create a control group that is as similar as possible to the treatment group. In the RCT of paper IV, block randomisation in sections of six were applied, i.e. each randomisation block contained three boxes with active treatment and three boxes with placebo in different combinations. To further facilitate blinding the randomisation was outsourced to a third-party to ensure allocation concealment (102). In addition, the knowledge about treatments was unknown to the study participants and the research team until after the study was finished and the statistical analysis was completed. Blinding may be undertaken to increase the quality of an RCT (103). Blinding refers to a process of preventing the people involved in an RCT to be influenced by their knowledge of the treatment allocation. Since knowledge of the received treatment could influence the

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outcome of an RCT in terms of an increased placebo effect9 among the study participants, differences in management of the participants during the trial or a priori knowledge when undertaking the statistical analysis.

Statistics The statistical analyses in paper II and III were performed using GraphPad Prism version 6 for Mac (GraphPad Software, San Diego California, USA) and for paper IV r version 3.0 (Auckland, New Zeeland) was used. Median values were reported together with their inter-quartile range (IQR) and mean values together with standard deviation (SD). The statistical tests used in each study are presented in Table 1. In case of statistical testing for between-group differences a p value of 0.05 was considered to be statistically significant. Missing questionnaire values were replaced with the arithmetic mean of all the completed items, in accordance with the instructions for each questionnaire. If missing values occurred in proportions that did not allow for imputation the individual questionnaire was excluded from analysis.

Ethical considerations All studies presented in this thesis have been conducted in accordance to the Declaration of Helsinki. The Regional Ethical Committee in Uppsala, Sweden, approved the research (dnr. 2012/309 and 2013/37) and written informed consent was obtained from all study participants. All participants were provided with a study code to avoid individual recognition throughout the process of data collection and analysis. Concerning the focus groups discussions (FGD) in paper II a verbal commitment to keep all information about the FGD content, as well as the personal information of all participants, confidential was made before the interviews started. The participants were also instructed that they had the right to refuse to answer to any question or withdraw from the interview at any time. The clinical trial, presented in Paper IV, was registered on clinicaltrials.gov (ID: NCT01837940).

‘Placebo effect’ is a term describing improvements in individuals receiving placebo, i.e. experiences of positive treatment effects from the inactive treatment. 9

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Public outreach As a part of the knowledge development all researchers are obliged to engage in public outreach, i.e. to communicate science to the general public. In Sweden this commitment is stipulated in The Higher Education Act (Högskolelagen 1992:1434), declaring that interplay with the surrounding society is crucial, as is communication of information about the research produced at each university to make the knowledge publicly available and useful. Working with this thesis me and my co-supervisor, Ida Schoultz, have had regular contacts with the research communication department at Örebro University. This has resulted in several popular scientific contributions to newspapers and interviews in national radio, as well as appearances on national television. Further, we have organised multiple meetings to present the results to our study participants and also kept them updated on the progress of our studies by email or postal communications.

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Results This thesis is based on four papers, each of which gives a different perspective on older adults health. Briefly, Paper I explored the concept of optimal functionality in old age, as a new approach to put the older adult’s own perspectives on health in focus. According to its results a plethora of factors related to the body, the self and the external environment needs to be acknowledged in order to create a comprehensive understanding of the older adults experience of health and function. Among the key factors found were: mental health, physical function and social relations. The results from this paper fuelled the idea to explore senior orienteering athletes as a new model of healthy ageing, since they are likely to fulfil the key factors of optimal functionality and successful ageing. Hence, in Paper II senior orienteering athletes were characterised as a new potential model of healthy ageing. Their health was found to be significantly better than that of other free-living older adults; in particular they displayed a better gut-health. Since the gut has been proposed as an important player in the health maintenance and immune function of older adults this result raised the curiosity to whether or not the superior guthealth of senior orienteers would be reflected in some common measures of inflammation and oxidative stress. Thus, paper III explores inflammation and oxidative stress among senior orienteering athletes, and older adults with gut complaints. The paper reports low levels of inflammation and oxidative stress in the orienteering athletes. However, few differences were found in comparison to the older adults reporting gut problems, suggesting that the high health and low gut complaints among the senior orienteering athletes were not particularly well reflected in the studied parameters. In Paper IV the health of free-living older adults was further investigated and a RCT was performed in order to evaluate the effect of a probiotic supplement on gut symptoms and self-reported health. Despite a relatively good health status two-thirds of the older adults included in the study reported gut problems. In addition, the RCT failed to show any significant effects on either perceived gut-health or wellbeing of the older adults. In the sections below follows a summarised description of the results from each of the included papers, for more detailed information please see the respective paper.

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PAPER I. Optimal functionality in old age is a multifaceted concept Paper I suggests that the concept of optimal functionality is comprised of a multitude of factors that may be organised into three major themes, i.e. self-related, body-related, and external factors; all of which needs to be considered in the context of the older adult’s health experience. An overview of the structure of the concept is provided in Figure 3. The three aspects of mental health, capability and adjustment were found to comprise the self-related factors of optimal functionality. The mental health aspects were represented by factors such as psychological wellbeing, absence of worry, feeling valued and having an extroverted personality. The capability aspects consisted of factors such as having a problem-based coping style, previous achievements in life, spirituality and religiosity. Moreover, factors such as keeping a positive attitude, thriving, enjoying small things and focusing on positive events represented the adjustment aspects.

Main%concept%

Environment'

Demographics'

Social'rela7ons'

External%factors%

' e.g.'social'network,' Social'ac7vi7es''' ' e.g.'educa7onal'level,' economy' ' e.g.'feeling'at'home,' living'in'a'homeBlike'environment' '

Adjustment'

Capability'

Body5related%factors% ' e.g.'independence,' Autonomy' func7on'' ' e.g.'gastrointes7nal'complains,'' Physical'health' musculoskeletal'complaints' ' e.g.'physical'ac7vity,' maintained'condi7on''' Physical'ac7vity' '

Factors%

' e.g.'psychological'health,' absence'of'worry' ' e.g.'problemBbased'coping,' previous'achievements'' ' e.g.'posi7ve'aCtude,' enjoying'small'things' '

Aspects%

Self5related%factors% Mental'health'

Major%themes%

OPTIMAL%FUNCTIONALITY%IN%OLD%AGE%

Figure 3 Overview of the optimal functionality structure

The body-related factors were comprised of activity aspects, health aspects and autonomy aspects. Among the activity aspects, factors such as 40

LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

being physically active and maintaining physical condition were found. While the health aspects comprise the factors of gastrointestinal disturbances, comorbidity, musculoskeletal complaints, exhaustion, fractures and being on medications. Further, independence, functioning as one would like and maintaining dignity were factors representing the autonomy aspects. Furthermore, the three aspects of social relations, demographics and environment were found to comprise the external factors of optimal functionality. The social aspects were represented by factors such as having a well-established social network, receiving emotional support from family and friends, and participating in social activities and cultural events. The demographic aspects were age, sharing ones life with someone (e.g. a spouse), education level and economy, while the environmental aspects covered factors such as feeling at home and living in a home-like environment. In particular, individuals living in ordinary housing show greater life satisfaction than nursing home residents, as do individuals living in supportive neighbourhoods. In summary, the results from Paper I indicate that maintained mental health together with a problem-focused coping style and the ability to adjust are essential for experiencing optimal functionality in old age. As is, being physically active, having maintained function and being independent in everyday life. Furthermore, a well-established social network influences perceived health in a positive way, while experiencing disease or gastrointestinal disturbances have a negative influence. Demographic factors, e.g. marital status, educational level, and financial stability were further of importance to the experience of optimal functionality. Taken together the factors presented in paper I make up the current knowledge about optimal functionality in old age.

