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Is the Hygiene Hypothesis True?

Did Covid shutdowns stunt kids' immune systems?

Caitlin Rivers

The hygiene hypothesis is the idea that kids need to be exposed to germs in order to develop healthy immune systems. We know that many common viruses did not circulate as widely during the pandemic, thanks to social distancing, masking, and other COVID mitigation measures. Are there downsides to those missed infections?

In this Q&A, Caitlin Rivers speaks with Marsha Wills-Karp, PhD, MHS , professor and chair of Environmental Health and Engineering , about the role of household microbiomes, birth, and vaccines in the development of kids’ immune systems—and whether early exposure really is the best medicine.

This Q&A is adapted from Rivers’ Substack blog, Force of Infection .

I think there’s some concern among parents who have heard about the hygiene hypothesis that there is a downside to all those stuffy noses that didn’t happen [during the COVID-19 pandemic]. Are there any upsides to viral infections? Do they help the immune system in some meaningful way?

I don’t think so.

You mentioned the hygiene hypothesis, which was postulated back in the ‘80s. German scientists noticed that families with fewer children tended to have more allergic disease. This was interpreted [to mean] that allergic disease was linked to experiencing fewer infections. I have explored this idea in my research for a couple of decades now.

This phenomenon has helped us to understand the immune system, but our interpretation of it has grown and expanded—particularly with respect to viruses. Almost no virus is protective against allergic disease or other immune diseases. In fact, infections with viruses mostly either contribute to the development of those diseases or worsen them.

The opposite is true of bacteria. There are good bacteria and there are bad bacteria. The good bacteria we call commensals . Our bodies actually have more bacterial cells than human cells. What we’ve learned over the years is that the association with family life and the environment probably has more to do with the microbiome. So one thing I would say is sanitizing every surface in your home to an extreme is probably not a good thing. Our research team showed in animals that sterile environments don’t allow the immune system to develop at all. We don’t want that.

What does contribute to the development of the immune system, if not exposure to viruses?

There are a number of factors that we’ve associated with the hygiene hypothesis over the last 20 years, and these exposures start very early in life. Cesarean sections, which do not allow the baby to travel through the birth canal and get exposed to the mother’s really healthy bacterial content, is a risk factor for many different immune diseases. Getting that early seeding with good bacteria is critical for setting up the child going forward. Breastfeeding also contributes to the development of a healthy immune system.

There are other factors. Our diets have changed dramatically over the years. We eat a lot of processed food that doesn’t have the normal components of a healthy microbiome, like fiber. These healthy bacteria in our gut need that fiber to maintain themselves. They not only are important for our immune system but they’re absolutely critical to us deriving calories and nutrients from our food. All these things contribute to a healthy child.

We’ve also noticed that people who live on farms have fewer of these diseases because they’re exposed to—for lack of a better term—the fecal material of animals. And what we have found is that it’s due to these commensal bacteria. That is one of the components that help us keep a healthy immune system. Most of us will probably not adopt farm life. But we can have a pet, we can have a dog.

I think all the pet lovers out there will be pleased to hear that.

There’s a lot of evidence that owning a pet in early childhood is very protective.

What about the idea that you need to be exposed to viruses in early life because if you get them as an adult, you’ll get more severely ill? We know that’s true for chickenpox, for example. Do you have any concerns about that?

We should rely on vaccines for those exposures because we can never predict who is going to be susceptible to severe illness, even in early childhood. If we look back before vaccines, children under 4 often succumbed to infections. I don’t think we want to return to that time in history.

Let me just give you one example. There’s a virus called RSV, it’s a respiratory virus. Almost all infants are positive for it by the age of 2. But those who get severe disease are more likely to develop allergic disease and other problems. So this idea that we must become infected with a pathogenic virus to be healthy is not a good one.

Even rhinovirus, which is the common cold, most people recover fine. But there’s a lot of evidence that for somebody who is allergic, rhinovirus exposures make them much worse. In fact, most allergic or asthmatic kids suffer through the winter months when these viruses are more common.

And that’s particularly salient because there is a lot of rhinovirus and enterovirus circulating right now.

From my point of view, right now, avoiding flu and COVID-19 is a priority. Those are not going to help you develop a healthy immune response, and in fact, they can do a lot of damage to the lungs during that critical developmental time. Data [show] that children that have more infections in the first 6 months to a year of life go on to have more problems.

It’s always surprising to me when I look at the data of the fraction of time that young children spend with these common colds—and this is pre-pandemic—it’s not uncommon for kids to be sick 50% of the time. That feels right as a parent, but it’s startling.

The other thing people don’t know is that the GI tract is where you get tolerized to all of your foods, allergens and things. Without those healthy bacteria in your gut, you can’t tolerate common allergens.

How does that relate to the guidance that’s changed over the years—that you should withhold peanuts in early life and now you’re supposed to offer them in early life?

The guidance to delay exposure to peanuts didn’t consider the fact that oral exposure to peanuts was not the only exposure kids were getting. There were peanut oils in all kinds of skin creams and other things. So kids got exposed through their skin, but they had no gut protection—and the GI tract is important for a tolerant system. If you have a healthy immune response, you get tolerized in early life.

This concept is a little bit different for those families who may already have a predisposition to allergies. But for the general public, exposure is key to protecting them in early life.

I think some parents look at the guidance that you should now offer peanuts in early life and say, “Are we not doing that with rhinovirus by masking kids or improving ventilation?” How should people think about the development of the immune system for food allergies compared to infections?

The thing about rhinoviruses is that after recovering, you’re not protected from the next infection. There is no real immune protection there. Most of us suffer from colds throughout our whole life. Like I said, bacterial exposure is what’s key to priming the immune response.

Also, we forget that a lot of kids die from the flu. Unlike COVID-19, where younger kids are not quite as susceptible to severe illness, that’s not true for flu. RSV, too, can be quite severe in young children and older adults.

Caitlin Rivers, PhD, MPH , is a senior scholar at the Johns Hopkins Center for Health Security and an assistant professor in Environmental Health and Engineering at the Johns Hopkins Bloomberg School of Public Health.

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Revisiting the hygiene hypothesis in the context of autoimmunity.

1 Université de Paris, Paris, France
2 INSERM U1151, Institut Necker-Enfants Malades, Paris, France
3 Academie des Sciences, Paris, France

Initially described for allergic diseases, the hygiene hypothesis was extended to autoimmune diseases in the early 2000s. A historical overview allows appreciation of the development of this concept over the last two decades and its discussion in the context of evolution. While the epidemiological data are convergent, with a few exceptions, the underlying mechanisms are multiple and complex. A major question is to determine what is the respective role of pathogens, bacteria, viruses, and parasites, versus commensals. The role of the intestinal microbiota has elicited much interest, but is it a cause or a consequence of autoimmune-mediated inflammation? Our hypothesis is that both pathogens and commensals intervene. Another question is to dissect what are the underlying cellular and molecular mechanisms. The role of immunoregulatory cytokines, in particular interleukin-10 and TGF beta is probably essential. An important place should also be given to ligands of innate immunity receptors present in bacteria, viruses or parasites acting independently of their immunogenicity. The role of Toll-Like Receptor (TLR) ligands is well documented including via TLR ligand desensitization.

Introduction

The hygiene hypothesis is a counterintuitive concept. While it is well known that infectious agents are potentially responsible for many diseases beyond infectious diseases, the idea emerged that they could in some cases have a favorable effect on non-infectious and sometimes very serious illnesses. The original report by Strachan in 1989 was based on an observation that might seem anecdotal: hay fever and atopic dermatitis are less frequent in families with many children than in families with only one or two children ( 1 ). It was to Strachan’s credit that he then proposed the hypothesis that common childhood infections may reduce the frequency of atopic diseases. It was only a little later, in 2000, that he proposed that the increase in the frequency of allergic diseases observed in the three or four preceding decades could be ascribed to the decrease in the frequency of infectious diseases ( 2 ). It was also in the early 2000s that the hygiene hypothesis was extended to autoimmune diseases ( 3 ). At that time there were already data obtained in experimental models showing that infections, particularly parasitic infections, could prevent the occurrence of autoimmunity ( 4 ). Since then, compelling evidence has been gathered to support the hygiene hypothesis. We review it here stressing the importance of causal relationships, since it is not sufficient to show a correlation between two events to affirm causality. We will highlight the importance of experimental models, particularly those concerning spontaneous diseases, the closest to human diseases.

Like any scientific hypothesis, the hygiene hypothesis has elicited conflicting opinions. By examining several hundred articles devoted to the subject, we find a majority supporting the hypothesis. There are, however, a number of articles expressing reservations or even, more directly, questioning the hypothesis. These challenging reports relate particularly to allergic diseases. Many allergists are more inclined to explain the increase in the frequency of allergic diseases, which nobody denies, by changes in the non-infectious environment, even going so far as to incriminate the increase in the dissemination of pollens. Such claims are difficult to accept when one considers that the increase in the frequency of allergic diseases affects all clinical forms ranging from atopic dermatitis to hay fever and even food allergies. In addition, the parallel evolution of autoimmune diseases does not support the hypothesis of changes in the allergenic environment. Another source of questioning is linked to the fact that all autoimmune diseases are not concerned by the hypothesis, without knowing why some of them are and others not, a subject of very great interest per se . Also, it is very difficult to know which infections are involved in the hypothesis. The study of experimental models makes it possible to identify infectious agents, bacteria, viruses and especially parasites, which prevent the occurrence of allergic and autoimmune diseases. Analysis is much more difficult in humans. The lower incidence of allergic and autoimmune diseases in large families mentioned above suggests that common childhood infections play a role. The mirroring chronological course of the decrease in major infectious diseases and the increase in allergic and autoimmune diseases argues for serious infectious diseases being also involved in the hypothesis. The problem is further complicated by the fact that certain infectious agents can cause acute autoimmune diseases as rheumatic fever and Guillain–Barré syndrome.

Autoimmune diseases are multifactorial and polygenic. The predisposing factors for autoimmune diseases are both genetic and environmental ( Figure 1 ). Among genetic factors major histocompatibility genes (HLAs) play a major role, variable depending on the disease yet sometimes highly significant as in the case of type 1 diabetes, ankylosing spondylitis and narcolepsy. The role of a very large number of chromosomal regions identified by GWAS is certainly important, although the multitude of regions in question and the very low risk factor associated with each of them makes their priority ranking uncertain. Other genes could be involved in particular rare variants. One must also mention the potential role of epialleles that control epigenetic modifications participating to certain autoimmune diseases. Concerning the environment, a distinction must be made between factors which contribute to the onset of autoimmune diseases and those which prevent them. One must cite the hypothesis, still not proven in humans, according to which viruses could contribute to the triggering of autoimmune diseases such as type 1 diabetes and multiple sclerosis, secondarily to inflammation of the target organ by a local viral infection. The role of the gut microbiota is also relevant, although much remains to be done to demonstrate causality. To all this, we must add the possible implication of stochastic events, the existence of which is well proven in cancer (somatic mutations), still unknown for autoimmune diseases.

Figure 1 Etiological Factors for Autoimmune Diseases.

At this point it is important to clarify what is intended by “hygiene” when referring to the hygiene hypothesis. When speaking about hygiene, one normally thinks of classic hygiene as ensured by hand washing or social distancing rules that have reappeared in the foreground during the recent SARS-CoV-2 pandemics. The hygiene hypothesis we are discussing here is something else. It is about the environmental infectious burden which relies more on the socio-economic context specific to each industrialized country, each region or the family social context than to personal hygiene. Most of the factors that contribute to the hygiene hypothesis are collective and not individual. This infectious burden depends, to a large extent, on the quality of the drinking water, respect for the cold chain, the extensive use of antibiotics but also the generalization of vaccines. A development, obviously favorable because it prevents the occurrence of serious infectious diseases, but once again, independently of personal hygiene. The possible solutions to reduce the frequency of allergic and autoimmune diseases will obviously not come from the reintroduction of certain infections but rather from the use of “substitutes” for these infections which will retain their protective benefits.

