Stress has often been called the psychological problem of the 21st century. Though stress can have some advantageous effects when it comes to productivity, it can also be significantly detrimental to physical and mental health. As the immune system becomes dysregulated in situations of stress, a number of immune markers are elevated if the person is chronically stressed. Investigating microbiota in patients suffering from depression and stress can be performed in a number of different ways.
Dr. Ted Dinan, the Medical Director at Atlantia Clinical Trials, speaks in this interview about the optimum design of a study if looking at the activity of a product, the typical biomarkers, and how the trials performed by Atlantia Clinical trials are contributing to the development in this area.
What impact does stress generally have on the body?
In many ways, stress can be something that produces an optimal outcome. Arguably human beings are not going to work or study effectively if they are not stressed. There is a pivot point beyond which stress is known to produce deleterious effects. It was Hans Selye, the Canadian endocrinologist, who first linked stress with a variety of medical disorders. It is now known that there are a vast array of psychological conditions associated with excessive stress – of which undoubtedly depression and anxiety are at the top of the list.
When we look at stress, there are biomarkers of stress which can be incorporated into clinical trials. Those biomarkers can be divided most optimally into three main areas. There are inflammatory biomarkers, endocrine biomarkers, or hormonal biomarkers, and over the past decade, it has become obvious that there are markers of microbiota abnormalities that occur in stress-related conditions.
What are the humoral or endocrine anomalies that occur with stress?
The stress axis that has been most extensively studied in relation to stress is unquestionably the hypothalamic pituitary adrenal axis. The adrenal axis - the adrenal glands - sit on top of our kidneys, and they secrete a number of hormones - one of which is cortisol, which is pivotal for appropriately adapting to chronic stress.
Unfortunately, if cortisol levels remain high over extended periods of time, this can notably negative effect on general health and wellbeing. The hypothalamic pituitary adrenal axis is, therefore, one axis that enables us to choose markers in clinical trials that are reliable and can be used in stress and distress-related conditions.
The immune system is also a system that becomes dysregulated in situations of stress, and a number of immune markers are elevated if we are chronically stressed. One good example of this is irritable bowel syndrome. Within irritable bowel syndrome, many inflammatory markers or cytokines become elevated. Similar elevations are seen in patients who are suffering from depressive illness. What can be seen is that interleukin-8 and the soluble interleukin-6 receptor are both elevated very significantly in many patients who suffer from irritable bowel syndrome. There are also elevations in the acute-phase protein, CRP, in patients with depressive illness. CRP is a relatively easy protein to measure, and it can be examined throughout the course of patient treatment. When depressed patients are effectively treated, their CRP levels actually decrease.
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Why investigate microbiota in patients with depression?
One of the first studies to demonstrate that the gut microbiota – otherwise known as the collection of bacteria within the intestine - might be altered in patients with depression emanated from a study in animals. It was a study that was published in biological psychiatry several years ago that investigated the effects of separating their rat pups from their mothers for a few hours each day: when those animals grew to maturity, they had an alteration in their gut microbiota, and their microbiota was less diverse. In other words, rat pups separated from their mothers for a few hours each day had a less diverse range of bacteria in the intestine than animals who had not been subjected to that particular stressor.
Therefore, this seemed significant enough that it was reasonable for us to look at patients attending my clinic at the university hospital in Cork and see what the microbiota in patients with depression actually looked like. A number of patients volunteered to provide fecal samples, and we then profiled the gut microbiota in those individuals, comparing them with the group of age- and sex-matched healthy controls.
What can be seen is that the microbiota of depressed patients differs quite considerably from that of healthy individuals: that there is a decreased diversity and richness of bacteria in the gut microbiota in patients with depression. The reasons for that are complex and not fully understood at this particular point in time. It must be noted that this finding has been replicated in several studies and different laboratories on both sides of the Atlantic.
There is, therefore, an interplay of humoral changes in stress-related conditions, largely where cortisol levels are excessively high. Inflammatory changes can be observed where cytokine levels - the key inflammatory molecules - are also elevated, particularly interleukin-6 and interleukin-8. There are changes in the diversity of the microbiota with decreased diversity in chronic stress-related conditions. Now, these three manifestations of stress are very much interrelated.
What are the most appropriate ways of recording stress in studies of stress or distress?
When looking at stress in a healthy population, Cohen’s Perceived Stress Scale is an excellent instrument that analyzes how individuals perceive the stress in their lives at any particular point in time. This can be examined before commencing a particular treatment, during the course of treatment, and after treatment.
The Holmes-Rahe Stress Inventory is another useful inventory that monitors the levels of stress in healthy individuals. If one wants to look at depressive symptoms or anxiety symptoms in a normal population, there are a number of instruments that can be used. The Hospital Anxiety and Depression Scale is particularly useful. The Hamilton Anxiety Rating Scale is also useful, as is the Beck Anxiety Inventory.
If one wishes to look at a psychiatric population or a population of patients who are suffering from depression or anxiety, it is important to create a diagnosis using the DSM-5 criteria, which the American Psychiatric Association puts forward. The MINI is an instrument that, with appropriate training, can lead to a diagnosis of a depressive or anxiety disorder.
If somebody is suffering from clinical depression, it may be important to rate the severity of the depressive symptoms, and there are a variety of scales out there that would enable one to do that. The Hamilton Depression Rating scale is probably the oldest and most well recognized of these, but there is also the Montgomery-Asberg scale and other scales that measure the severity of depression.
When we look at stress biomarkers, there are many different options, but several would be particularly recommended in trials where one is interested in stress. For over 20 years, I have been looking at salivary cortisol levels, which is an easy measure of stress. The level of cortisol in one’s saliva parallels the level in the bloodstream.