PAPER II. Senior orienteering athletes may be considered a new model of healthy ageing Paper II suggests that senior orienteering athletes may be considered a new model of healthy ageing. The orienteers included in this study had been actively orienteering for an impressive amount of years, ranging between 30-57 years. None of the included orienteers smoked at the time of the study and their number of medications was low. As expected the physical activity among the senior orienteering athletes were significantly higher than that of the general LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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population of older adults. In addition, they reported to exercise on the same high level throughout the whole year, despite orienteering being a “summer sport”. Many of the orienteers described how they for example replaced the running with skiing in the winter. The orienteers further displayed favourable scores on all the included health-related questionnaires (see Table 1 and 2 for further information on the included questionnaires), which in general were significantly higher in comparison to a population of free-living older adults. In particular the significantly lower gut complaints among the senior orienteering athletes as compared to the freeliving older adults stood out, as gut problems are common among the general population of older adults. The only questionnaire that did not display any significant differences between the two groups was the Hospital Anxiety and Depression Scale. The high subjective health displayed among the senior orienteers was further confirmed in the focus groups discussion (FGDs) about health and physical activity. The qualitative findings shed further light on the senior orienteers experience of health and factors that drives their continuous engagement in physical activity. For example, the orienteers pointed out that their perceived health is strongly connected to the way they interpret their physical fitness. Experiencing body-related health was directly dependent on their ability to be physically active, including feeling physically strong and performing outdoor activities. The orienteers also discussed the importance of health promotion, i.e. preventing ill-health and injuries by having regular health check-ups. Furthermore, maintained gut-health was described as an important factor to experience health, as a bad gut may largely impact the overall health experience of anyone. In addition, the senior orienteering athletes talked about the importance of mental health, i.e. feeling mentally fit as compared to other older adults. Experiencing health was further described as being able to stay positive in life, feeling the desire to move and eat, keeping a positive attitude and maintaining the will to experience new things. The orienteers also talked about the importance of keeping your brain active, for example by taking university courses or doing crossword puzzles. The orienteers also stated a number of external factors of importance for their health experience, e.g. eating well-tasting food and experiencing cultural activities. Also, social engagement was considered vital for experiencing health. For example, the orienteers discussed the importance of having a social network in different areas of life. They all agreed on the importance of expanding one’s social network outside of orienteering, in case they would no longer be able to 42

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continue their active lifestyle. Furthermore, being outside in nature was described as particularly important for their health experience. But also the indoor environment was of significance, i.e. avoiding unhealthy environments and habits, such as visiting smoky bar rooms and drinking alcohol, were regarded as important. During the FGDs the orienteers also talked about their motivation to stay physically active; factors such as having good genes and feeling physically stronger than your peers were discussed, as was the importance to be driven by passion. The orienteers meant that your sport should bring energy and thus can’t be “a must”, but on the contrary ought to be driven by will and desire. Competitiveness was also discussed as a reason for staying engaged in orienteering. The orienteers claimed to have a ’competitive instinct’, at least, to some degree. However, solely focusing on competition was not viewed upon as something positive, instead the fact that you don’t have to win to be appreciated by your fellow orienteers was thought to be the beauty of orienteering. An additional reason for staying engaged with orienteering was that it gives the endurance to cope with other things in life and it improves mood. Having fun was also described as an important prerequisite to stay continuously engaged in the sport; practicing your sport should make you happy. Furthermore, the athletes talked about the significance of growing up in a supportive environment and being surrounded by friends and family that are also engaged in physically activity. In addition, childhood experiences such as having positive memories from the physical education in school, enjoying informal competitions with friends, and the fact that alternative entertainment options were scarce in their youth, was mentioned as prerequisites for staying physically active through life. In addition, the importance of making the physical activity a routine was discussed, as it then becomes an essential part of your life. The orienteers further viewed the opportunity for social interactions as a very important motivating factor. In particular they regard orienteering as a unique sport in that it promotes vast opportunities for inter-generational interaction, i.e. orienteers between 4 and 90 years old are commonly competing at the same events and practice their sport together. The senior orienteers also brought up the importance of being part of a community, referring to the significance of doing things together and belonging to a club. Orienteering was further described as unique in that it is a lifelong sport, particularly promoting prolonged continuous engagement, as there is no age-limit to its practice.

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When subjected to a multiple choice question, developed on basis of the FGD results, the orienteers rated the most important reason to stay physically active as the improvement of physical health, while the least important factor was found to be to perform well at competitions. Furthermore, improvement in mental health was reported as being a stronger motivator to stay physically active than being part of a social context. It should, however, be noted that the majority of the orienteers reported all offered alternatives as equally important for their motivation to staying physical active, except for performing well at competitions, which was substantially less frequently chosen than the other response options. In summary, orienteering is described as a lifelong sport that is more about enjoyment, social interaction and closeness to nature, rather than performance and exertion. The regular engagement in physical activity and map-reading, as “brain-exercise”, together with the unique social climate points towards orienteering as an ideal senior sport. Supported by the low medication and the high levels of self-reported health among the senior orienteering athletes studied in paper II.

PAPER III. The levels of inflammation and oxidative stress are low in senior orienteering athletes Paper III reports low levels of inflammation and oxidative stress in senior orienteering athletes. However, few differences were found in comparison to older adults experiencing gut discomfort. Suggesting that the high health and low number of gut complaints among the senior orienteering athletes was not particularly well reflected in the studied parameters. The senior orienteering athletes enrolled in this study reported a median physical activity corresponding to 1-2 hours per week of physically exercise, such as running. The median was the same for both summer and winter scores, indicating that the senior orienteers preform the same amount of physical activity throughout the whole year, irrespective of seasonal changes. Concerning gut-health the total Gastrointestinal Symptoms Rating Scale (GSRS) score was reported at a low level, i.e. the senior orienteers display a good gut-health with few reported complaints. The systemic inflammation, as measured by CRP, was reported within the range of what is considered normal, indicating that there is no increased inflammation among the senior orienteering athletes despite their high age. The circulating free radicals corresponded to an intermediate level of oxidative stress, suggesting that this population of older adults 44

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display oxidative stress at a relatively low level. Further, the median level of gastrointestinal inflammation, as measured by calprotectin, was below the assay detection limit as indicative of low levels of intestinal inflammation; corresponding well to the high gut-health reported in this population. Correlation analyses of the measured parameters only showed modest relationships: systemic inflammation was found inversely correlated to the level of physical activity, while positively correlated to the GSRS reflux domain and to oxidative stress load. In addition, oxidative stress load was found positively correlated to intestinal inflammation. A group of older adults with gut problems was also included in the study to investigate if this health difference would be reflected in levels of inflammation and/or oxidative stress. The older adults displayed significantly higher levels of systemic inflammation as compared to the senior orienteering athletes, however, their median value was still below the cutoff for what is considered normal. Despite the poorer gut-health among the older adults, their levels of oxidative stress and faecal calprotectin did not differ significantly in comparison to the orienteers. In summary, senior orienteering athletes display low levels of systemic and intestinal inflammation, as well as oxidative stress. The senior athletes also demonstrate a high level of self-reported physical activity in combination with low gut complaints, as previously shown in Paper II. In comparison to older adults with gut problems they show significantly lower systemic inflammation, however, there were no differences found in intestinal inflammation or oxidative stress. Moreover, only modest correlations were found between the investigated parameters, the strongest one being the positive relationship between CRP and oxidative stress, as well as between oxidative stress and calprotectin.

PAPER IV. Probiotic supplementation shows no significant effects on gut complaints or self-reported health in free-living older adults Paper IV shows that Swedish free-living older adults display a relatively high self-reported health, in combination with a high prevalence of gut problems. Twelve-week daily supplementations with the probiotic strain L. reuteri did, however, not improve these gut complaints and had no further effects on self-reported health.

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In the descriptive part of this study, investigating the self-reported health in a sample of Swedish free-living older adults, 2/3 of the enrolled participants reported problems in at least one of the GSRS domains. Analysis per symptom domain revealed that half of the participants had problem with indigestion (i.e. discomfort related to the upper parts of the gastrointestinal system), approximately one third reported problems with constipation or diarrhoea, while a fourth suffered from abdominal pain, and a sixth were identified to suffer from reflux. The subjects displaying the most severe gastrointestinal problems predominantly suffered from diarrhoea or constipation. In addition, it was common in this population to suffer from 2 or more gastrointestinal symptoms at the same time. Cognitive function was evaluated in a subset of the population, namely the first 100 enrolled subjects. These study participants reported a mean value indicating adequate cognitive function on one of the included screening instruments, the Mini Mental State Exam. However, the mean value on the Montreal Cognitive Assessment (MoCA) score was found to be just under the cut-off indicative of mild cognitive impairment. In addition, the study population was divided into three age categories (age category 1: 6570 years; 2: 71-80 years; 3: >80 years), in order to evaluate if any of the measured health parameters were affected by age. No significant differences were found between the three age groups, however, a tendency toward more severe gut complaints, as measured by total GSRS score, as well as lower cognitive function, as measured by MoCA score, was observed with increasing age. For the randomised controlled trial (RCT), investigating the effect of a probiotic dietary supplement on self-reported health among older adults, 290 older adults were randomised to 12-weeks of daily treatment with L. reuteri or placebo. Retention to primary outcome was 85% (249 analysed; 125 probiotic, 124 placebo treatment, respectively); 37 subjects were excluded due to loss to follow up. The primary outcome of the intervention, i.e. the total score of GSRS, did not change markedly in either of the treatment arms during the study. Similarly, no significant differences were observed in the separate GSRS domains, except for the reflux domain where a significant difference between groups occurred at week 8; the reflux symptoms decreased in the placebo group while increasing in the probiotic group. This trend did not, however, persist at week 12. In addition, no significant differences were found for either of the secondary outcomes. If selecting only the participants suffering from gastrointestinal problems at baseline, some small but significant effects were seen 46