Interest and Limitations of Epidemiology

The hygiene hypothesis in its dynamic aspect is based on the negative correlation observed between the decrease in the frequency of infectious diseases and the increase in that of allergic and autoimmune diseases. The question arises as to whether the trends reported twenty years ago persist today ( 3 ). The answer is clearly positive concerning infectious diseases which, under the effect of hygiene, vaccinations and antibiotics, continued to decrease in industrialized countries. However, many common childhood infections persist and new pandemics such as COVID 19 occur. The question is more complex concerning allergic and autoimmune diseases. Unfortunately, there is relatively little recent epidemiological data with no international databases on these diseases as they exist for the major infectious diseases. It can be noted, however, that for T1D, as well as for allergic diseases, the frequency has continued to increase in recent years ( 5 , 6 ) with, for T1D, affecting very young children ( 7 ). The question is more open for other diseases such as multiple sclerosis for which it seems, at least in some countries, that a plateau has been reached.

The main aim of the epidemiological approach to the hygiene hypothesis is to show the existence of a direct relationship between the number of infections and the frequency of allergic and autoimmune diseases. Unfortunately, it is very difficult to count infections because if one usually remembers serious infections it is much more difficult to memorize common infections which, as we have seen, probably play a significant role. Like others, we ourselves have tried to study this problem in the setting of atopic dermatitis and reached the conclusion that reliable enumeration of infections is almost impossible ( 8 ). Indirect markers must be used. The most often used concerns the socio-economic environment and family composition, in particular the number of children in families ( 1 , 2 ). It is very interesting to note, as has been done for decades, that allergic and autoimmune diseases are more common in high socio-economic backgrounds and in families with few children. We find this conclusion in the study of the geographical disparity of allergic and autoimmune diseases on the one hand and infectious diseases on the other ( 3 , 9 ). Several hypotheses have been put forward to explain this phenomenon ( 3 , 9 ). Genetic factors do not have a determining role because migrants from countries with a low incidence of allergic or autoimmune diseases to countries with a high incidence develop these diseases with the same frequency as in host countries from the first generation ( 10 – 14 ). It suffices that the migration takes place before the age of 5 years for allergic diseases ( 14 ) or fifteen years for multiple sclerosis ( 12 , 13 ) for the increase in incidence to manifest. This last observation, which suggests that the protective effect of infections develops over a fairly long period of childhood should be taken into consideration when discussing the role of intestinal dysbiosis insofar as the composition of the gut microbiota is fixed very early in life (2 or 3 years of age) (see below). Another interpretation calls for climatic differences. This hypothesis, which could explain the role of parasitic infections that are more frequent in tropical regions, must be considered with caution when we know that the frequency of T1D and allergies is four to six times greater in Finland than in Karelia which differ for socio-economic level while the climate and genetic factors are basically the same in these two contiguous countries ( 15 , 16 ). Incidentally, one should highlight that the difference of T1D incidence between Finland and Karelia does not apply to islet-cell autoantibodies suggesting that the effect of hygiene applies more to the progression than to the triggering of the autoimmune process ( 17 ). In brief, all this suggests that it is the socio-economic factors with all the consequences on health conditions which primarily explain the differences in the frequencies of allergic and autoimmune diseases in the different regions of the world.

It would, however, be interesting to find other indirect markers of infections. This was done by analyzing the prevalence of stigmata of infections by bacteria, viruses or parasites widely distributed in the population. For example, atopy has been shown to be more common in some parts of the world when the rate of seropositivity against hepatitis A virus is low ( 3 , 18 ). Regarding autoimmune diseases multiple sclerosis is associated with a lower seropositivity for cytomegalovirus (CMV) ( 19 , 20 ) or Helicobacter pylori ( 21 ). The same observation was made for CMV in T1D ( 22 ). Also, multiple sclerosis is associated with an abnormally low exposure to Toxoplasma gondii ( 23 ).

Finally, another extremely original approach results from the analysis of the repertoire of the antigen receptor of T lymphocytes (TCR) in subjects presenting allergy. It has in fact been shown that the diversity of this repertoire was restricted which was interpreted as the reflect of a lesser solicitation of the immune system by infectious agents ( 24 ).

Causal Relationship

It is not sufficient to observe a negative correlation between the decrease in infections and the increase in allergic and autoimmune diseases to affirm a cause and effect relationship. It is difficult to prove this in humans, although there are many arguments in favor of such interpretation. The best answer will undoubtedly come from therapeutic trials in which it will be shown directly with statistically interpretable results that the suppression of certain infections increases the frequency of allergic and autoimmune diseases or conversely that the administration of certain infectious agents or parasites, needless to say preferably in the form of extracts ( 25 , 26 ), prevent their occurrence. Some elements of response have been obtained for allergic diseases, in particular worsening of asthma in patients who have been subjected to antiparasitic treatments ( 27 ). We can also mention, although the data are contradictory, the improvement in atopy observed after administration of probiotics ( 28 ). Far fewer arguments exist for autoimmune diseases. At most, one can note therapeutic trials of limited size suggesting a favorable effect of the infestation of patients suffering from multiple sclerosis by a live parasite Tricuris suis ( 29 , 30 ). The best arguments come from studying spontaneous experimental models of autoimmunity such as the non-obese diabetic (NOD) mouse and the lupus B/W mouse. It is necessary to set aside the models of induced autoimmune diseases upon administration of autoantigens. These models use adjuvants which are known to themselves induce protection from autoimmunity ( 31 ) and therefore complicate the interpretation of a potentially preventive effect by infections. A large number of infectious agents (bacteria, viruses or parasites) prevent autoimmune disease in NOD and BW mice. We refer the reader to a recent review for the NOD mouse ( 9 ). With regard to B/W mice, kidney disease and survival can be considerably improved by viral ( 32 ) or parasitic ( 33 ) infections. It is, in fact, in this model that was published the first convincing observation of the prevention of autoimmune diseases by a parasite, in this case Plasmodium berghi ( 4 ). Other studies have confirmed this favorable action of parasites ( 25 , 26 , 34 – 36 ).

As we will see below, the use of these models, in addition to providing the necessary proof of concept for the hygiene hypothesis, have shed light on the underlying mechanisms.

To conclude this data clearly shows that numerous pathogenic infectious agents protect from autoimmune diseases independently of any relationship with the gut microbiota.

Hygiene Hypothesis and Evolution

The epidemiological observations on which the hygiene hypothesis is based date back some fifty years. It is obvious that the increase in the frequency of allergic and autoimmune diseases does not have a genetic basis within populations in which changes in the frequency of these diseases have been observed, except in the case of the migrants mentioned above. These phenotypes reflect an adaptation of the organism, more particularly of the immune system, to the environment and more specifically to the infectious environment. It is, however, interesting to note that this deviance implies that the organism has adapted to changes in the environment by creating a phenotype that had not been the object of natural selection during evolution. Other examples of diseases come under the same commentary such as obesity and type 2 diabetes which are linked to overeating in individuals who have been selected to develop energy storage mechanisms ( 37 ).

On the other hand, we can ask the question of an interaction between the occurrence of infectious diseases and inflammatory diseases during evolution. This possibility has been considered in particular by L. Quintana-Murci ( 38 , 39 ). This author highlighted the delicate situation in which the immune system found itself between establishing a strong inflammatory response to fight against pathogens in an environment with a high pathogen load while avoiding the harmful consequences of acute and chronic inflammation, which could lead to inflammatory and/autoimmune diseases ( 38 ). Interestingly, Genome Wide Association Studies (GWAS) studies have shown a community of single nucleotide polymorphisms (SNPs) associated with a strong anti-infective immune response and those associated with a predisposition to inflammatory and autoimmune diseases ( 40 – 42 ).

We can also wonder if the composition of the intestinal microbiota which, as we will discuss below, contributes to the hygiene hypothesis is not also subject to evolution, more precisely to a co-evolution of the immune system and commensal bacteria. One can imagine that the bacteria that resisted evolution did so because they had no pathogenicity and did not endanger people’s lives and could even have a favorable influence. A kind of immune tolerance has thus been created, the consequences of which are difficult to perceive and especially the relationship with the role of the microbiota in the hygiene hypothesis.

Asking this question leads us to evoke the selective pressure that has weighed on the human species since the appearance of Homo-sapiens ( 38 , 39 ). In recent years, population genetics work has provided major information thanks to advances in genomics. First, it should be noted that modern Homo-sapiens arose from crosses between African humans and Neanderthals. Neanderthals contributed little (about 2%) to the Homo-sapiens genome. Nevertheless, it has been repeatedly shown that some of the haplotypes which persist in Homo-sapiens and which originate from Neanderthals include genes important for immune responses. It has recently been shown that certain genes predisposing to COVID 19 are derived from Neanderthals ( 43 ). Subsequently, over the past 100,000 years, genes involved in immune reactions have evolved through a process of natural selection, positive or negative (purifying). Evolution has taken place under the selective pressure of environmental conditions, first the shift from humans hunters/collectors to humans cultivators, then the migration of humans from Africa to Asia and then to Europe. Numerous studies carried out on this subject, in particular by L. Quintana-Murci, have made it possible to identify the genes in question ( 38 , 39 ). These are essentially genes controlling innate immunity. We note more particularly the presence of genes encoding toll-like receptors (TLRs). It thus appears, which was intuitive, that the evolution gave rise to an improvement of the immune responses against infectious agents under their pressure. The case of epidemics illustrates this point. Thus, subjects with a mutation in the NOD-2 gene, associated with inflammatory bowel diseases, seem to be over-represented in populations that have been severely affected by plague epidemics ( 44 ).

How, then, can these observations be linked to the hygiene hypothesis? It is important to note that the changes in the genes of immune responses during evolution have been made at a particularly rapid rate. These changes did not span hundreds of thousands of years as is the case for other genes but often only a few thousand years, and sometimes even less in major epidemics or extreme environmental situations. In contrast, the hygiene hypothesis is based on adaptation rather than selection. We thus find ourselves in a situation where the time scale associated with the selective pressure which influenced natural selection intersects with that of the hygiene hypothesis which only applies to the last 50 years. Going further, one can wonder whether the reduction in the infectious burden, on which the hygiene hypothesis is based, will not influence the evolution of the genes controlling the immune responses. Conversely, even if these diseases were serious enough to reduce the number of offsprings in affected subjects, the genes which control them are so numerous and interactive that it is difficult to see how they could influence natural selection, especially since they are rarely deleterious mutations but more often polymorphisms whose isolated presence in healthy subjects has no consequence.

Intestinal Dysbiosis, Cause, Consequence, or Modulation

The emergence of metagenomics, which has made it possible over the past fifteen years to characterize the composition of the intestinal microbiota, has opened a new page in the hygiene hypothesis. Several arguments, suggest that a decrease in the diversity of the intestinal microbiota could contribute to the occurrence of autoimmune diseases as well as many other pathologies including allergic diseases, type 2 diabetes and obesity.

The mainstay is the existence of a dysbiosis, that is to say an imbalance of the commensal bacteria that make up the intestinal microbiota in the diseases in question. One observes, indeed, in these different diseases a reduction in the diversity of the microbiota, more particularly in certain species, with often a decrease in lactobacilli ( 45 ), in particular in type 1 diabetes ( 9 ), multiple sclerosis ( 46 – 48 ) and systemic lupus erythematosus ( 49 – 51 ).

At the same time, destruction of the gut microbiota by administration of broad-spectrum oral antibiotics to mothers and newborns has been shown to increase the frequency of T1D in NOD mice ( 52 ) and experimental asthma ( 53 ).