Providing a simple, non-invasive saliva sample is a very useful way of monitoring stress. Generally speaking, the salivary cortisol level in the morning is the best indicator of stress. If you were interested in looking at a particular product and whether it impacted stress levels, the awaking cortisol response is probably the best measure of that, from a humoral perspective.
Are there any other ways of looking at or monitoring stress?
There are other ways of looking at or monitoring stress as well. The galvanic skin response has been around for a long time, and it is based on the principle that the resistance in our skin is altered if we are significantly stressed. Other ways in which the individual can be stress-tested and look at their hormonal response to that stress would be the cold pressor test, where we ask the individual to put their hand into a bucket of ice. Obviously, after a few moments, this becomes distressing and will elevate cortisol levels.
A stressed population can be compared to a non-stressed population, or one can look at an individual throughout therapy. This is a slightly more complex scenario, which is relatively easy to conduct is the Trier Social Stress Test. For most people, speaking in public is a stressful event – so the process within the Trier Social Stress Test is a subject unfamiliar with speaking in a public scenario to give a presentation in front of 2-3 (typically stern-looking) individuals. For most people, this is stressful and does elevate their cortisol levels.
This is another way of looking at how somebody deals with stress by looking at their hormonal response to that stress.
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What are some situations in which one might want to look at cognitive responses?
There are a variety of computerized test batteries out there - CANTAB is probably the most well-known test battery. It is a comprehensive test battery that enables one to look at memory, attention, and a variety of psychological measures. The CANTAB has the advantage that each test has been associated with a functional image of the brain, so we know exactly what areas of the brain are activated with the particular test.
Paired associate learning is the name given to the association of one thing with another. For example, you remember that you left your car keys on the hall table or the kitchen table, so you are pairing the car keys with the kitchen table. This is just one form of learning which involves the prefrontal cortex and the temporal lobes. Cognitive tasks can be very useful in certain types of studies.
What is the optimal design of any particular study if one is looking at the activity of a product?
The optimal design is a random allocation design; it is probably a parallel group and involves a placebo. The advantages of a placebo-controlled parallel group design are obvious. The disadvantage of this is that it usually requires a very large sample size to demonstrate the efficacy of a particular product, but it clearly is the optimal design.
There are cheaper alternative designs. The crossover design with the placebo control can be useful. This is particularly useful where a product does not have a hangover effect. In other words, when you discontinue the product, its effects are not going to ‘hang over’ for a period of time. This is a cheaper alternative. Open label studies can also be useful to give some hint of efficacy, but they are rather limited in most scenarios.
There are occasions when one can see a good design but also when one sees very poor quality design. Poor design is often associated with scenarios where the individuals have not done a power calculation.
It is amazing how many people go into a study without doing a power calculation: often, people enter a study and simply pluck a dose as though they had some evidence that this was the appropriate dose without doing a dose-response study. This is often seen in probiotic studies where somebody might choose 109 as the appropriate dose, but that figure has been arbitrarily selected without any dose response curve being identified. Of course, one also sees studies where these subjects are very poorly-phenotype. It is extremely important that subjects are well phenotyped. Without good phenotyping, it is likely that users are going to end up with a very poor quality study.
What are the common reasons that some studies fail?
Many studies fail - and they fail for all sorts of reasons. There was a series of studies in animals suggesting that lactobacillus rhamnosus, the JB-1 strain, was exceedingly effective in dealing with stress-related conditions, but when a large-scale study was done in humans, no effect was found whatsoever. Studies do not always translate well from rodents to humans, and one must bear that in mind.
It is possible to obtain some efficacy from an open-label study, and it may have little in the way of efficacy when one moves to a proper placebo-controlled trial - so you can end up with a very erroneous conclusion from an open label study. Of course, one of the big problems in the literature is that many negative studies are never published for all sorts of reasons. They are not particularly attractive to journals, and sometimes the companies who do the studies do not wish to publish negative findings.
Stress is clearly a major problem in the 21st century. In many situations, it leads to illnesses like depression or anxiety. It is a symptom or a manifestation that can be treated in a number of ways, but to demonstrate the efficacy of any product requires optimal trial design. At Atlantia Food Clinical Trials, we pride ourselves on being able to do trials in a cost-effective manner, which demonstrate either the product’s efficacy or the product’s lack of efficacy.
About Atlantia Clinical Trials
Atlantia Clinical Trials Ltd is a CRO that specializes in conducting studies on foods, beverages, and supplements for companies worldwide that want to scientifically validate their functional ingredients to support an: EFSA (European Food Safety Authority) Health Claim; FDA (Food & Drug Administration) Structure Function Claim; or General Product Marketing Claim.
Atlantia works with world-leading scientists (among the top cited 1% internationally, in the areas of digestive health and functional foods) at the: APC Microbiome Institute in University College Cork, Ireland; Teagasc, Moorepark, Ireland, and recognized centers of excellence globally.
Atlantia runs and operates its own clinic sites and conducts all studies to ICH-GCP standard (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use - Good Clinical Practice). Its team includes physician experts in digestive health, mental health (psychological stress and cognition), cardiovascular health, sports performance, metabolic disease, bone health, immune health, and healthy ageing. The clinical team also includes project managers, research nurses, nutritionists, certified sports trainers and lab researchers.
Atlantia manages all elements from protocol design, placebo manufacture, recruitment, and study execution, to sample and data analysis, statistics, and report/dossier preparation to provide a service that is technically, scientifically, and clinically superior.
The clinical studies cover a broad spectrum of functional food and beverage categories, such as dairy, cereal, probiotics, different protein forms, infant-specific foods, vitamins/minerals, plant or marine extracts, and medical foods.
The brain-gut microbiota axis: Impact on mental health & potential treatment avenues