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on anxiety at week 8, however, not at week 12. Further, an arbitrary cut off was chosen in order to investigate whether subjects suffering from elevated stress levels at baseline would experience a decrease in stress over the study period. A non-significant trend was found for the probiotic treatment to decrease symptoms of stress at week 12. Concerning general health status as measured by the EQ-5D-index, both the probiotic treatment and the placebo was associated with a significant increase in subjective wellbeing at the 12-week follow-up. In summary, the health of free-living older adults in Örebro County may be considered as good, as estimated by self-reported questionnaires. However, a majority of the older adults tend to suffer from gut discomfort. A tendency towards more severe gut complaints, as well as lower cognitive function, was observed with increasing age. A twelve-week supplementation with the probiotic bacteria L. reuteri could not alleviate the gut complaints and showed no further significant effects on the self-reported health of the older adults.

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Discussion The overall aim of this thesis was to further explore the health and gut function of older adults, by 1) exploring the concept of optimal functionality to increase the understanding of perceived health in old age (paper I), 2) characterising a new potential model of healthy ageing (paper II-III) and 3) investigating the health status of Swedish older adults, in particular their prevalence of gut symptoms, and also to evaluate the effect of a dietary supplement containing a well-characterised probiotic bacterial strain (paper IV). According to paper I the factors of importance to the older adults optimal functionality relates to the body, the self and the external conditions of the older adult. Perceiving one’s health as poor, for example when suffering from poor mental health and decreased physical function or experiencing gastrointestinal discomfort, has a negative effect on the older adult’s health experience. Furthermore, paper II suggests senior orienteering athletes as a new model of healthy ageing on basis of their high level of perceived health. For example, the senior orienteers display a significantly better self-reported health status, and reports less gut problems, than other free-living older adults. However, the difference in gut-health was not particularly well reflected in some common biological measures of inflammation and oxidative stress, as presented in paper III. Furthermore, older adults in general show a high prevalence of gut problems, despite an otherwise relatively good health status. Unfortunately, a non-invasive and easy-to-use treatment strategy to alleviate the gut symptoms, i.e. supplementation with the probiotic bacterium L. reuteri, showed no effect, as reported in Paper IV.

Health in relation to old age This thesis has defined older adults as individuals’ ≥65 years, an age cutoff which have been applied in all four studies, paper I-IV. In most developed countries this age cut-off is commonly accepted for defining an older adult and also correspond to the first opportunity to receive general pension benefits here in Sweden. Although this age cut-off is often used it is, however, not entirely without dispute. The definition of “old age” varies across different settings and over time. In the Friendly Societies Act from 1875 the definition of old age is defined as any age after 50 (104). While, in more recent years, the United Nations and other international agencies have decided on ages >60 years. However, arguments have been raised 48

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that the ages >65 years, or even older, are more appropriate to use, at least in industrialised countries (23,105) where the life-expectancy has increased, such as in Sweden. Choosing a higher age cut-off is likely to generate a sample of older adults displaying poorer health status, as indicated by paper IV, that might be less receptive to health interventions. Such a sample may not be preferable when the goal is to study health with the aim to increase independence and decrease the health-care need in a long-term perspective, as in the current thesis. An alternative to the conventional age cut-offs may be classifying older adults as “young olds” (60-69 year), “old olds” (70-79 years) and “oldest olds” (>80 years). However, all use of chronological age cut-offs to define old age assumes their equivalence with biological age, yet it is quite accepted that they do not necessarily correspond to each other (23). A notion that is supported by this thesis, for example, the senior orienteering athletes investigated in paper II and III display a significantly higher health status as compared to other older adults in the same age range. In general there is a great variance in functional capacity and health status between individuals of any age and, hence, chronological age may be seen as a poor estimator of health status. Particularly individuals’ ≥65 years differ considerably in health status; some suffer from multiple disorders and substantial loss of functionality while others, like the senior orienteers, manage to maintain health and functional capacity late in life. Older adults are thus not a homogenous group in terms of health status, on the contrary, old people seem to become even more different from one another as they grow old, as briefly outlined in the introduction of this thesis. Thus, alternatives such as dividing older adults in to a “third age”, i.e. the period of personal achievement following working life and a “fourth age”, defined as a time of frailty and dependency (106), without any specific age categorisation might be more appropriate as a future approach.

Interpreting health from an individual perspective Since the health status and functional capacity of older adults varies greatly on an individual basis, strategies to address health from an individual perspective are crucial. The concept of optimal functionality, as explored in paper I, is based on the notion that the older adults themselves should be involved in defining the areas of importance to experience the highest achievable level of health, i.e. to optimise ones function on the basis of individual preferences and prerequisites. Which is well in line with the Public Health Agency of Sweden stating that healthy ageing is ‘a process LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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where the opportunities for physical, social and psychological health are optimised so that older adults can take active part in society and enjoy autonomy and a good quality of life, independent of high age’ (107). Furthermore this is supported by Nordenfelts (25) holistic theory of health arguing that the health of a person is to be considered in terms of the individual’s ability to live life as she wishes. Hence, a person’s ability to experience health can only be valued in relation to her goals and preferred way of life. On basis of the papers presented in this thesis (paper I-IV) such specific health goals and preferences are deemed to vary between individuals, for example a senior orienteering athletes is likely to evaluate health based on the ability to run and read maps, while an older adult with gut problems may perceive health as satisfactory when she experience a decrease in her gut discomfort. The senior orienteering athlete thus put higher demands on functional capacity and will interpret health based on the goal of being able to maintain physical and cognitive function as preferred. The intention behind developing the concept of optimal functionality (paper I) is to facilitate the identification of the older adult’s existing demands on functionality. Charting the areas of importance to the individual health experience on the basis of preferences and current health status may be important in a number of health-care related situations, for example in rehabilitation. Re-establishing the experience of health after a bone fracture is likely to be dependent on whether or not the older person is able to live life as before the injury. Thus, mapping the areas of importance for the older adult to function as usual is pivotal to promote the return to a normal life. If enjoying the nature in the company of fellow sportsmen were identified as one of the most important aspects of an older person’s life, the opportunity to still do so will be essential for this individual’s experience of health. Ergo, the concept of optimal functionality is intended to provide a comprehensive overview of the areas and factors that may be of importance to consider in relation to an older individual’s health. However, more work is needed to fully develop this concept.

A new model of healthy ageing In addition to developing means to describe the older adult’s health from a more individual perspective, the steadily increasing segment of the worldwide population reaching the older ages motivates the search for new models to study and elucidate the healthy ageing process. Previously used models, such as centenarians whom have managed to reach the impressive age of a 100 years (22,105), have been criticised as they are often studied 50