The link between these observations on the hygiene hypothesis quickly became apparent. We know, in fact, that the composition of the intestinal microbiota is different depending on the level of hygiene. This has been demonstrated in particular by comparing the microbiota of subjects living in Italy or Burkina Faso ( 54 ), an observation which is, however, not conclusive because many other elements differ between such countries, in particular diet which influences the composition of the microbiota. A more direct argument is the observation that pigs reared in a clean facility have a different microbiota than those reared in a conventional barn ( 55 ).

These observations aroused great enthusiasm. Numerous studies have attempted to characterize commensal bacteria which could be responsible for regulating autoimmunity and whose absence or decrease could contribute to the occurrence of autoimmune diseases. It must be recognized, however, that so far, few conclusive results have been reported.

To these fairly convincing arguments it is necessary to mention other elements which incite more reticence. The main question is that of the causal relationship between dysbiosis and the occurrence of the diseases under consideration. Does said dysbiosis play a role in triggering the disease or in its progression or is it the consequence of the disease. To answer this question, we should not be content to study the composition of the microbiota at the time when the disease is already declared. The microbiota should be studied before the onset of the disease. In fact, this has so far only been done extensively in T1D, where cohorts of subjects with a high inheritance of diabetes have been followed from birth. Dysbiosis has been observed at the time of disease onset ( 56 ). However, if we carefully examine the timing of the onset of dysbiosis, we find that it takes place after the development of the autoimmune response against the beta-cells of the islets of Langerhans, suggesting either that it is secondary to the inflammation associated with the onset of diabetes or it contributes to the transformation of respectful insulitis into malignant insulitis which marks the onset of clinical diabetes.

The conclusions from the use of probiotics remain uncertain. The probiotics used were not really calibrated and the number of bacteria administered was very low compared to the number of intestinal bacteria, posing the problem of their mode of action: modification of the composition of the microbiota, but then for how long, pharmacological effect or other mechanisms.

In brief, there are interesting arguments to suggest the role of dysbiosis in the occurrence of autoimmune diseases, but they are fragile. In any case, data suggests in view of the results obtained in animal models that pathogens also play an important role, in particular agents that have no relation to the intestine such as mycobacteria.

Multiple and Complex Underlying Mechanisms

Many publications have been devoted to the mechanisms underlying the hygiene hypothesis namely, how to explain that infections can reduce the frequency of allergic or autoimmune diseases. It appears very clearly that no univocal explanation can be presented for all the protective effects of infections. Several major mechanisms appear to operate. The problem is complicated by the fact that the mechanisms can be different depending on the infection. We will briefly discuss the main data available by referring the reader who would like more details and a more documented bibliography to a general review recently published ( 9 ).

It has been known for many decades that concomitant immune responses compete, a phenomenon termed antigenic competition which could well be applied to the hygiene hypothesis by supposing that very strong immune responses against infectious agents could compete with immune responses directed against weak antigens such as allergens or autoantigens due to increased consumption of homeostatic factors. In fact, this mechanism has been studied very little in the case of the hygiene hypothesis. This may be explained by the poor knowledge that we still have today on the molecular basis for antigenic competition, even if converging data seem to give a major role to homeostatic factors, in particular interleukin (IL)-2, IL-7 and IL-15. In any event, as attractive as it is, this hypothesis remains very poorly documented.

Before attempting to present a unitary hypothesis, it is important to mention that certain mechanisms appear to be relatively specific to certain infectious agents. Thus, the protective effect of live mycobacteria or Freund’s complete adjuvant involves CD4+CD25+FOXP3+ regulatory T lymphocytes like other infectious agents but also, more unexpectedly, natural killer or NK cells ( 57 ). Lipopolysaccharide (LPS) contained in Escherichia coli similarly stimulates regulatory T lymphocytes and it also stimulates a particular subset of IL-10-producing B lymphocytes which play an important immunoregulatory role ( 58 ).

The commensal bacteria of the intestinal microbiota also have a protective effect against autoimmune diseases. This has been shown globally with probiotics ( 9 ) but also with well-identified commensal bacteria (lactobacilli). In this case too, various mechanisms are involved. As far as probiotics are concerned, a predominant role has been attributed to IL-10. In other situations, such as Clostridium, a major role has been ascribed to CD4+CD25+ FOXP3+ regulatory T cells ( 59 ). It should also be noted that many infectious agents modulating autoimmune responses involve the immunoregulatory cytokine TGF beta. This is the case of a gram-positive bacterial extract which protects the NOD mouse from diabetes, an effect which is reversed by the administration of antibodies neutralizing TGF beta but not IL-10 ( 60 ). Moreover, a key role of TGF beta has also been shown in the protective mechanisms mediated by various parasites with the exception of schistosomes which appear to act through IL-10 production ( 61 ). A role for interferon gamma (IFN γ ) has been proposed for both allergy ( 62 ) and autoimmunity ( 63 ). In brief, multiple mechanisms are involved that mostly rely on immunoregulatory circuits.

At the molecular level, many arguments suggest that both pathogens, bacteria or viruses and also parasites as commensal bacteria exert their protective effects by primarily involving their molecular interactions with TLR receptors. These different infectious agents indeed contain TLR ligands, both pathogens and commensals. Above all, the systemic administration of chemically characterized ligands of the various TLRs reproduces the protective action of the infectious agents mentioned above ( 64 ). It should also be noted that it is not necessary for the infectious agents in question to be alive to prevent the onset of autoimmune diseases: they can be substituted with bacterial ( 60 ) or parasitic extracts which have the same effect ( 25 , 26 ). The different TLR ligands could have distinct mechanisms of action depending on the specific receptor involved. For example, TLR4 ligands appear to act through FOXP3+ regulatory T cells, while TLR3 ligands involve invariant NKT cells ( 64 ).

It is now well known that the desensitization of macrophages to the pro-inflammatory effect of LPS, also called “endotoxin tolerance”, is mediated by a joint action through TLR4 and TLR2 receptors and their common signaling pathways ( 65 ). Another example of desensitization is the prevention of type 1 diabetes in NOD mice by TLR2 ligands, called “TLR2 tolerance” ( 66 ). In an adoptive transfer model, it was shown that the repeated treatment with the TLR2 agonist Pam3CSK4 of NOD mice receiving diabetogenic T lymphocytes inhibited the development of the disease ( 66 ). This same TLR2 desensitization could also be involved in the prevention of experimental allergic encephalomyelitis (EAE) ( 67 ). Administration of low doses of two different TLR2 ligands, Pam2CSK4 or Lipid 654 (L654), to naive recipients of encephalitogenic (EAE-inducing) T cells decreased the level of TLR2 signaling at the same time as it attenuated EAE ( 67 , 68 ). Interestingly, L654 is a TLR2 ligand derived from a commensal of the microbiota which is present in healthy human serum, but whose concentration is significantly reduced in the serum of patients with multiple sclerosis ( 67 , 68 ). In another mouse model of EAE, repeated administration of a synthetic TLR7 ligand has been reported to significantly decrease the severity of disease as well as the expression of chemokines in the target organ ( 69 ). Finally, it is interesting to note that NOD mice genetically deficient in TLR4 and MyD88 show an acceleration in the severity of diabetes and experimental asthma respectively, again suggesting a protective role for TLR signaling ( 64 , 70 ).

In humans, the role of desensitization by TLRs has been demonstrated in an elegant work by the group of B. Lambrecht concerning children raised on dairy farms (an environment rich in LPS) who present a low incidence of allergy ( 62 , 71 ). Epithelial cells in the lungs have shown reduced production of cytokines that normally activate dendritic cells to induce TH2-type lymphocyte responses. The TLR4 desensitization induced by LPS which could be responsible for this effect targets the pulmonary epithelium ( 71 ).

In conclusion, although very interesting, this data do not suffice to give a complete picture of the mechanisms underlying the hygiene hypothesis. It is therefore necessary to consider the overall response to pathogens or commensals as the result of the integration of positive and negative signals delivered via the TLRs. This concept paves the way for a TLR targeted immunopharmacology.

Conclusions

The hygiene hypothesis, misnamed as it is, teaches us a lot about immunity, immunopathology, epidemiology and evolution. It indicates how flexible the immune system is, under constant control by immunoregulation under the influence of the environment. It sheds light on the mechanisms underlying many immune related diseases, in particular autoimmune diseases but also probably many other diseases which involve an uncontrolled differentiation of lymphocytes, whether they are allergic diseases or certain malignant lymphoproliferative disorders ( 72 ). It opens new perspectives on the etiological factors of autoimmune diseases by distinguishing those that could trigger or on the contrary protect ( Figure 1 ). Finally, when it comes to evolution, it provides a particularly bright illustration of the relationships that may exist between natural selection and adaptation under the control of infectious agents.

Author Contributions

The author confirms being the sole contributor of this work and has approved it for publication.

The laboratory of the author was supported by an advanced grant from the European Research Council (ERC, Hygiene N°: 250290).

Conflict of Interest

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords: hygiene hypothesis, autoimmune diseases, type 1 diabetes, non-obese diabetic mouse, Toll-Like Receptor, gut microbiota, evolution, migrants

Citation: Bach J-F (2021) Revisiting the Hygiene Hypothesis in the Context of Autoimmunity. Front. Immunol. 11:615192. doi: 10.3389/fimmu.2020.615192

Received: 08 October 2020; Accepted: 07 December 2020; Published: 28 January 2021.

Reviewed by:

Copyright © 2021 Bach. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Jean-François Bach, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Asthma: The Hygiene Hypothesis

What do clean houses have in common with childhood infections.

One of the many explanations for asthma being the most common chronic disease in the developed world is the “hygiene hypothesis.” This hypothesis suggests that the critical post-natal period of immune response is derailed by the extremely clean household environments often found in the developed world. In other words, the young child’s environment can be “too clean” to pose an effective challenge to a maturing immune system.

According to the “hygiene hypothesis,” the problem with extremely clean environments is that they fail to provide the necessary exposure to germs required to “educate” the immune system so it can learn to launch its defense responses to infectious organisms. Instead, its defense responses end up being so inadequate that they actually contribute to the development of asthma.

Scientists based this hypothesis in part on the observation that, before birth, the fetal immune system’s “default setting” is suppressed to prevent it from rejecting maternal tissue. Such a low default setting is necessary before birth—when the mother is providing the fetus with her own antibodies. But in the period immediately after birth the child’s own immune system must take over and learn how to fend for itself.

The “hygiene hypothesis” is supported by epidemiologic studies demonstrating that allergic diseases and asthma are more likely to occur when the incidence and levels of endotoxin (bacterial lipopolysaccharide, or LPS) in the home are low. LPS is a bacterial molecule that stimulates and educates the immune system by triggering signals through a molecular “switch” called TLR4, which is found on certain immune system cells.

The science behind the hygiene hypothesis

The Inflammatory Mechanisms Section of the Laboratory of Immunobiochemistry is working to better understand the hygiene hypothesis, by looking at the relationship between respiratory viruses and allergic diseases and asthma, and by studying the respiratory syncytial virus (RSV) in particular.

What does RSV have to do with the hygiene hypothesis?

RSV is often the first viral pathogen encountered by infants.
RSV pneumonia puts infants at higher risk for developing childhood asthma. (Although children may outgrow this type of asthma, it can account for clinic visits and missed school days.)
RSV carries a molecule on its surface called the F protein, which flips the same immune system “switch” (TLR4) as do bacterial endotoxins.

It may seem obvious that, since both the RSV F protein and LPS signal through the same TLR4 “switch,” they both would educate the infant’s immune system in the same beneficial way. But that may not be the case.

The large population of bacteria that normally lives inside humans educates the growing immune system to respond using the TLR4 switch. When this education is lacking or weak, the response to RSV by some critical cells in the immune system’s defense against infections—called “T-cells”—might inadvertently trigger asthma instead of protecting the infant and clearing the infection. How this happens is a mystery that we are trying to solve.

In order to determine RSV’s role in triggering asthma, our laboratory studied how RSV blocks T-cell proliferation.

Studying the effect of RSV on T-cells in the laboratory, however, has been very difficult. That’s because when RSV is put into the same culture as T-cells, it blocks them from multiplying as they would naturally do when they are stimulated. To get past this problem, most researchers kill RSV with ultraviolet light before adding the virus to T-cell cultures. However we did not have the option of killing the RSV because that would have prevented us from determining the virus’s role in triggering asthma.

Our first major discovery was that RSV causes the release from certain immune system cells of signaling molecules called Type I and Type III interferons that can suppress T-cell proliferation (Journal of Virology 80:5032-5040; 2006).

The hygiene hypothesis suggests that a newborn baby’s immune system must be educated so it will function properly during infancy and the rest of life. One of the key elements of this education is a switch on T cells called TLR4. The bacterial protein LPS normally plays a key role by flipping that switch into the “on” position.

Prior research suggested that since RSV flips the TLR4 switch, RSV should “educate” the child’s immune system to defend against infections just like LPS does.

But it turns out that RSV does not flip the TLR switch in the same way as LPS. This difference in switching on TLR, combined with other characteristics of RSV, can prevent proper education of the immune system.

One difference in the way that RSV flips the TLR4 switch may be through the release of interferons, which suppresses the proliferation of T-cells. We still do not know whether these interferons are part of the reason the immune system is not properly educated or simply an indicator of the problem. Therefore, we plan to continue our studies about how RSV can contribute to the development of asthma according to the hygiene hypothesis.

Further research

This finding that Type I and Type III interferons can mediate the suppression of T-cells caused by RSV generated two significant questions that our laboratory is now addressing:

Interferons are important molecules that enhance inflammation, so why--in the context of RSV--do they suppress T-cells?
Interferons are clearly not the only way RSV suppresses T-cells. What are the other mechanisms that may depend upon T-cells coming in direct contact and communicating with other immune cells?

Related Research

Assessing the Mechanism of Immunotherapy for Allergy and Allergic Asthma: Effect of Viral Respiratory Infections on Pathogenesis and Clinical Course of Asthma and Allergy Ronald Rabin, MD

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Introduction, section snippets, references (144), cited by (42).

Acta Tropica

The hygiene hypothesis at a glance: early exposures, immune mechanism and novel therapies, the hygiene hypothesis, the role of gut microbiome, the influence of helminth infections, the influence of protist infections, the impact of other infections, the controversial studies: when hygiene hypothesis failed, the hygiene hypothesis and the cross-talk with the immune system, the future of hygiene hypothesis: insights into therapeutic implications, final remarks, acknowledgments, fecal bacteriotherapy for recurrent clostridium difficile infection, treatment of ulcerative colitis by implantation of normal colonic flora, cesarean delivery may affect the early biodiversity of intestinal bacteria, early-life gut microbiome composition and milk allergy resolution, j. allergy clin. immunol., the impact of parasite infections on the course of multiple sclerosis, j. neuroimmunol., global issues in allergy and immunology: parasitic infections and allergy, allergy and parasites reevaluated: wide-scale induction of chronic urticaria by the ubiquitous fish-nematode anisakis simplex in an endemic region, allergol. immunopathol., helminth therapy and multiple sclerosis, int. j. parasitol., secretory products of helminth parasites as immunomodulators, mol. biochem. parasitol., foxp3+ t regulatory cells and immunomodulation after schistosoma mansoni egg antigen immunization in experimental model of inflammatory bowel disease, cell. immunol., endotoxin exposure in allergy and asthma: reconciling a paradox, (self-) infections with parasites: re-interpretations for the present, trends parasitol., the effect of neonatal bcg vaccination on atopy and asthma at age 7 to 14 years: an historical cohort study in a community with a very low prevalence of tuberculosis infection and a high prevalence of atopic disease, cesarean versus vaginal delivery: long-term infant outcomes and the hygiene hypothesis, clin. perinatol., intestinal parasites infection: protective effect in rheumatoid arthritis, rev. bras. reumatol., not infection with parasitic worms, but rather colonization with therapeutic helminths, immunol. lett., cellular and molecular immunology, helminth-derived immunomodulatory molecules, adv. exp. med. biol., the presence of serum anti-ascaris lumbricoides ige antibodies and of trichuris trichiura infection are risk factors for wheezing and/or atopy in preschool-aged brazilian children, respir. res., bacteriotherapy for chronic constipation - a long term follow-up, gastroenterology, inverse association between skin response to aeroallergens and schistosoma mansoni infection, int. arch. allergy immunol., characteristics of the immune response in protozoan infections, med. pregl., infant gut microbiota and the hygiene hypothesis of allergic disease: impact of household pets and siblings on microbiota composition and diversity, allergy asthma clin. immunol., the hygiene hypothesis: an explanation for the increased frequency of insulin-dependent diabetes, cold spring harb. perspect. med., association between previous enterobiasis and current wheezing: evaluation of 1018 children, allergy asthma proc., fecal microbiota transplantation for refractory crohn’s disease, intest. res., filarial infection or antigen administration improves glucose tolerance in diet-induced obese mice, j. innate immun., too clean, or not too clean: the hygiene hypothesis and home hygiene, clin. exp. allergy, time to abandon the hygiene hypothesis: new perspectives on allergic disease, the human microbiome, infectious disease prevention and the role of targeted hygiene, perspect. public health, heligmosomoides polygyrus bakeri induces tolerogenic dendritic cells that block colitis and prevent antigen-specific gut t cell responses, j. immunol., bacteriotherapy for chronic fatigue syndrome: a long-term follow up study, cfs national consensus conference, fecal microbiota transplantation and emerging applications. nature reviews, gastroenterol. hepatol., bowel-flora alteration: a potential cure for inflammatory bowel disease and irritable bowel syndrome, med. j. aust., treatment of ulcerative colitis using fecal bacteriotherapy, j. clin. gastroenterol., reversal of idiopathic thrombocytopenic purpura (itp) with fecal microbiota transplantation (fmt), am. j. gastroenterol., fecal microbiota transplantation (fmt) in multiple sclerosis (ms), environmental exposure to endotoxin and its relation to asthma in school-age children, n. engl. j. med., the hygiene hypothesis and its inconvenient truths about helminth infections, plos negl. trop. dis., disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse, nat. genet., the ige response to ascaris molecular components is associated with clinical indicators of asthma severity, world allergy organ. j., microbiota regulates type 1 diabetes through toll-like receptors, proc. natl. acad. sci. u.s.a., the developing intestinal ecosystem: implications for the neonate, pediatr. res., wao white book on allergy: update 2013, executive summary, clonorchis sinensis infection is positively associated with atopy in endemic area, infection with schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice, parasite immunol., association between parasite infection and immune responses in multiple sclerosis, ann. neurol., a proof of concept study establishing necator americanus in crohn's patients and reservoir donors, effect of hookworm infection on wheat challenge in celiac disease - 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The Hygiene Hypothesis and Autoimmune Disorders

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The hygiene hypothesis is a hypothesis that suggests that the increased incidence of allergic and autoimmune disorders are linked to the tremendous changes in sanitation standards and practices that occurred in industrializing countries throughout the industrial revolution of the 19th and 20th centuries.

Throughout the last century, striking increases in the incidence of autoimmune diseases such as Type 1 diabetes mellitus and multiple sclerosis were evident.

The same holds good for allergic conditions such as atopic dermatitis, allergic rhinitis and asthma.

Although many factors are likely to be involved, including genetics and other triggering mechanisms, the rapidity of the changes appear to indicate the input of other changes, such as those seen in the environment.

This is supported by the finding that emigrants from a country with a low incidence of autoimmune disease to one with a high incidence of such acquire a high incidence of such conditions in the very first generation.

The associated changes in these countries that have witnessed drastic rises in the incidence of such diseases include the widespread use of antibiotics, milk pasteurization, vaccination against common childhood preventable diseases and a supply of almost sterile water.

In particular, the presence of certain microbes is thought to have a salutary effect on the robust functioning of the human immune system.

Throughout the industrial revolution, drastic changes in sanitation standards led to reduced or almost no exposure to these vital bacteria.

As a result, the function of the immune system was compromised, and the incidence of allergic and autoimmune disease began to rise.

Hygiene Hypothesis and “Old Friends” Hypothesis

Strachan first proposed the hygiene hypothesis in 1989, although some observations of the relationship between sanitation and autoimmune disorders had been noted previously.

An earlier observational study of more that 17,000 children in Britain in 1958 found an inverse relationship between allergic diseases such as hay fever, type 1 diabetes and asthma, and the number of older siblings.

Having attended a day care center early in life, within the first 6 months, protected against the development of asthma and atopy in children.

Another study in 1966 found a relationship between sanitation and the prevalence of multiple sclerosis. However, these findings were later extended to asthma and autoimmune diseases.

Relationship with Autoimmune Diseases

Diverse mechanisms have been proposed to explain the relationship between microorganisms and the prevention of autoimmune diseases.

The “old friends” microbes and the human immune system, including the distinctive antigens of the microbes, may work together in a reciprocal relationship. These antigens have been suggested to stimulate stronger immune responses, especially as compared to the autoantigens associated with autoimmune disorders.

Competition for cytokines, major histocompatibility complex (MHC) receptors and growth factors that are required for an immune response to occur is likely to be an important mechanism of protection against autoimmune disease.

The weak self-antigens and allergic antigens cannot compete successfully with the strong antigens which elicit immune responses in the case of other infections and parasitoses. Additionally, immunoregulatory interactions with the host toll-like receptors (TLRs) have been proposed as another mechanism.

Type 1 Diabetes

Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM), is becoming increasingly more prevalent worldwide, in both industrialized and developing countries.

This trend began in the 1970s in industrialized countries and continues today to become a public health problem in some countries, such as Finland. Additionally, younger children are now being seen to be affected by IDDM, including children under the age of 2 years, which was not before noted.

Multiple Sclerosis

In 1966, Leibowitz published an epidemiological study that observed a positive relationship between the prevalence of multiple sclerosis and levels of sanitation. It appeared that high levels of sanitation, such as those in the temperate areas of Israel, were associated with a higher risk of multiple sclerosis when compared to areas of lower sanitation, such as in tropical areas. This relationship has been further supported by other epidemiological studies designed to investigate the impact of the hygiene hypothesis on autoimmune diseases.

Inflammatory Bowel Diseases

The incidence of Crohn’s disease, ulcerative colitis and primary biliary cirrhosis is also increasing. This rise may be due in part to improved medical access and diagnostic techniques, but cannot be linked solely to these explanations. For this reason, an environmental link and the hygiene hypothesis is also thought to be involved.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841828/
http://www.nejm.org/doi/full/10.1056/NEJMra020100
http://link.springer.com/article/10.1007%2Fs12016-011-8285-8
http://perspectivesinmedicine.cshlp.org/content/2/2/a007799.full

Yolanda Smith

Yolanda graduated with a Bachelor of Pharmacy at the University of South Australia and has experience working in both Australia and Italy. She is passionate about how medicine, diet and lifestyle affect our health and enjoys helping people understand this. In her spare time she loves to explore the world and learn about new cultures and languages.

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The hygiene hypothesis in autoimmunity: the role of pathogens and commensals

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1 Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
2 INSERM U1151, Institut Necker-Enfants Malades, Hôpital Necker-Enfants Malades, Paris, France.
3 CNRS UMR 8253, Institut Necker-Enfants Malades, Hôpital Necker-Enfants Malades, Paris, France.
PMID: 29034905
DOI: 10.1038/nri.2017.111

The incidence of autoimmune diseases has been steadily rising. Concomitantly, the incidence of most infectious diseases has declined. This observation gave rise to the hygiene hypothesis, which postulates that a reduction in the frequency of infections contributes directly to the increase in the frequency of autoimmune and allergic diseases. This hypothesis is supported by robust epidemiological data, but the underlying mechanisms are unclear. Pathogens are known to be important, as autoimmune disease is prevented in various experimental models by infection with different bacteria, viruses and parasites. Gut commensal bacteria also play an important role: dysbiosis of the gut flora is observed in patients with autoimmune diseases, although the causal relationship with the occurrence of autoimmune diseases has not been established. Both pathogens and commensals act by stimulating immunoregulatory pathways. Here, I discuss the importance of innate immune receptors, in particular Toll-like receptors, in mediating the protective effect of pathogens and commensals on autoimmunity.