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without consideration of the many disabilities commonly displayed in this population (108). Healthy ageing is not exclusively about reaching a high age, but rather about experiencing health and maintaining optimal functionality (109). Hence, identifying cohorts of older individuals experiencing health and maintained functional capacity, at their present age, would seem as a crucial step on the way to identify factors of importance to maintain health through life. This thesis suggests senior orienteering athletes to represent such a group of older individuals (paper II and III). Orienteering is an endurance-running sport, performed outdoors, involving navigation with the help of a map and a magnetic compass, often through a highly diverse terrain. Hence, practicing orienteering demands both excellent physical health and considerable cognitive skills from its sportswomen and men. Orienteering athletes have previously been found to adopt a healthier lifestyle and experience lower frequency of illness compared to the general population (110). In paper II orienteering was described as unique in that it is a lifelong sport, promoting prolonged engagement; long-term practice of leisure-time physical activities may increase the survival of older persons (11). In addition, the orienteering sport is known for its familiar and social atmosphere and harbours a substantial proportion of aged athletes. Thus it is evident that the orienteering sport holds a unique cohort of aged individuals that may be particularly suitable as a new model of healthy ageing. Especially since the ageing process has been suggested as the end-product of disuse and physical inactivity (9), which makes it logical to study groups of older adults that have maintained a high level of physical activity. The orienteering athletes investigated in this thesis (paper II and III) further display a number of factors that are important to experience optimal functionality (109) as well as successful ageing (5–7). One of the strongest predictors of optimal functionality is maintained mental health (paper I), which the senior orienteers likewise identifies as a main driver to be engage in orienteering. The orienteers further discussed the importance of being able to make adjustments if the day comes when they can no longer run, for example building a social network outside of the orienteering sport. The process of adaptation and adjustment has previously been described as an important part in optimal functionality (109) (paper I), as well as in successful ageing (111). In addition, the orienteers talked about the importance of eating healthy food and enjoying mealtimes, which may be an interesting matter to study further since the diet is of importance to healthy ageing. For example, a high intake of fruits and vegetables and low intake of meat may support cognitive LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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functioning in older adults (112) and also habitual dietary patterns may drive gut microbiota alterations in varying rates of health decline in ageing (60) . The orienteering sport further seems to facilitate a number of other factors of importance to maintain health and optimal functionality, for example the regular engagement in physical activity, maintained physical functioning and gut-health, as well as access to an extensive social network. The orienteers particularly points out the significance of interacting over the “generation borders”, i.e. enjoying the company of younger orienteers that shares the same interest. This might be an attractive factor to investigate further in relation to health maintenance and optimal functionality. Moreover, sharing similarities with our proposed model of healthy agers are master athletes, primarily as they maintain a high physical activity late in life. It has been suggested that these aged athletes provide the best model for understanding the essentials of human ageing (9). However, this fact is not without dispute, as former elite athletes also show elevated risk of a number of health aberrations, as outlined in the introduction. Hence, prolonged high intensity exercise into the late ages might have adverse health consequences. According to the orienteers investigated in paper II physical exercise must be driven by passion and enjoyment, and the body must be carefully looked after. The orienteers further emphasized that it is of importance to maintain the balance between pleasure and physical exercise. In addition, their reported median physical activity level, i.e. 1-2 hours strenuous exercise per week, corresponds quite well to previous epidemiological data suggesting that 150 minutes a week of moderate exercise is enough to gain a survival advantage (14,113). Thus, the senior orienteers is likely to exercise enough to experience the benefit of physical exercise, without putting themself at risk for adverse effects. In addition, the senior orienteering athletes investigated in paper II talked about the importance of avoiding unhealthy environments and none of the orienteers smoked. This might be a particularly important feature in this group of healthy agers as non-smoking is associated with increased survival and independence in old age (11). Altogether these factors may speak in favour of senior orienteering athletes as a preferable model of healthy ageing. Their regular engagement in “brain exercise”, i.e. map reading, is further a component that is unique to this group of athletes that may have important consequences for maintaining cognitive function into the older ages, however, this matter need further investigation. Moreover, on the basis of this thesis senior orienteers should not be regarded as an “elite” 52

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group of healthy agers. Rather, they put focus on the social aspects of their sport and maintain a moderate level of physical activity based on enjoyment and pleasure. In this sense they may represent a way of life that is more transferable and accessible to the general population of older adults, as compared to master athletes and centenarians.

The gut as an important denominator of health According to the results of paper I and II (exploring the concept of optimal functionality and senior orienteers as a model of health ageing) maintained gut-health is an important factor for optimal functionality and perceived health in old age. Supported by the fact that gastrointestinal problems have been described to negatively affect functional status and quality of life in older adults (34). In addition, the gut and it’s microflora have lately attracted a lot of attention as a main player in human health (36). Hence, gut-health may be considered a particularly important denominator of wellbeing and health among older adults. Two-thirds of the older adults included in paper IV reported gut complaints. Indicating that poor guthealth is a common problem among older adults, which has also been supported by previous studies in other populations of older adults (31,33,43). Despite the high prevalence of gut problems in this age group the treatment options are rather scarce and only a few non-pharmaceutical treatments are available. Thus, new treatment strategies are warranted, preferably that are non-invasive and easy-to-use. The probiotic organism L. reuteri is an endogenous organism (114,115) able to modulate gut-health via the intestinal microbiota through the production of reuterin, a potent antibacterial compound capable of inhibiting a wide spectrum of microorganisms (116). As described in the introduction section of this thesis L. reuteri has previously been identified as a promising therapy in various gastrointestinal disorders (70,71,117) making it a promising treatment of gastrointestinal discomfort. L. reuteri can easily be supplied to older adults as a dietary supplement containing freeze-dried bacteria. This easy-to-use, non-invasive, strategy to improve gut-health among older adults was investigated in paper IV of this thesis. However, the intervention failed to show a health promoting effect.

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Methodological considerations and limitations The RCT outcome in paper IV There are a number of factors to be considered in relation to the negative outcome of the RCT investigating the effect of L. reuteri on older adults health. It may be a question of dosage, as it is possible that the daily amount of bacteria was not enough to exert a beneficial response from the host. Yet, the dosing was identical to a previous trial reporting a positive effect of L. reuteri in constipated adults (74). There is also a possibility that the targeted probiotic organism itself was not able to provoke a response. It has been shown that L. reuteri is more prevalent in subjects over 66 years of age (118), which might limit a positive treatment effect. A further consideration is that the present study might have been underpowered, for example a rather large number of subjects had to be excluded due to influences of medication. This resulted in a substantial loss of power, which we did not initially account for. It is also important to note that several subjects did not experience any gastrointestinal symptoms at the time of inclusion, and thus did not have the possibility to improve during the intervention. This was an effect of recruiting a study population that was supposed to resemble the general population, in order to increase result generalizability. However, focusing on a population with more well-defined gut problems might have been preferable in this case. Furthermore, probiotic administration can be expected to elicit rather modest treatment effects, as a rather limited number of probiotic bacteria will encounter an environment that is already colonised with an overwhelmingly vast microflora. Hence, including subjects with too severe gut problems may equally present a difficulty; as such subjects might not be responsive to the modest effects of the probiotic supplementation. In support of this, a sub-analysis of the data in paper IV comprising only those subjects displaying moderate and severe gastrointestinal complaints also failed to find differences between the two treatment arms. Moreover, it is possible that our primary outcome parameter, the GSRS, was not able to detect subtle changes in the gastrointestinal health of the study participants. Alternative instruments, for example those specifically developed for Irritable Bowel Syndrome (119), might have increased the sensitivity in this outcome. This may also be the case for the questionnaires selected as secondary outcomes. However, all instrument used to evaluate the probiotic intervention in paper IV have all been suggested as appropriate for executing longitudinal measurements. In addition, the he 54

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GSRS, HADS and PSS have previously been used in probiotic trial reporting positive outcomes (120,121). Furthermore, there were indications that individuals suffering from elevated stress at baseline improved their mood during the study and hence there may be further aspects to consider in relation to L. reuteris potential effects on older adults health. Possibly another sample selection, better defined in terms of health status and gastrointestinal symptoms, in combination with other outcome measures may bring further clarity to this matter. For example, investigating the effects of L. reuteri on metabolic changes in the gut microflora of stressed subjects may increase the understanding of the potential benefits of supplementing such individuals with this particular bacterium. In general, including more biological outcome measures might have favoured this trial.