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Cover image: The juxtaposed images of nucleopores, which are channels in the nuclear membrane, testify to advances in biological imaging over the past half century. The electron micrograph on the left was taken in 1953 by Joseph G. Gall and reveals a field of nucleopores. In the image on the right taken in 2016, Joseph G. Gall, Steven L. McKnight, and colleagues used super resolution microscopy to visualize binding of a proline:arginine poly-dipeptide to the central channel of nucleopores (red). The toxic poly-dipeptide is produced in the most common form of heritable amyotrophic lateral sclerosis. The periphery of nucleopores is visualized by antibody staining to the annular protein, gp210 (blue). The study illuminates the role of nucleocytoplasmic transport in the pathogenesis of the disease. See article by Kevin Y. Shi et al. on pages E1111–E1117 . Images courtesy of Zehra F. Nizami and Joseph G. Gall.

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Published: 21 October 2022

Disease stages and therapeutic hypotheses in two decades of neurodegenerative disease clinical trials

Meredith A. Mortberg 1 , 2 ,
Sonia M. Vallabh 1 , 2 &
Eric Vallabh Minikel 1 , 2

Scientific Reports volume 12 , Article number: 17708 ( 2022 ) Cite this article

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Clinical trial design

Neurodegenerative disease is increasingly prevalent and remains without disease-modifying therapies. Engaging the right target, at the right disease stage, could be an important determinant of success. We annotated targets and eligibility criteria for 3238 neurodegenerative disease trials registered at ClinicalTrials.gov from 2000 to 2020. Trials became more selective as the mean number of inclusion and exclusion criteria increased and eligible score ranges shrank. Despite a shift towards less impaired participants, only 2.7% of trials included pre-symptomatic individuals; these were depleted for drug trials and enriched for behavioral interventions. Sixteen novel, genetically supported therapeutic hypotheses tested in drug trials represent a small, non-increasing fraction of trials, and the mean lag from genetic association to first trial was 13 years. Though often linked to disease initiation, not progression, these targets were tested mostly at symptomatic disease stages. The potential for disease modification through early intervention against root molecular causes of disease remains largely unexplored.

Observational studies in Alzheimer disease: bridging preclinical studies and clinical trials

Redefining the hypotheses driving Parkinson’s diseases research

Temporal order of clinical and biomarker changes in familial frontotemporal dementia

Introduction.

Neurodegenerative disease is on the rise globally due to aging populations 1 , highlighting a need for effective therapeutic interventions. Developing new drugs is incredibly difficult, with only 8–14% of all drug-indication pairs that enter clinical trials ultimately succeeding 2 , 3 , 4 . Success has been particularly limited in adult-onset neurodegenerative diseases, for which no disease-modifying drug yet exists. Patients, scientists, regulators, and public health experts have called for prioritizing preventive approaches to neurodegeneration, citing potential benefits to quality of life and alleviation of economic burden, as well as improved prospects for success if intervention against core molecular drivers of disease occurs before downstream pathology takes hold 1 , 5 , 6 , 7 , 8 , 9 , 10 . Yet there exist considerable barriers to achieving prevention, including the potential cost and duration of trials, need for deeper validation of biomarker endpoints, and the requirement for careful protections and counseling of at-risk subjects in trials 5 , 7 , 11 .

In this study, we sought to understand the disease stages and therapeutic hypotheses studied in clinical trials conducted in neurodegenerative diseases to date. Current drug development pipelines have been catalogued elsewhere 12 , 13 , 14 , 15 , but we found that none of these reports answered our key questions. First, experimental drugs are often broadly categorized as “disease-modifying” if the hypothesis is one of disease modification, regardless of the quality of that hypothesis. We wished to examine the share of drugs with molecular targets underpinned by human genetic associations, which can inform drug development strategy 16 and have been shown empirically to double success rates in drug development 17 , 18 . Second, while reports sometimes categorize trials as preventive versus symptomatic, we wished to examine more quantitative metrics of disease stage or severity. Third, while snapshots of the present pipeline are illuminating, we sought a longitudinal view of drug discovery over the past 2 decades. Finally, prior studies did not make their full datasets publicly available to facilitate re-analysis. Therefore, combining automated annotation and deep manual curation of clinical trial registrations from ClinicalTrials.gov, we set out to map the landscape of clinical trials and their therapeutic interventions and to test correlations and trends over the past 2 decades.

Study design

Our goals were to characterize clinical trials across major neurodegenerative disease indications, identifying correlations and temporal trends, particularly with regards to disease stages and molecular targets of drugs. We chose ClinicalTrials.gov as a data source because (i) it is publicly available, allowing us to release our reviewed and annotated dataset and source code and thus make our analyses fully reproducible, (ii) it has existed since 2000, thus providing 2 decades of data, and (iii) compliance with Food and Drug Administration Amendments Act (FDAAA) mandates have made it a fairly comprehensive listing of trials 19 , 20 . Trial results are not always posted or published in a timely fashion 21 , 22 , 23 , but our analysis focused on the design, not results, of trials. We determined that the ClinicalTrials.gov data, downloadable in XML format, necessitated a hybrid approach using both scripting and manual curation. Some variables, such as study start and end dates, enrollment, and phase are captured in specific fields and can be trivially extracted with scripts. Other variables require manual curation either because they are described using variable diction within free text fields (eligibility criteria), contain large numbers of synonyms, typos, and qualifiers (intervention names), or require cross-referencing to diverse external data sources (drug target). We therefore used a Python script to extract fields of interest into separate trials and interventions tables which were manually reviewed in Google Sheets.

Search strategy and selection criteria

We included 4 major neurodegenerative diseases: Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia / amyotrophic lateral sclerosis (FTD/ALS) and Huntington’s disease (HD). On April 16, 2020, we searched ClinicalTrials.gov for alzheimer OR huntington OR parkinson OR als OR amyotrophic OR frontotemporal OR ftd OR ftld with start date on or before March 31, 2020, yielding 4542 NCT identifiers. Full ClinicalTrials.gov registration data for these trials were then pulled from https://clinicaltrials.gov/AllPublicXML.zip on April 16, 2020 for manual curation as described below. In order to estimate the extent to which our strategy might under-sample trials that lacked a specific disease focus, we conducted additional searches between September 16–22, 2022 for the terms dementia, movement disorders, and mild cognitive impairment. For the time period considered here, 2041/4010 (51%) of dementia trials, 1994/3545 (56%) of movement disorder trials, and 302/853 (35%) of mild cognitive impairment trials were in our original dataset. See Discussion for more details.

Trial curation

Because our research question centered on patient populations and therapeutic hypotheses, we sought to include all trials that tested an intervention hypothesized to modify the patient’s disease or symptoms, in a disease-relevant population (Fig. 1 ). We excluded trials lacking a therapeutic intervention, such as studies of biomarkers, diagnostics, patient data, or imaging agents, where the goal was to evaluate diagnostic or prognostic value rather than to confer therapeutic benefit. We also excluded trials that enrolled only healthy volunteers, did not study one of these four neurodegenerative diseases, or targeted the intervention to caregivers rather than patients. Finally, we excluded trials where data were incomplete or contained errors: examples included trials where inclusion required participation in a prior trial for which inclusion criteria had never been publicly posted, or where drugs listed in intervention arms did not match the title or description of the trial. We performed internet searches to identify the sector to which trial sponsors belonged (industry or “other” including academia, government, non-profits). We defined disease stage 0 (“at-risk”) as individuals at risk of disease due to genotype, age, or other risk factors. Generalizing FDA Alzheimer’s guidance 7 across diseases, we defined stage 1 (“molecular”) as individuals with molecular evidence of disease pathology, stage 2 (“detectable”) as individuals without functional impairment but where a sensitive neuropsychological test could discern disease-related phenotypic changes, stage 3 (“mild”) as indicating mild detectable functional impairment not yet meeting criteria for disease diagnosis, and stage 4 (“diagnosed”) as individuals diagnosed with dementia or other changes (such as motor impairment) meeting diagnostic criteria for neurodegenerative disease. For each trial, we manually determined the earliest and latest disease stage of patients eligible to enroll based on reading the inclusion and exclusion criteria. We also manually extracted the eligible score ranges on tests from these criteria, prioritizing the Mini-Mental State Examination (MMSE) 24 and Hoehn and Yahr 25 in instances where more than one test was used. For trials that specified only a minimum score, we inferred the maximum to be the maximum possible score; and vice versa. Trial duration was calculated as study completion date minus start date. Patient-years of enrollment were calculated as trial duration times enrollment, though we acknowledge this is an imperfect approximation, as in reality participants accrue (and withdraw) gradually rather than all participants being on board for a trial’s full duration. We used only enrollment numbers described as “Actual”; patient-years for “Anticipated” enrollment values were set to missing. The number of inclusion/exclusion criteria was calculated by counting the total number of items in numbered or bulleted lists under the headings “Inclusion Criteria” and “Exclusion Criteria”, or manually entered for 75 trials without numbered/bulleted lists.

Flowchart of trial inclusion and curation. Trials returned by our search strategy were sequentially excluded based on launch year, lack of therapeutic intervention, patient population, or other. See Methods for explanation.

Intervention curation

Intervention arms extracted included trials were converted to lower case and stripped of invalid text characters, yielding unique values for curation. The “intervention type” in ClinicalTrials.gov is specified inconsistently even for the exact same intervention (for instance, “deep brain stimulation” was categorized alternatively as “device”, “procedure”, “behavioral” or “other”), so we manually assigned every intervention to an intervention class: drug, device, procedure, behavioral, placebo, other, and none. We defined “drugs” as molecular interventions regardless of modality (synthetic drug, biological) or regulatory status (experimental, approved/repurposed, supplement). We considered as “behavioral” any interventions intended to alter patient behavior (exercise, diet, drug compliance) even if they utilized a device. We classified as “procedures” surgery, acupuncture, cell transplants, radiation, and changes in care protocols. In contrast, intervention arms that consisted solely of blood draws, lumbar punctures, or other events defined as “procedures” by Institutional Review Boards but not intended to confer therapeutic benefit to the patient, were classified as “none”. We classified as “placebo” explicit placebo arms as well as standard of care, normal saline, no treatment, and similar variations. In addition, often, a single unique intervention name value (“donepezil”) would appear across dozens of trials, where sometimes it was the active therapeutic agent being tested, while at other times it served as the standard-of-care arm. For trials with > 1 intervention arm listed, if any arm was an experimental intervention while another arm was a drug that was already FDA approved for the disease in question in the year the trial occurred, we counted the latter as a placebo arm. Names of intervention arms often included information such as dose level (“riluzole 50 mg”) or formulation (“rosiglitazone xr (extended release) oral tablets”), the same drug would be assigned various generic names assigned at different stages of development (“ly3002813” is “donanemab”), and commercial names, typos, and other variations also occurred. We therefore assigned for each drug intervention arm the best generic name for the molecular entity being tested. For the 27 drugs approved by FDA for treatment of these 4 neurodegenerative diseases, years of initial approval were extracted from Drugs@FDA database searches. For the purposes of classifying experimental drugs (those not approved for these neurodegenerative diseases) as novel versus repurposed, we considered as “approved” any drug with full marketing approval in any jurisdiction worldwide. We considered only full approvals, thus aducanumab, which received Accelerated Approval in 2021, was classified as experimental. We searched several data sources to identify any available evidence as to the molecular target of each drug, and assigned gene symbols of molecular targets based on annotations in DrugBank 26 (N = 993), articles in PubMed (N = 278), Alzforum (N = 244), company press releases (N = 46), information provided directly in ClinicalTrials.gov submissions (N = 19), or other (N = 18).

Human genetic associations

Gene-disease links established by human genetics up through the end of the study period (March 2020) were identified through manual searches of Online Mendelian Inheritance in Man (OMIM) 27 for Mendelian forms of disease, and Open Targets Genetics (OTG) 28 for genome-wide association studies (GWAS) of common/complex forms of disease. For GWAS loci mapping to multiple potentially causal genes, the gene with the highest locus-to-gene score 28 was generally used unless additional GWAS studies of the same trait supported a different causal gene at the same locus or unless Mendelian or other compelling evidence implicated another gene (example: due to its association with familial ALS, C9orf72 was selected despite ranking 2nd after MOB3B in locus-to-gene score for an ALS GWAS 29 ). Where OTG data were missing or indeterminate, the gene highlighted by the study’s original authors was used.

Role of the funding source

This work was supported by the National Institutes of Health (R21 TR003040 and R01 NS125255). The funder had no role in study design, analysis, interpretation, or decision to publish.

Statistics, source code, and data availability

Scripted extraction of ClinicalTrials.gov and DrugBank data used scripts in Python 3.8.9; data analysis and visualization were performed in R 4.2.0. All statistical tests were two-sided and nominal P values less than 0.05 were considered significant. Enrichment analyses used Fisher’s exact test. Tests for temporal trends used linear regression, except for the testing of a temporal trend in disease stage (an ordinal variable), which used ordinal logistic regression (polr from the R MASS package). Loess fits were additionally used for visualization of potentially non-linear temporal trends. Error bars represent 95% confidence intervals of the mean (± 1.96 standard errors of the mean). Distributions were compared using Kolmogorov–Smirnov test, which does not assume normality. All data and source code used in this study are publicly available at http://github.com/ericminikel/nd_trials and are sufficient to reproduce the figures and statistics herein.

Characteristics of neurodegenerative disease trials

Of 4542 trials returned by our search strategy, 3238 met inclusion criteria and were reviewed and annotated (Fig. 1 ). In order to understand the landscape of neurodegenerative disease trials, we first considered both the simple count of trials (Fig. 2 A), as well as the total patient-years of enrollment (Fig. 2 B) as potentially a better proxy for R&D spend, in a univariate breakdown of trials by disease area, sector, intervention type, phase, and control group status. The majority of trials were non-industry-sponsored, lacked a placebo or standard of care (SOC) arm, and for a plurality, phase was other/unspecified. But whereas only a minority of trials were industry-sponsored (N = 1239, 38%), these trials were larger on average, and so accounted for a majority (64%) of all patient-years. Drug interventions accounted for a majority of trials (N = 1864; 58%) but an even larger majority of patient-years (76%). In a bivariate cross-tabulation (Fig. 2 C), drug trials accounted for not only the largest number of trials but were also the most intense (patient-years/trial), especially for Alzheimer’s disease, Phase III, placebo/SOC-controlled, and industry-sponsored. Overall, industry-sponsored drug trials accounted for 61% of all patient-years (Fig. 2 D). The remainder—non-industry and/or non-drug trials—were highly skewed in terms of size: 34% enrolled ≤ 20 patients, while the 6 largest trials accounted for 33% of all patient-years, indeed, a single trial of medication adherence reminder devices 30 that included PD patients comprised 13%. Industry-sponsored drug trials were less variable in size: it took the top 25 to comprise 33% of patient-years, and only 15% off trials enrolled ≤ 20 patients (Fig. 2 D). Industry-sponsored drug trials also differed from other trials in being 3.1 times more likely to have a placebo or standard of care (SOC) control arm, 2.2 times as likely to complete, and > 100 times as likely to have a specified phase (Fig. 2 E). Industry-sponsored drug trials grew much more selective over the 2 decades considered here, with the mean number of inclusion and exclusion criteria per trial rising from ~ 9 to ~ 17 ( P = 3e-10, linear regression; Fig. 2 F), with no corresponding trend for non-industry and/or non-drug trials P = 0.31, linear regression; Fig. 2 F). The number of inclusion and exclusion criteria rose for Phase I, II, and III trials, but not for other/unspecified phase trials (Fig. 2 G).

Characteristics of neurodegenerative disease clinical trials. ( A ) Univariate count of trials by disease, sponsor, intervention class, phase, or placebo/SOC control status. ( B ) Total Univariate total of patient-years (see Methods) by the same variables. ( C ) Bivariate cross-tabulation of number of trials and patient-years. Intensity (patient-years per trial) is expressed as a color palette from transparent yellow to opaque blue; number of trials is expressed as the size of each circle. ( D ) Pie chart of patient-years of enrollment, with industry drug trials shown in 3 alternating shades of pink and all other trials in 3 alternating shades of green. Each wedge represents one trial, and trials are sorted by number of patient-years. ( E ) Barplot representation of contingency tables for whether trials are (purple) or are not (gray) placebo/SOC-controlled (top), completed (middle), or have a specified phase (bottom) depending on whether they are industry-sponsored drug trials (right) or other (left). ( F – G ) Mean total number of inclusion and exclusion criteria per trial as a function of ( F ) industry-sponsored drug trials versus all other, and ( G ) phase. Loess curves were fit on the raw individual values, but due to the large number of trials, individual values are not shown; instead the average for each year is shown as a semitransparent horizontal bar. Statistical significance was evaluated by linear regression, see Results text.

Disease stage of participants in trials

Reading the inclusion and exclusion criteria, we manually annotated which disease stages, numbered 0–4 (see Methods), were eligible for each trial. 89% of trials required a diagnosis of the neurodegenerative disease in question, corresponding to disease stage 4 (Fig. 3 A). Another 7.8% permitted patients with mild cognitive impairment (MCI) or an analogous level of other functional impairment, who did not yet meet diagnostic criteria for their diseases (Fig. 3 A). The 2.7% of trials (N = 89) that permitted pre-symptomatic patients, corresponding to stages 0–2 (Fig. 3 A), differed from other trials in several respects. Trials open to pre-symptomatic individuals were less likely to be industry-sponsored and less likely to test a drug or device; they were much more likely to test a behavioral intervention (Fig. 3 B). They trended less likely to have a specified phase and had a non-significantly lower completion rate, although the proportion that included placebo/SOC arms was similar. Trials open to pre-symptomatic individuals were significantly longer on average ( P = 0.0004, Kolmogorov–Smirnov test; Fig. 3 C) and trended slightly larger in enrollment, though the difference was not significant ( P = 0.09, Kolmogorov–Smirnov test). The proportion of trials enrolling earlier disease stages rose slightly in recent years ( P < 1e−10, ordinal logistic regression), although in absolute terms, the proportion of trials enrolling at stages < 4 rose only from 8% in the first 4 years of the data to 15% in the final 4 years of data (Fig. 3 D). We also examined whether quantitative measures of impairment changed over time, regardless of nominal disease stage. A majority of trials (57%, N = 1833/3238) used a disease severity scale as one inclusion or exclusion criterion, most often the Mini-Mental State Examination 24 (MMSE; 28%, N = 911) or Hoehn & Yahr 25 (23%; N = 757). For MMSE, on average, the maximum (least impaired) admissible score rose from 25.6 in 2000 to 27.6 in 2020 ( P = 2e−5, linear regression), while the minimum (most impaired) admissible score rose from 13.6 to 17.7 ( P = 1e−5; Fig. 3 E). Thus, trials using MMSE focused on less impaired patients over time, and because the exclusion of too-advanced patients became stricter more rapidly than the inclusion of less advanced patients, the size of the eligible window shrunk over time. Analogously, for Hoehn & Yahr, where higher scores correspond to more advanced disease, the average minimum (least impaired) admissible score dropped from 1.6 to 1.2 over the 20 years ( P = 0.002), while the average maximum (most impaired) limit dropped from 3.8 to 3.1 ( P = 6e−10), again reflecting a shift towards less impaired patients together with a shrinking of the eligible window (Fig. 3 F).

Disease stages eligible for trials. ( A ) Barplot of trial count by earliest disease stage (legend at left, see Methods for details) eligible to enroll. ( B ) Forest plot of odds ratios (Fisher’s exact test) for properties of preventive (stage 0–2) versus symptomatic (stage 3–4) trials. ( C ) Scatterplot of study duration (x axis) and enrollment (y axis) for preventive (stage 0–2, cyan) versus symptomatic (stages 3–4, gray) trials, with marginal histograms on both axes. Crosshairs represent mean and 95% confidence intervals of the mean on both dimensions. ( D ) Stacked area plot of the number of trials per year by earliest eligible disease stage. For 2020, only 3 months of data were included, so the raw number of trials was scaled by a factor of 4 to yield trials/year. ( E ) Eligible MMSE score ranges by year, N = 911. Each trial is displayed as a purple rectangle of 10% transparency stretching from the lowest to highest eligible score on the y axis and staggered by ± 0.5 years on the x axis, such that darker shades of purple indicate a greater density of trials recruiting patients in a given score range in a given year. Green lines represent best fits from linear regression models. Lower scores indicate greater impairment. ( F ) As in ( E ), but for Hoehn and Yahr, N = 757; note that on this scale, higher scores indicate greater impairment.

Therapeutic hypotheses tested in drug trials

We identified N = 748 unique molecular entities (including unique combinations) tested across N = 1864 drug trials. Based on regulatory status and molecular target(s), we classified these trials into 7 categories (Fig. 4 A). There exist 27 drugs that have full approval and are labeled by FDA specifically for the treatment of the neurodegenerative diseases considered here. Trials in support of these approved drugs, for their approved indications, comprised 18% of patient-years. Only a minority, however, were launched in years prior to FDA approval (Fig. 4 A). Trials of these same molecular entities occurring after first approval—for example, those seeking to expand the label, meet regulators’ requirements in other countries, understand drug effects on additional endpoints, or test new formulations or delivery routes of the same molecular entity—outnumbered the trials preceding initial approval by a factor of > 4 (Fig. 4 B). Indeed, donepezil, whose approval for Alzheimer’s (1996) pre-dates the time range considered here, was the single most intensely studied drug in this entire dataset (N = 55 trials). For each of the drugs approved for these diseases prior to 2017, there were at least as many post-approval as pre-approval trials (Fig. 4 B).

Therapeutic hypotheses tested in drug trials. ( A ) Area-scaled barplot of 7 categories of drug interventions tested. The length of each rectangle on the x axis is the number of trials; thickness on the y axis is proportional to the number of patient-years per trial. Thus the total area occupied by each rectangle is proportional to the total patient-years invested in each category, for which percentages of the total are overlaid in white text. ( B ) Barplot of number of trials per year for drugs FDA approved for the treatment of the indicated disease before (cyan) and after (gray) initial FDA approval. Trials of these same drugs in other neurodegenerative diseases, for which they are not yet labeled by FDA, are not included in this plot; trials where these drugs served as SOC arms are also excluded. Black triangles indicate years of first FDA approval; approvals prior to 2000 are left-aligned. ( C ) Proportional area Euler diagram of genes that encode targets of drugs approved for any indication (gray), targets tested in neurodegenerative disease clinical trials in this dataset (red), or targets supported by human genetic evidence (yellow). ( D ) Stacked area plot of cumulative number of genes associated to these 4 diseases by year. Association of ACE to AD is counted in 1999 acknowledging candidate gene studies 31 which replicated by GWAS in 2018 32 . ( E ) Stacked area plot of categories of trials as defined in ( A ) by year. ( F ) Scatterplot of genetically supported target-indication pairs pursued in drug trials, displayed as year of first reported genetic association (x axis) versus year of first trial in the genetically linked neurodegenerative disease (y axis), color-coded by disease. *Gamma secretase is represented here by PSEN1 ; members PSEN2 and APH1B also have genetic association to AD risk. ( H ) Barplot of number of trials for each genetically supported target, in its genetically supported indication, tested clinically.