Validity and reliability of questionnaire data in paper II-IV A substantial part of the results presented in this thesis is based on selfreported questionnaire data, paper II-IV. All the included questionnaires have been previously used in research studies and are validated for this purpose (Table 2). In addition, all instruments were pilot tested prior their inclusion, both among senior orienteering athletes and among other older adults, to assess their applicability. Based on the pilot testing two of the instruments originally considered for inclusion were found non-applicable to our targeted populations and was thus replaced by other instruments: the International Physical Activity Questionnaire (IPAQ) (122) was replaced in favour of FGAS and the Short Form-36 (SF-36) (123) was replaced in favour of HI. The new instruments were likewise evaluated, prior their inclusion in the studies, by the pilot populations to assure that their applicability. The FGAS and the HI are undoubtedly the least commonly known instruments included in this thesis. The reason for including these two questionnaires was the difficulties for the pilot population to complete the IPAQ and the SF-36 questionnaires originally considered for inclusion. The FGAS and the HI were judged as straightforward instruments, with response alternatives that are easy for the older adults to interpret and were thus regarded as more suitable for our targeted populations. However, we might have lost some of the more detailed information about physical activity and functional health that IPAQ and the SF-36 could have provided. If including self-reported data in research studies the accuracy of the results are always dependent on the respondents’ honesty, correct understanding and interpretation of the questions asked. In addition, at least in LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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the case of health-related questionnaires, the results are also likely to be dependent on how the respondents interpret their current health status. As suggested in this thesis the health experience of older adults may vary greatly on the basis of personal goals and demands on function. For example, a senior orienteer with a common cold might perceive the cold as a much greater obstacle to live life as she wishes, i.e. not being able to perform exercise as preferred, as compared to the older adult who is not as active and may be satisfied with her physical function when she can take a walk around the block. Hence the common cold may have a much larger impact on the senior orienteer’s experienced health. Therefore, healthrelated questionnaires are prone to be sensitive to the respondent’s interpretation of the questions on the basis of their present health experience. Furthermore, individual health interpretation may particularly have affected the results of paper III and IV; the two papers intended to investigate some more biological perspectives of health, i.e. whether gut-health would be reflected in blood plasma and faeces of senior orienteers, and older adults with gastrointestinal discomfort, and whether the probiotic bacteria L. reuteri would exert biological effects robust enough to be reflected in self-rated health. In multiple conditions gut-health and psychological wellbeing seem to go hand-in-hand, as briefly mentioned in the introduction of this thesis. For example Irritable Bowel Syndrome (an aberrant gut function with unknown aetiology) is commonly considered as a manifestation of psychosomatic origin, at least in part (124,125). Although, very uncomfortable and problematic for functioning in everyday life, this disorder is poorly displayed in biological measures. The fear of not having control over the occurrence of pain or need to defecate often hinders these patients to live a normal life. This behavioural pattern may cause a vicious circle, whereby the patient becomes increasingly sensitised to the experience of gut discomfort (126). In the same way older adults perceiving themselves as hindered by their gastrointestinal disturbances is likely to interpret these disturbances as more severe than a person who is maintaining an active life style. In addition, a less active older adult might become more attentive to gastrointestinal discomfort and eventually the symptoms may receive more attention than is reasonable, setting of a vicious circle similar to patients with Irritable Bowel Syndrome. Such differences in health interpretation may produce a bias in self-reported data. Hence, solely characterising individuals based on self-reported data when designing or evaluating research studies, like the ones investigating gut-health in this thesis, may not be the most successful strategy. In addition, it has been 56

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suggested that especially aged individuals are subject to reporting bias due to social desirability, which might have introduce subject bias in the collected data (127). Still, we judged questionnaires to be the most suitable way to collect data from our study populations, as questionnaires have a number of advantages, such as accessibility, data structure, limited time and resource requirements.

Comparing senior athletes to older adults with gut problems in paper III In the paper III we compared senior orienteering athletes to older adults displaying gut problems, defined on the basis of self-reported data. A part from the difference in gut problems, these two groups of older adults also displayed differences in terms of physical activity level and sex distribution. These between-group differences generated multiple issues that may have an impact on the investigated parameters. The group of senior orienteers displayed a higher physical activity level and comprised both women and men, while the group with gut problems consisted of women only. The study failed to enrol men as only women with gut problems declared their interest to participate in the study. This might indicate that gut problem is more common among females, a notion that has been previously raised in the literature (31,128). However, the variation in sex distribution was controlled for by stratifying the data for the senior orienteering athletes into groups of men and women. This made, however, no difference for the obtained results. To thoroughly investigate the impact of guthealth on our selected parameters, the optimal reference group would have been older adults experiencing gut problems but still engaging in physical activity to the same level. From previous pilot studies we know that such a group is difficult to find as gut discomfort often results in less physical activity (129). In addition, poor gut-health has been identified as a central aspect in the decline of subjective wellbeing often occurring among older adults. Here, we compare senior orienteering athletes to a group of older adults suffering from gut discomfort as a first step to identify the mechanisms behind a good gut-health. The gut has for example previously been suggested to play an important part in the inflammatory network in old age (37) as well as in the health status of older adults (60). Moreover, an additional limitation to this paper was the choice of biological markers to capture the inflammatory status and oxidative stress, as considered in the method section (titled ‘Considerations of the biological parameters assessed’). LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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Trustworthiness of the focus group discussions in paper II Focus group discussion (FGD) trustworthiness is critical, and can be difficult to guarantee. Reliability of the FGDs performed in this thesis was ensured by the use of pre-constructed, semi-structured interview guide and maintaining the interview environment consistent between groups (127). In addition, a person that attended the sessions did the transcriptions of the recorded material in close conjunction to the FGDs. The language and expressions used during the FGDs were adjusted to the respondent group in order to support the respondents understanding of the interview questions and to increase the dependability of the data. Further, the experienced moderators were observant to the respondents’ reaction during the interview and could clarify if any ambiguities arose. The role of the moderators was to stimulate the participants to engage in an active discussion were each individual would contribute and to direct their focus towards the topics of interest. In addition, the authors checked and evaluated the results tables together in order to achieve agreement on the presentation of the qualitative findings and whether these truly reflected the FGD dialogues. As a step to confirm the credibility (78) and ‘face validity’ (127) the FGD findings were also presented to the respondents prior publication. Considerations of the database search in paper I A limitation to paper I is the use of ‘personal satisfaction’ as a substitute term for optimal functionality, which has influenced the collection of articles retrieved in the search. Thus, personal satisfaction has shaped the results and the initial exploration of the concept of optimal functionality. However, using the term optimal functionality in our trial search made evident that this would provide a very limited picture of the area of interest and also retrieve a high number of articles judged as unrelated to the topic, for example studies dealing with optimal functionality of plant organs cell growth, surgical tools, dental care, genetics and cellular therapy in cancer. Turning to a sample of older adults to support the definition of a proper search term seemed as the appropriate way to tackle this obstacle. This was further confirmed by the results of the final literature search providing a more holistic and extensive picture of the area. Moreover, only two electronic databases were chosen for the literature search: PubMed and CINAHL. In an initial search PsycINFO (American Psychological Association, Washington, DC, USA) was also included, however, the search retrieved a substantial number of duplicates with the other two 58

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databases and PsycINFO was thus removed as a source. PubMed and CINAHL provided a high diversity among the retrieved papers, as judged by their few duplicates, and were thus concluded to have attained proper coverage of the targeted area.

Originality and representativeness of paper I-IV In terms of originality Paper I is the first scientific publication on the concept of optimal functionality in old age. However, a limitation to this work is that we were confined to an available MeSH term to enable the literature search, which affected the collection of papers retrieved and thus the representativeness of the results. Further work to establish the accurate meaning of this concept is under way and so far one scientific paper has been accepted for publication (as listed in ‘Related publications’ in the beginning of this thesis). Paper II and III are the first scientific studies to explore senior orienteering athletes as a potential model of healthy ageing. This group of particularly healthy older adults adopts a way of life that corresponds well to the aspects of optimal functionality and successful ageing. Senior orienteers are not an ‘elite’ group of healthy agers, making them an intriguing model of healthy ageing as it may be more transferable to the general population of older adults as compared to previously suggested models, such as master athletes, octo- and centenarians. Furthermore, the representativeness of the local orienteering athletes recruited for FGDs, in paper II, was judged as a suitable match to the national sample of O-Ringen orienteers from which the questionnaire data was collected. No statistical differences between the two groups were found on either of the parameters investigated and to further support the representativeness of the FGD sample, all 14 FGD participants reported participation in one or more O-Ringen competitions over the years. Regarding the originality of paper IV, this is the first scientific study investigating the effect of L. reuteri on self-reported health of free-living older adults. In the targeted age group of this thesis, L. reuteri supplementation has proven effective in reducing the prevalence of high Candida counts in the oral cavity of older adults (75) and has also shown modest effects on constipation in institutionalised elderly (130). The treatment randomisation in the current RCT was considered adequate, as there were few statistical differences between the two treatment groups. Representativeness of the sample, in comparison to the true general population of older adults, was considered as satisfactory. However, in considering the LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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aim, i.e. to investigate the effects of a probiotic bacterium on older adults health with particular focus on gut-health, there is a possibility that this study attracted somewhat more of older adults presenting gut problems than what would be expected in the true general population.

Gender perspective All the four studies included in this thesis have dealt with sex-mixed populations. In terms of generalizability to the general population of older adults this may be seen as a strength, however, this also entailed an increased heterogeneity of the study sample. The sex, or gender, differences have not been extensively addressed in my papers; no effects of sex were found on the quantitative data presented in paper II, III and IV. In paper II the female and male orienteers were interviewed separately from each other in order to allow for gender specific views on health and physical activity to come through. The FGD results were described on basis of these two groups, however, no particular efforts were made to discuss differences between the two groups further. The matter remains to be addressed in future studies and may present an interesting approach for further analysis of the qualitative material presented in paper II.