Another 34% of patient-years were spent on trials of either repurposed drugs, new drugs for established targets (targets with a drug approved for any disease), or combinations of 2 or more therapies where all are either approved drugs or supplements. In all, trials explored 272 different targets of approved drugs (Fig. 4 C). Of repurposing efforts, 55 trials used a drug approved for one neurodegenerative disease and tested its efficacy in a different neurodegenerative disease (for example, the Alzheimer’s drug memantine was trialed for Parkinson’s, Huntington’s, and FTD/ALS). Ten times as many trials (N = 557), however, tested drugs approved for other indications, chiefly in neurology (N = 195), metabolic (N = 72), and cardiovascular disease (N = 47); the most studied repurposed drug was botulinum toxin A (N = 17).

Trials comprising 48% of patient-years tested novel therapeutic hypotheses—molecular entities not yet approved and whose molecular targets are either unknown, or are not yet targeted by any other approved drug. Curating associations from both Mendelian forms and genome-wide association studies for these 4 diseases (see Methods), we identified N = 101 gene-disease pairs linked by human genetic evidence (Fig. 4 D). We asked which of these therapeutic hypotheses had been tested clinically. Approximately three-quarters (429/577) of trials of novel therapeutic hypotheses lacked direct human genetic association implicating the target in the disease. The two most intensely studied targets in this group were those with functional evidence for disease relevance ( MAPT and BACE1 in AD, N = 23 trials each). For the majority of trials in this category (72%, N = 307), however, we were unable to identify any known molecular target. The remaining (148/577) trials tested N = 16 target-indication pairs backed by human genetics. Their share of all drug trials did not increase over time ( P = 0.59, linear regression; Fig. 4 E) despite the increased number of genetic associations reported (Fig. 4 D). Instead, the only two categories of trial whose share increased significantly were repurposed targets and novel hypotheses without genetic support ( P = 0.02 and 0.008 respectively). For 14 genetically supported targets, the first clinical trial followed the discovery of the genetic association, with a mean lag time of 13 years, and a minimum lag time of 5 years ( TREM2 ; Fig. 4 F). All (4/4) targets with both a genetic association and a pre-existing approved drug had been trialed (Fig. 4 C): 1 with a novel drug ( CD33 in AD), 2 apparently coincidentally ( BST1 33 and SCN2A in PD) and 1 with a repurposed drug selected at least in part based on genetic evidence ( ACE in AD 34 ).

Overall, trials of genetically supported hypotheses only comprised half as many patient-years as novel hypotheses without genetic support (Fig. 4 A), and no genetically supported agent was studied as intensely as gingko biloba, to which the two largest trials in the dataset were devoted. Investment in genetically supported target-indication pairs was highly skewed, with 68% of trials and 84% of patient-years devoted to targeting Aβ ( APP ) in Alzheimer’s disease (Fig. 4 G). Just 3 trials tested genetically supported hypotheses in a preventive paradigm, enrolling individuals at disease stages 0–2: A4 35 , API 36 , and DIAN-TU 37 , all of which tested Aβ antibodies.

Here we used 2 decades of clinical trial registration data to analyze the characteristics of trials in 4 major neurodegenerative diseases. We were motivated by evidence from other disease areas showing that drug programs whose therapeutic hypotheses are supported by human genetic associations enjoy doubled success rates 17 . We analyzed trial types, disease stages and therapeutic hypotheses being tested to assess to what degree this opportunity has been utilized in major neurodegenerative diseases.

Our study has several limitations. By design, our search and curation strategy focused on four specific neurodegenerative diseases. Trials on other neurodegenerative diseases, or trials that lacked a specific disease focus, may not be included here. Our dataset included about half of trials matching search terms “dementia” and “movement disorders” but only a third for “mild cognitive impairment”, which may suggest that our search strategy leads to undersampling of early symptomatic stage trials. Other limitations include non-exhaustive capture of trials by ClinicalTrials.gov, limited amount and types of data available in trial registrations, human error in the curation process, and the inherently retrospective nature of the analysis.

Pre-symptomatic intervention offers the promise of preserving quality of life before patients are impaired, but is very rare in clinical trials. To the extent that trial enrollment has shifted toward less impaired patients over the past 2 decades, this has come at the expense of screening more patients out, with proliferating inclusion and exclusion criteria and narrower acceptable score ranges. Trials in pre-symptomatic patients remain vanishingly rare, comprising just 2.7% of all trials, and this small fraction is enriched for behavioral interventions and depleted for drug trials and industry sponsorship. Industry-sponsored drug trials, despite representing a minority of all trials in our dataset, accounted for a majority of patient-years of enrollment, and were much more likely than other trials to complete, to have placebo/SOC control arms, and to have a specified trial phase. It is reasonable to conclude that these industry-sponsored drug trials likely represent a large majority of the “shots on goal” for well-powered demonstrations of clinical efficacy. Such trials have very seldom aimed at pharmacologic prevention of neurodegenerative disease.

Trials testing hypotheses rooted in human genetics are a minority and have not become more common despite a proliferation of genetic associations. More common types of trials include those of agents without any known molecular target, post-approval trials of approved symptom-managing drugs, and repurposing trials of drugs approved for other indications. Across the pharmacopeia, of the 729 targets corresponding to drugs approved for any condition, 272 (37%) were tested for neurodegenerative disease, while of 101 hypotheses nominated by human genetics, just 16 were tested. The average time from genetic discovery to first human trial was more than a decade, and the majority of trials and an even larger majority of patient-years focused on Aβ in AD, with limited attention paid to other potential targets.

Our findings suggest the risk of a missed opportunity. Most of the genetic studies that have nominated new molecular targets are familial linkage or case–control studies, and thus are best suited to identify the initial triggers of disease. There appears to be an imperfect overlap between the molecular drivers of neurodegenerative disease initiation and the molecular drivers of subsequent progression 38 , 39 , 40 , 41 . For some targets nominated by these types of genetic studies, pre-symptomatic populations might represent the best, or in some cases only, opportunity for efficacy. Yet trials are conducted overwhelmingly in symptomatic patients. On one hand, GWAS of disease progression rates may help to identify targets more likely to yield efficacy in symptomatic patients. Meanwhile additional investment in well-powered, well-controlled trials at earlier disease stages may be needed to realize the potential of targets involved in disease initiation, and ultimately improve our odds of not only extending life, but extending quality of life.

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Funding was provided by National Institutes of Health (R21 TR003040 and R01 NS125255).

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Meredith A. Mortberg, Sonia M. Vallabh & Eric Vallabh Minikel

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S.M.V. and E.V.M. conceived and designed the study. M.A.M., S.M.V., and E.V.M. curated the dataset. E.V.M. analyzed the data and drafted the manuscript. M.A.M., S.M.V., and E.V.M. reviewed, edited, and approved the final manuscript.

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Mortberg, M.A., Vallabh, S.M. & Minikel, E.V. Disease stages and therapeutic hypotheses in two decades of neurodegenerative disease clinical trials. Sci Rep 12 , 17708 (2022). https://doi.org/10.1038/s41598-022-21820-1

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Developmental Origins of Health and Disease: Brief History of the Approach and Current Focus on Epigenetic Mechanisms

Pathik d. wadhwa.

1 Department of Pediatrics, University of California, Irvine, School of Medicine, Irvine, California

2 Department of Psychiatry & Human Behavior, University of California, Irvine, School of Medicine, Irvine, California

Claudia Buss

Sonja entringer, james m. swanson.

“Barker’s hypothesis” emerged almost 25 years ago from epidemiological studies of birth and death records that revealed a high geographic correlation between rates of infant mortality and certain classes of later adult deaths as well as an association between birthweight and rates of adult death from ischemic heart disease. These observations led to a theory that undernutrition during gestation was an important early origin of adult cardiac and metabolic disorders due to fetal programming that permanently shaped the body’s structure, function, and metabolism and contributed to adult disease. This theory stimulated interest in the fetal origins of adult disorders, which expanded and coalesced ~5 years ago with the formation of an international society for developmental origins of health and disease (DOHaD). Here we review a few examples of the many emergent themes of the DOHaD approach, including theoretical advances related to predictive adaptive responses of the fetus to a broad range of environmental cues, empirical observations of effects of overnutrition and stress during pregnancy on outcomes in childhood and adulthood, and potential epigenetic mechanisms that may underlie these observations and theory. Next, we discuss the relevance of the DOHaD approach to reproductive medicine. Finally, we consider the next steps that might be taken to apply, evaluate, and extend the DOHaD approach.

FROM EPIDEMIOLOGICAL OBSERVATIONS TO THE FETAL ORIGINS HYPOTHESIS

The Developmental Origins of Health and Disease (DOHaD) approach evolved from epidemiological studies of infant and adult mortality. A trio of articles in The Lancet by Barker and colleagues 1 – 3 represent perhaps the most influential early publications in this area that led to the fetal origins hypothesis (often called “Barker’s hypothesis”). There are many reviews of Barker’s hypothesis, and one of the best is provided by Barker himself, who summarized the genesis of the developmental origins hypothesis. 4 The main points are best described by quotes from these original sources, which we provide later.

Barker 4 gives a personal account of a program of epidemiological research of the geographic distributions of diseases across local authorities of England and Wales, which provided the countrywide data used by Barker et al 1 to show a large positive geographic correlation (~0.7) for standardized rates for infant mortality from 1921 to 1925 and ischemic heart disease from 1968 to 1978. An interpretation of this relationship was based on several factors: the association of neonatal deaths in the 1920 with low birthweight, the dependence on adverse intrauterine rather than postnatal factors, the paradoxical rise in heart disease with rising prosperity but lower rates in the most prosperous locations, a review of the literature on maternal and infant nutrition, and other factors. This led to the insight and hypothesis that the geographic relationship of infant and adults death rates “reflects variations in nutrition in early life, which are expressed pathologically on exposure to later dietary influences” (p. 1081).

According to Barker’s 4 account, the next step of investigation “required studies of a kind that had not hitherto been carried out” (p. 415). This was initiated within a sample of adults (men born from 1911 to 1930 in Hertfordshire) with good records of size at birth, weight in infancy, and death from ischemic health disease, which Barker et al 2 used to confirm (in individuals) the deductions from the geographic study: Men with the lowest birthweights had the highest death rates, those with the highest birthweights had the lowest death rates, and standardized death rates fell steeply with increasing weight at 1 year of age. This led to the hypothesis that “an environment which produces poor fetal and infant growth is followed by an adult environment that determines high risk for ischemic heart disease” (p. 579).

To develop the hypothesis further, Baker et al 3 reviewed how fetal undernutrition at different stages of gestation can be linked to different birth phenotypes, each linked to adaptations associated with changes in concentrations of placental and fetal hormone and later with different metabolic abnormalities in adulthood. This integration proposed that “undernutrition during gestation reprograms the relationship between glucose and insulin and between growth hormone and IGF [insulin-like growth factor]” (p. 940), which permanently changes the body’s structure, function and metabolism that increases risk for coronary heart disease in later life.

FROM FETAL ORIGINS OF ADULT DISEASE TO DEVELOPMENTAL ORIGINS OF HEALTH AND DISEASE

Barker’s hypothesis stimulated a great deal of worldwide interest and activity in the area of developmental plasticity, Gillman et al 5 summarized in a report of the meetings of the World Congress on Fetal Origins of Adult Disease that were convened in 2001 (Mumbai, India) and 2003 (Brighton, United Kingdom) and the transition to DOHaD that was formed subsequently “to recognize the broader scope of developmental cues, extending from the oocyte to the infant and beyond, and the concept that the early life environment has widespread consequences for later health” (p. 625). The DOHaD society has sponsored meetings in 2005 (Toronto, Ontario, Canada), 2006 (Utrecht, The Netherlands), 2007 (Perth, Western Australia), and 2009 (Santiago, Chile), and these international congresses provided an important forum for exchange of ideas and progress in this rapidly expanding field ( www.dohadsoc.org ).