Future perspectives In this thesis we have explored different perspectives of health and gut function in older adults. Generally, the thesis have adopted two main focuses, 1) the importance of interpreting health on the basis of the older adult’s own perception of health, and 2) the gut as an important denominator of older adults health status, perceived as well as objective. The thesis presents an initial exploration of the concept of optimal functionality in old age, as an approach to characterise older adults health based their personal preferences. Concluding that there is a lack of qualitative knowledge in relation to the concept of optimal functionality. Thus, research of qualitative nature is needed in order to increase the understanding of what constitutes optimal functionality at an individual level to develop the concept further. This work is already on going and a first qualitative study has been accepted for publication in the BMC Geriatrics journal (as listed under ‘Related publications’ in this thesis). Furthermore, senior orienteering athletes are put forward as a new model of healthy ageing. However, the biological parameters assessed here were not sufficient to separate this group of healthy agers from a group of older adults presenting gut complaints. Further research using more sensi60

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tive measures of inflammation and oxidative stress may be performed to more thoroughly investigate the importance of the gut in healthy ageing, preferably in conjunction with methods to evaluate the intestinal microbial environment. The exploration of senior orienteering athletes gut microbial composition is currently underway at the Nutrition-Gut-Brain Interaction (NGBI) research centre, Örebro, Sweden. In addition, the “brain exercising”, i.e. map reading, performed by the senior orienteers is further a component that is unique to this group of aged athletes. This regular practice of cognitive functions may have important consequences for maintaining cognitive health into the older ages; a matter that need further investigation. Moreover, this thesis investigated the health status of older adults, finding that two-thirds reported gut complaints. Suggesting that gut discomfort is a common problem among older adults. In addition, experiencing gut discomfort has a negative impact on the general health experience of older adults, as indicated by this thesis and supported by previous research. Yet, so far few research studies seeking the key to their resolution has been completed. To identify the relationship between common gut symptoms and physiological or microbial changes in the gastrointestinal tract of otherwise healthy older adults would be helpful in further determining their pathophysiology. In particular the impact of ageing on the intestinal barrier may be of interest to study further, as declines in barrier function is likely to have a great influence on the gastrointestinal milieu, as well as on inflammatory status and immune function. Hence, the gastrointestinal barrier might be a key player in the intricate network of factors with significance to the human ageing process in general and increased systemic inflammatory activity specifically. In addition, studies collecting detailed information on dietary intake to further investigate the relations between diet and gastrointestinal status, are needed to increase our understanding of the association between diet, gut microbiota and health status among older adults. Furthermore, new treatment strategies to alleviate gut discomfort are warranted. Unfortunately, despite of promising indications from previous research, supplementation with the probiotic bacterium L. reuteri was not proven effective in this matter. Hence, further interventions investigating other probiotic bacteria or prebiotic10 substances may be a next step toPrebiotics are complex carbohydrates that favour the growth of beneficial bacteria in the gastrointestinal tract. 10

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wards resolving the gut complaints of older adults, targeting microbial dysbiosis. In addition, more studies investigating the effect of pro- and prebiotic dosage and intervention duration will be important in order to personalise the treatment and improve treatment outcomes. Studies of prebiotic effects on microbiota composition and gastrointestinal permeability is currently undertaken within the NGBI research centre, as well as studies of older adults gut complaints in relation to barrier function and inflammation.

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Conclusions The overall aim of this thesis was to further explore the health and gut function among older adults, applying a down-up approach by exploring health on basis of the older adults own perception. The thesis provides additional perspectives on older adults health and gut function by presenting four scientific papers that uses a broad set of methods to explore this matter further. Paper I strived towards describing the health status of older adults on basis of individual preferences by exploring the concept of optimal functionality. In addition, paper II characterised a new model of healthy ageing in senior orienteering athletes, by exploring their views on health and optimal functioning. In both these papers the gut came forward as an important element for experiencing health and optimal functionality. Based on these findings, and previous suggestions of the gut as a major player in human health maintenance and perceived quality of life, this thesis proposes that the gut may be a particularly important denominator of health in old age. To further explore gut-health, paper III investigated whether perceived gut-health would be associated to inflammatory status among older adults, as an increase in inflammatory status have been previously suggested as driving element of human ageing. Subsequently, paper IV investigated self-reported health and gut function among free-living older adults, concluding that a majority of these individuals report mild to severe gut problems. As a strategy to promote the health of the older adults the gut was selected as target for a probiotic intervention, however, a twelve-week daily supplementation with L. reuteri did not improve selfreported health or alleviated the gut discomfort perceived by this population. This may be due to loss of power during the intervention or possibly the probiotic organism of choice did not exert strong enough effects on the gut to elicit any clinical manifestations. Overall this thesis concludes that new strategies are needed to put the older adult’s own preferences in focus when evaluating health, and in addition, new targets are warranted for early and effective interventions to maintain health and independence in this age group. The gut is suggested to represent such a target, as it is an organ that is of significance to older adults health on several levels - biologically as well as subjectively. More specifically this thesis concludes that: • In order to promote an individualised approach to older adults health the three areas of body, self and the external environment needs to be considered. LINA ÖSTLUND-LAGERSTRÖM Health and gut function in older adults

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• Senior orienteers represent a suitable model of healthy ageing, fulfilling the main aspects of optimal functionality and successful ageing. • The health of free-living older adults in Örebro County, Sweden, is relatively good, however, a majority of the population suffers from gut complaints. • Gastrointestinal health can be considered an important denominator of health among older adults and, thus, health-promoting interventions targeting the gut may be particularly important in this age group. • A twelve-week supplementation with the probiotic bacteria L. reuteri did not improve gut-health among older adults and showed no further effects on their general wellbeing. • Future research is needed to: a) develop the concept of optimal functionality further, especially in terms of increasing the qualitative understanding of individual preferences in relation to health. b) more thoroughly characterise senior orienteers as a model of healthy ageing. c) elucidate the relationship between manifestations of gut problems among older adults and physiological changes in the gastrointestinal tract, as well as structural and metabolic changes in the gut microbiota. d) put further efforts into discovering new treatment strategies to improve older adults gut-health, possibly through controlled interventions using pro- or prebiotic supplements.

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Acknowledgements Being a PhD student has been quite an experience, mostly to the positive, and here I have saved some space to recognise all the people that have supported and helped me to finalise this thesis. I am ever so grateful to you! The scientific work presented in this thesis was performed at the School of Medical Sciences, Örebro University, Sweden. I wish to express my gratitude to each and every staff member at Örebro University and the Örebro University Hospital who has in any way contributed to making this thesis possible. I hope you know who you are, thank you! I would like to present my special thanks to my main supervisor, Robert J. Brummer, who has given me this opportunity. I’m grateful for your support and the help you have provided me along the way. The way you always manage to see the bigger picture in everything impresses me. Thank you for the opportunities to go abroad to present my research and for always maintaining a positive mind-set. I wish to show my gratitude to my co-supervisor, Ida Schoultz, who has guided me through these four years. Your help with planning, performing, prioritising, reading and writing have been indispensable. A special thanks for investing time in me even when you formally have been on maternity leave. I will always remember our 1990 km round-trip to 2013 years ORingen, in Boden, to collect data from senior orienteers (why couldn’t it have been somewhere south in Sweden, near a nice summer resort, like it was the year before?!). Thanks to my co-supervisor Wilhelm Tham for your concerns for me during this time and all your interesting and encouraging emails. My warmest appreciations to my NUPARC co-workers: my PhD colleagues, Samal Algilani, Stina Engelheart, Cecila Bergh, Carren Melinder and Dara Rasoal – the journey has been easier with you by my side! Annica Kihlgren, thanks for all the collaboration both in and outside work. Bengt Björkstén, thanks for your encouragements and your comments on my work. Thank you, Karin Blomberg, for your help with prepping me

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before my half-time defence, as well as your help with my manuscripts and “kappa” review. A special thanks to Samal Algilani for fruitful companionship and professional collaboration. I look forward to further joint work and friendship. My sincere thanks to all my colleagues within the NGBI research centre: John-Peter Ganda Mall (thanks for all the joint work and travelling), Dirk Repsilber (thanks for helping me with the statistics), Julia König (thanks for the nice stay in south of Germany), Hanna Edebol (thanks for introducing me to the choir), Julia Sabet (thanks for reading my manuscripts), Rebecca Wall (thanks for reading my manuscript), Johanna Sundin (thanks for the awesome stay in New York) Savanne Holster, Sezin Günaltay, Carolin Kremp, Niklas Evertsson, Tatiana Marques and all of you other newly recruited masterminds that I have yet to get to know. Anette Oskarsson, Johnny Karlsson and Åsa Berglind, thank you so much for all the administrative and practical help that have taken my thesis forward. Thank you Anders Zachrisson and BioGaia AB for the opportunity to work within the ELROY project. Thank you Rolf Andersson and SCA for the research updates and the opportunity to join the DDW conference in Washington DC. Thank you, Maria Jenmalm and Ammi Fornander, for welcoming me into your lab and advising me regarding IgA analyses (even tough I didn’t get the chance to present any of the results in this thesis). Thank you, Mårten Lindqvist, for supporting Ida in supporting me and for your explanations of next generation sequencing methods in conjunction with my half-time defence (I’m quite sad that I couldn’t integrate any NGS results in my thesis, but at the same time a bit relieved that I wont have to defend them). Thank you all Gimiicum fellows that have facilitated the inspiring and enjoyable stays during the courses and thanks to the executive group that gave me the opportunity to partake. 66

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Thank you all colleagues working at the X-building, even tough we haven’t had the chance to get particularly close, I appreciate seeing you in the corridor or chatting a bit over lunch or a cup of tea in the fika-room. I would also like to express my gratitude to all my study participants, who made this thesis possible, and a special thanks to the senior orienteers Ingbritt and Arne Nordström who were kind enough to join me in the lab when the TV 4 news crew came to do their reportage. Last, but certainly not least, I want to thank and express my love to my family whom have supported me in so many ways during these years. Thank you, Christian, for your encouragements and patience, I love you. A tremendous thanks to you, mom, for all your support and help with things regarding my thesis and all other five-hundred-eleven, uncountable, areas in my life that you have engaged in to make everything a little bit easier! Thanks dad, without you I wouldn’t have set foot on this planet. Thank you, sis and Linda, for just being who you are, I appreciate you both very much. Thank you Linn for being part of my life, I wish we had more time to spend together. I would also like to thank all of my other family members, existing and future, for making my life into what it is, I enjoy it greatly! Love to you all. The Knowledge Foundation, Stiftelsen Olle Engkvist Byggmästare, BioGaia AB and the Faculty of Medicine and Health at Örebro University have financially supported this thesis work. Now, if I have forgotten someone I hereby convey my deepest apologies… please notify me of my wrong and I will thank you in person!

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81

Publications in the series Örebro Studies in Medicine 1.

Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term sequelae from closed head injury.

2.

Jansson, Kjell (2004). Intraperitoneal Microdialysis. Technique and Results.

3.

Windahl, Torgny (2004). Clinical aspects of laser treatment of lichen sclerosus and squamous cell carcinoma of the penis.

4.

Carlsson, Per-Inge (2004). Hearing impairment and deafness. Genetic and environmental factors – interactions – consequences. A clinical audiological approach.

5.

Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis.

6.

Jatta, Ken (2006). Inflammation in Atherosclerosis.

7.

Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on Malignant Diseases in Childhood.

8.

Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods.

9.

Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance.

10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing on their Fear and on how their Fear is Handled. 11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in vascular smooth muscle cells. 12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry in children. 13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and diagnostic aspects. 14. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies. 15. Andrén, Ove (2008). Natural history and prognostic factors in localized prostate cancer. 16. Svantesson, Mia (2008). Postpone death? Nurse-physician perspectives and ethics rounds.

17. Björk, Tabita (2008). Measuring Eating Disorder Outcome – Definitions, dropouts and patients’ perspectives. 18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery. 19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes. 20. Berglund, Carolina (2008). Molecular Epidemiology of MethicillinResistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals. 21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in persons with Multiple Sclerosis. 22. Johansson, Ann-Christin (2008). Psychosocial factors in patients with lumbar disc herniation: Enhancing postoperative outcome by the identification of predictive factors and optimised physiotherapy. 23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco products. 24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study. 25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α and Methylenetetrahydrofolate Reductase Genes as Determinants of Plasma Homocysteine Concentrations. 26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence, treatment seeking behaviour, experiences and perceptions among persons with and without urinary leakage. 27.

Elfström, Peter (2009). Associated disorders in celiac disease.

28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment: Social background, drug use patterns and criminality. 29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment – Prevalence, Measurement and Long-term Outcome. 30. Vumma, Ravi (2009). Functional Characterization of Tyrosine and Tryptophan Transport in Fibroblasts from Healthy Controls, Patients with Schizophrenia and Bipolar Disorder. 31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens.

32. Allvin, Renée (2009). Postoperative Recovery. Development of a Multi-Dimensional Questionnaire for Assessment of Recovery. 33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia with Special Reference to Folate and Fibrinolysis. 34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide study of assiciated morbidity and mortality. 35.

Söderqvist, Fredrik (2009). Health symptoms and potential effects on the blood-brain and blood-cerebrospinal fluid barriers associated with use of wireless telephones.

36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives on parent–child interaction interventions and beneficial meetings. 37.

Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer and Interaction with the Health Care System through Different Phases of the Disease.

38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic characteristics. 39. Hammer, Ann (2010). Forced use on arm function after stroke. Clinically rated and self-reported outcome and measurement during the sub-acute phase. 40. Westman, Anders (2010). Musculoskeletal pain in primary health care: A biopsychosocial perspective for assessment and treatment. 41. Gustafsson, Sanna Aila (2010). The importance of being thin – Perceived expectations from self and others and the effect on self-evaluation in girls with disordered eating. 42. Johansson, Bengt (2010). Long-term outcome research on PDR brachytherapy with focus on breast, base of tongue and lip cancer. 43. Tina, Elisabet (2010). Biological markers in breast cancer and acute leukaemia with focus on drug resistance. 44. Overmeer, Thomas (2010). Implementing psychosocial factors in physical therapy treatment for patients with musculoskeletal pain in primary care. 45. Prenkert, Malin (2010). On mechanisms of drug resistance in acute myloid leukemia.

46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters in Obese Patients. 47.

Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological aspects.

48. Almon, Ricardo (2010). Lactase Persistence and Lactase NonPersistence. Prevalence, influence on body fat, body height, and relation to the metabolic syndrome. 49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis. 50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of Cystic Fibrosis. 51. Franzén, Karin (2011). Interventions for Urinary Incontinence in Women. Survey and effects on population and patient level. 52. Loiske, Karin (2011). Echocardiographic measurements of the heart. With focus on the right ventricle. 53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of Staphylococcus epidermidis isolated from prosthetic joint infections. 54. Eriksson Crommert, Martin (2011). On the role of transversus abdominis in trunk motor control. 55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal Sphincters. 56. Holländare, Fredrik (2011). Managing Depression via the Internet – self-report measures, treatment & relapse prevention. 57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding Properties in Neuropsychiatric Disorders using the Fibroblast Cell Model. 58. Vidlund, Mårten (2011). Glutamate for Metabolic Intervention in Coronary Surgery with special reference to the GLUTAMICS-trial. 59. Zakrisson, Ann-Britt (2011). Management of patients with Chronic Obstructive Pulmonary Disease in Primary Health Care. A study of a nurse-led multidisciplinary programme of pulmonary rehabilitation. 60. Lindgren, Rickard (2011). Aspects of anastomotic leakage, anorectal function and defunctioning stoma in Low Anterior Resection of the rectum for cancer.

61. Karlsson, Christina (2011). Biomarkers in non-small cell lung carcinoma. Methodological aspects and influence of gender, histology and smoking habits on estrogen receptor and epidermal growth factor family receptor signalling. 62. Varelogianni, Georgia (2011). Chloride Transport and Inflammation in Cystic Fibrosis Airways. 63. Makdoumi, Karim (2011). Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis. 64. Nordin Olsson, Inger (2012). Rational drug treatment in the elderly: ”To treat or not to treat”. 65. Fadl, Helena (2012). Gestational diabetes mellitus in Sweden: screening, outcomes, and consequences. 66. Essving, Per (2012). Local Infiltration Analgesia in Knee Arthroplasty. 67. Thuresson, Marie (2012). The Initial Phase of an Acute Coronary Syndrome. Symptoms, patients’ response to symptoms and opportunity to reduce time to seek care and to increase ambulance use. 68. Mårild, Karl (2012). Risk Factors and Associated Disorders of Celiac Disease. 69. Fant, Federica (2012). Optimization of the Perioperative Anaesthetic Care for Prostate Cancer Surgery. Clinical studies on Pain, Stress Response and Immunomodulation. 70. Almroth, Henrik (2012). Atrial Fibrillation: Inflammatory and pharmacological studies. 71. Elmabsout, Ali Ateia (2012). CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions. 72. Stenberg, Reidun (2012). Dietary antibodies and gluten related seromarkers in children and young adults with cerebral palsy. 73. Skeppner, Elisabeth (2012). Penile Carcinoma: From First Symptom to Sexual Function and Life Satisfaction. Following Organ-Sparing Laser Treatment. 74. Carlsson, Jessica (2012). Identification of miRNA expression profiles for diagnosis and prognosis of prostate cancer. 75. Gustavsson, Anders (2012): Therapy in Inflammatory Bowel Disease.

76. Paulson Karlsson, Gunilla (2012): Anorexia nervosa – treatment expectations, outcome and satisfaction. 77. Larzon, Thomas (2012): Aspects of endovascular treatment of abdominal aortic aneurysms. 78. Magnusson, Niklas (2012): Postoperative aspects of inguinal hernia surgery – pain and recurrences. 79. Khalili, Payam (2012): Risk factors for cardiovascular events and incident hospital-treated diabetes in the population. 80. Gabrielson, Marike (2013): The mitochondrial protein SLC25A43 and its possible role in HER2-positive breast cancer. 81. Falck, Eva (2013): Genomic and genetic alterations in endometrial adenocarcinoma. 82. Svensson, Maria A (2013): Assessing the ERG rearrangement for clinical use in patients with prostate cancer. 83. Lönn, Johanna (2013): The role of periodontitis and hepatocyte growth factor in systemic inflammation. 84. Kumawat, Ashok Kumar (2013): Adaptive Immune Responses in the Intestinal Mucosa of Microscopic Colitis Patients. 85. Nordenskjöld, Axel (2013): Electroconvulsive therapy for depression. 86. Davidsson, Sabina (2013): Infection induced chronic inflammation and its association with prostate cancer initiation and progression. 87. Johansson, Benny (2013): No touch vein harvesting technique in coronary by-pass surgery. Impact on patency rate, development of atherosclerosis, left ventricular function and clinical outcome during 16 years follow-up. 88. Sahdo, Berolla (2013): Inflammasomes: defense guardians in host-microbe interactions. 89. Hörer, Tal (2013): Early detection of major surgical postoperative complications evaluated by microdialysis. 90. Malakkaran Lindqvist, Breezy (2013): Biological signature of HER2positive breast cancer.

91. Lidén, Mats (2013): The stack mode review of volumetric datasets – applications for urinary stone disease. 92. Emilsson, Louise (2013): Cardiac Complications in Celiac Disease. 93. Dreifaldt, Mats (2013): Conduits in coronary artery bypass grafting surgery: Saphenous vein, radial and internal thoracic arteries. 94. Perniola, Andrea (2013): A new technique for postoperative pain management with local anaesthetic after abdominal hysterectomy. 95. Ahlstrand, Erik (2013): Coagulase-negative Staphylococci in Hematological Malignancy. 96. Sundh, Josefin (2013): Quality of life, mortality and exacerbations in COPD. 97. Skoog, Per (2013): On the metabolic consequences of abdominal compartment syndrome. 98. Palmetun Ekbäck, Maria (2013): Hirsutism and Quality of Life with Aspects on Social Support, Anxiety and Depression. 99. Hussain, Rashida (2013): Cell Responses in Infected and Cystic Fibrosis Respiratory Epithelium. 100. Farkas, Sanja (2014): DNA methylation in the placenta and in cancer with special reference to folate transporting genes. 101. Jildenstål, Pether (2014): Influence of depth of anaesthesia on postoperative cognitive dysfunction (POCD) and inflammatory marker. 102. Söderström, Ulf (2014): Type 1 diabetes in children with non-Swedish background – epidemiology and clinical outcome 103. Wilhelmsson Göstas, Mona (2014): Psychotherapy patients in mental health care: Attachment styles, interpersonal problems and therapy experiences 104. Jarl, Gustav (2014): The Orthotics and Prosthetics Users´ Survey: Translation and validity evidence for the Swedish version 105. Demirel, Isak (2014): Uropathogenic Escherichia coli, multidrugresistance and induction of host defense mechanisms 106. Mohseni, Shahin (2014): The role of ß-blockade and anticoagulation therapy in traumatic brain injury

107. Bašić, Vladimir T. (2014): Molecular mechanisms mediating development of pulmonary cachexia in COPD 108. Kirrander, Peter (2014): Penile Cancer: Studies on Prognostic Factors 109. Törös, Bianca (2014): Genome-based characterization of Neisseria meningitidis with focus on the emergent serogroup Y disease 110. von Beckerath, Mathias (2014): Photodynamic therapy in the Head and Neck 111. Waldenborg, Micael (2014): Echocardiographic measurements at Takotsubo cardiomyopathy - transient left ventricular dysfunction. 112. Lillsunde Larsson, Gabriella (2014): Characterization of HPV-induced vaginal and vulvar carcinoma. 113. Palm, Eleonor (2015): Inflammatory responses of gingival fibroblasts in the interaction with the periodontal pathogen Porphyromonas gingivlis. 114. Sundin, Johanna (2015): Microbe-Host Interactions in Post-infectious Irritable Bowel Syndrome. 115. Olsson, Lovisa (2015): Subjective well-being in old age and its association with biochemical and genetic biomarkers and with physical activity. 116. Klarström Engström, Kristin (2015): Platelets as immune cells in sensing bacterial infection. 117. Landström, Fredrik (2015): Curative Electrochemotherapy in the Head and Neck Area. 118. Jurcevic, Sanja (2015): MicroRNA expression profiling in endometrial adenocarcinoma. 119. Savilampi, Johanna (2015): Effects of Remifentanil on Esophageal Sphincters and Swallowing Function. 120. Pelto-Piri, Veikko (2015): Ethical considerations in psychiatric inpatient care. The ethical landscape in everyday practice as described by staff. 121. Athlin, Simon (2015): Detection of Polysaccharides and Polysaccharide Antibodies in Pneumococcal Pneumonia. 122. Evert, Jasmine (2015): Molecular Studies of Radiotheray and Chemotherapy in Colorectal Cancer.

123. Göthlin-Eremo, Anna (2015): Biological profiles of endocrine breast cancer. 124. Malm, Kerstin (2015): Diagnostic strategies for blood borne infections in Sweden. 125. Kumakech, Edward (2015): Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV) and Cervical Cancer Prevention in Uganda: Prevalence, Risk factors, Benefits and Challenges of PostExposure Prophylaxis, Screening Integration and Vaccination. 126. Thunborg, Charlotta (2015): Exploring dementia care dyads’ person transfer situations from a behavioral medicine perspective in physiotherapy. Development of an assessmement scale. 127. Zhang, Boxi (2015): Modulaton of gene expression in human aortic smooth muscle cells by Porphyromonas gingivalis - a possible association between periodontitis and atherosclerosis. 128. Nyberg, Jan (2015): On implant integration in irradiated bone: - clinical and experimental studies. 129. Brocki, Barbara C. (2015): Physiotherapy interventions and outcomes following lung cancer surgery. 130. Ulfenborg, Benjamin (2016): Bioinformatics tools for discovery and evaluation of biomarkers. Applications in clinical assessment of cancer. 131. Lindström, Caisa (2016): Burnout in parents of chronically ill children. 132. Günaltay, Sezin (2016): Dysregulated Mucosal Immune Responses in Microscopic Colitis Patients. 133. Koskela von Sydow, Anita (2016): Regulation of fibroblast activity by keratinocytes, TGF-β and IL-1α –studies in two- and three dimensional in vitro models. 134. Kozlowski, Piotr (2016): Prognostic factors, treatment and outcome in adult acute lymphoblastic leukemia. Population-based studies in Sweden. 135. Darvish, Bijan (2016): Post-Dural Puncture Headache in Obstetrics. Audiological, Clinical and Epidemiological studies. 136. Sahlberg Bang, Charlotte (2016): Carbon monoxide and nitric oxide as antimicrobial agents – focus on ESBL-producing uropathogenic E. coli.

137. Alshamari, Muhammed (2016): Low-dose computed tomography of the abdomen and lumbar spine. 138. Jayaprakash, Kartheyaene (2016): Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis. 139. Elwin Marie (2016): Description and measurement of sensory symptoms in autism spectrum. 140. Östlund Lagerström, Lina (2016): ”The gut matters” - an interdisciplinary approach to health and gut function in older adults.