Here we review some of the expanded themes, including: (1) the development of theory based on the concept of predictive adaptive responses of the fetus to a variety of environmental cues and consequences of mismatch between prenatal and postnatal environments, (2) the emphasis on the fetal origins of obesity and overnutrition as well as undernutrition during gestation and in infancy as pathways into obesity in childhood and adulthood, (3) the evaluation of psychobiological effects of stress during pregnancy on fetal development and later outcomes, and (4) the consideration of epigenetic mechanisms to account for some of the observations based on the DOHaD approach.

The field is now so large that a selective review is necessary. For relevance to the topic of reproductive medicine, we chose to focus our review on important details from three specialized research programs (the Southampton Women’s Survey (SWS), Project VIVA, and the Behavioral Perinatology Research Program) that emphasize prospective evaluations of fetal development during pregnancy.

The Southampton Women’s Survey: Background and Current Status

Barker and collaborators developed the first generation of theories to account for the observations of correlations of fetal and adult death rates across geographic locations 1 and birthweight and ischemic heart disease death rates across individuals, 2 which included the “thrifty phenotype” theory of Hales and Barker 6 and the “developmental plasticity” theory of Bateson et al. 7 With this scientific background (see www.mrc.soton.ac.uk ), the next stage of a program of research at the University of Southampton focused on data from cohorts of individuals born in the first half of the 20th century. Using data from one of these cohorts, Roseboom et al 8 investigated effects of timing of fetal undernutrition based on the Dutch cohort exposed to the 1944–1945 famine at the end of World War II and showed that fetal undernutrition may affect different organs of the body depending on different critical phases of development (i.e., in the Dutch famine, those individuals conceived before the famine and exposed to an energy-poor fetal environment late in gestation as adults had increased risk for insulin resistance and impaired glucose tolerance, but those conceived during the famine as adults had increased risk for high serum cholesterol and coronary heart disease). Using data from another cohort, Barker et al 9 investigated the trajectory of growth during infancy and childhood in the Helsinki 1934 to 1944 Birth Cohort (see Eriksson et al 10 ) and showed that adult outcomes were moderated by the tempo of growth in infancy and childhood as well as by fetal growth and birthweight (i.e., the risk of coronary events in adulthood was more strongly related to the tempo of childhood body mass index [BMI] gain from ages 2 to 11 years than to BMI itself at any other age).

This program of research also initiated a new a cohort study of contemporary births. Initial goals were to directly test some of the assumptions of the DOHaD approach (i.e., documentation of adaptations occurring in the fetus when undernourished, including changes in metabolism, alterations in hormone production and tissue sensitivity to these alterations, and changes in the relative growth rates of organs and structures of the body). The SWS started with interviews of 12,500 young female residents of Southampton to obtained measures of prepregnancy characteristics, followed by detailed, prospective evaluation of this cohort that was described by Inskip et al. 11 According to the SWS protocol, detailed measures were obtained of fetal development during conceptions and gestations that produced 3,000 live births, of birth phenotypes, and of outcomes in infancy and childhood. The ambitious goals of the SWS include evaluation of (1) influences of a mother’s diet, body composition, and endocrine profile on fetal growth, placental, and fetal adaptive responses, and (2) interactions of maternal and intrauterine factors with genes and postnatal environments of offspring that influence growth in infancy, pathways that lead to poor adult health, and risk factors for diseases in childhood (obesity, cardiorespiratory function, and asthma) and adulthood (coronary heart disease, type 2 diabetes, and osteoporosis).

An impressive list of publications from 2004 to 2009 is available from the SWS section of the MRC Epidemiology Resource Centre Web site ( http://www.mrc.soton.ac.uk/index.asp?page=4 ). Two examples from this research program are described here to address methodological issues that are critical for the evaluation of some basic assumptions of the DOHaD approach (e.g., the tracking of nutrients and oxygen supply by the measurement of fetal blood flow 12 and the characterization of infant size by measurement of body composition 13 ). These are crucial measures in the theory of developmental plasticity or predictive adaptive response, which predicts that maternal diet may regulate blood flow to developing organs (i.e., to the brain versus the liver) and may elicit fetal programming that affects body composition at birth (fat mass versus lean mass). Haugen et al 12 evaluated the effects of maternal adiposity and diet in 381 low-risk pregnancies in the SWS at 36 weeks of gestation using Doppler ultrasound to estimate blood flow in the umbilical cord and ductus venosus, which shunts well-oxygenated placental blood from the liver to the brain and heart. The low-risk group was selected to gain a better understanding of these factors in normal conditions rather than in extreme conditions in a high-risk group. Two independent effects were documented: the fetuses of women with low versus high central adiposity and operationally defined imprudent versus healthy diet had reduced ductus venosus shunting and increased liver blood flow. The observed fetal adaptations of cardiovascular responses to nutrient availability in this low-risk group suggested that these maternal characteristics were associated with liver-sparing response that “contrast with the brain-sparing response to fetal hypoxemia, which reduces hepatic flow and increases ductus venosus shunting” (p. 14). Harvey et al 13 reported on parental determinants of neonatal body composition in 448 births in the SWS, with dual-energy x-ray absorptiometry scan assessment of fat and muscle mass components of body composition in the offspring within 2 weeks of birth. With this rigorous measurement of neonatal body composition, this study documented that total fat mass was related to maternal lifestyle factors (smoking and physical activity) as well as maternal height, parity, and triceps skinfold thickness. One conclusion was that if these influences on fat mass have persisting effects, this information could point the way to early life interventions that may prevent later obesity. These examples of rigorous and prospective early evaluations in the SWS protocol show how modern methods can be used to assess fetal adaptations and their effects on structures and functions that may alter body composition (rather than just weight) at birth. This may provide improved estimates of the underlying DOHaD-related factors that contribute to risk for common adult disorders (e.g., obesity) later in life.

Collaboration between centers (see www.liggins.auckland.ac.nz/uoa/affiliations ) directed by the first two chairs of the DOHaD Society (Peter Gluckman from the Liggins Institute in Auckland and Mark Hanson from the MRC Epidemiology Resource Centre at the University of Southampton) led to the next generation of theory based on the concept of predictive adaptive response (see Gluckman and Hanson 14 ). Applications of the predictive adaptive response concept, presented and discussed in additional detail in a book by Gluckman and Hanson, 15 suggest that the fetus forecasts the future by sensing the current environment in utero and develops adaptively to match capabilities with expected demands. Gluckman et al 16 emphasized one of the premises of this hypothesis that the association of outcomes with birthweight is an epiphenomenon of the relationship between nutrient availability to the fetus and the predictive adaptive responses that this elicits. Gluckman et al 17 extended the concept of developmental plasticity by emphasizing that fetal programming may operate across the range from undernutrition to over-nutrition, with a U-shaped curve relating prenatal nutrition to adult metabolic disease.

Project VIVA: Background and Current Status

Gillman 18 noted limitations of undernutrition and low birthweight as markers of prenatal etiological pathways related to postnatal health outcomes. This led to consideration of fetal, infant, and child body composition as a phenotype and abnormalities anywhere in the maternal-fetal supply line of nutrients as a common final pathway that may alter body composition. Oken and Gillman 19 addressed the fetal origins of obesity and noted two different relationships with birthweight: a direct relationship held for birthweight with BMI in childhood and adulthood, but an inverse relationship held for low birth-weight with central adiposity, insulin resistance, and the metabolic syndrome. Gillman et al 20 , 21 used the life course approach to focus investigations on general in utero conditions and placental function and physiological processes regulating fetal development across a broad range of birth sizes, rather than on abnormal or pathological processes at one extreme.

Project VIVA was initiated in 1999 in eight offices of Harvard Vanguard Medical Associates, a large multi-specialty group practice in Massachusetts. Its initial goals were to identify women early in pregnancy and to enter them into a protocol for prospective assessments twice during pregnancy, within 3 days of birth, and at 6 months, and at 1, 2 and 3 years of age. 20 In 2006 it was extended to conduct follow-up through 7 years of age. The Web site for Project VIVA lists an impressive set of publications (see www.dacp.org/viva/publications.htm ), describing the initial evaluations of factors associated with blood pressure of the newborn, including maternal age 20 and maternal prenatal smoking, 22 and factors related to obesity at 3 years of age, including gestational weight gain 23 and weight in the first 6 months of life. 24 One example will be described in detail here that is particularly relevant to recent extensions of the DOHaD approach to consider more than birthweight as a predictor of early adaptations that are permanent and affect later outcomes by programming. Taveras et al 24 evaluated 559 children in Project VIVA to determine association of weight early in life (weight-for-length at birth and 6 months of age) with obesity (BMI >95th percentile) at 3 years of age. Weight early in life was directly associated with higher BMI at 3 years of age, but the association was larger for standardized weight at 3 years of age than for birth-weight. This suggest that increases in weight in the first 6 months of life may produce additional programming effects that increased risk for obesity in early childhood and thus may influence risk for later obesity more than birthweight alone that is assumed to reflect fetal programming.

Behavioral Perinatology Research Program: Background and Current Status

An early extension of the DOHaD approach was to go beyond nutrition hypotheses and to relate fetal development to exposure to other factors such as prenatal maternal stress and maternal-placental-fetal biological mediators of stress. Wadhwa 25 reviewed how psychoneuroendocrine processes in human pregnancy influence fetal development and health. Drake et al 26 reviewed the fetal glucocorticoid overexposure hypothesis as an alternative to the fetal undernutrition hypothesis to account for the relationship of the prenatal environment to adult disorders related to cardiovascular, metabolic, neuroendocrine, and behavioral phenotypes.

A multi-investigator research program at the University of California, Irvine, was initiated in 1993, and Wadhwa 25 reviewed the contributions of this program over the initial 12 years, which generated extensive information on the short-term effects of exposure to maternal psychosocial stress during pregnancy. This focused research documented that length of gestation and fetal growth are mediated, in part, by maternal-placental-fetal stress physiology, particularly placental corticotrophin-releasing hormone. Because simple birth phenotypes such as birthweight may represent a crude marker of intrauterine conditions that are likely to exert a causal role, this research program was recently extended based on the DOHaD approach to evaluate healthy young adults born with a normal birth-size phenotype (i.e., no low birthweight or preterm birth) but exposed during intrauterine life to maternal psychosocial stress, defined by a major stressful life event during pregnancy. Entringer et al 27 – 29 showed that compared with healthy young individuals without this history, these prenatal stress-exposed individuals exhibited primary insulin resistance and a lipid profile consistent with the metabolic syndrome, 27 altered immune function, 28 altered endocrine function, 29 and compromised cognitive function. 30 These findings suggest that in utero exposure to maternal stress may have long-term negative physiological consequences and may directly influence adult health even in the absence of adverse birth phenotypes such as low birthweight. Also directed by the DOHaD approach, Buss et al 31 evaluated brain morphology in young adults and revealed an association between birthweight and postnatal environment: Lower birth-weight was associated with smaller hippocampal volume (a well-established risk factor for depression and psychopathology) only in individuals exposed to postnatal adversity (low levels of parental bonding). Swanson and Wadhwa 32 have suggested that the DO-HaD approach also may be relevant to the origins of some child mental health disorders.

EPIGENETIC PROCESSES AND THE DOHaD APPOACH

In a review of the emerging science of epigenomics, Callinan and Feinberg 33 defined epigenetics as “the study of heritable changes other than those in the DNA sequence that encompass two major modifications of DNA or chromatin: DNA methylation, the covalent modification of cytosine, and post-translational modification of histones including methylation, acetylation, phosphorylation and sumoylation” (p. R95). They provide an excellent description of the potentially unique contributions of epigenetics, a glossary of terms, and a thorough description of methods used to detect genome-wide variation in DNA methylation and chromatin modification.

Outstanding reviews were published in 2007 that provide background on how the principles and concepts of epigenetics have been applied to the DOHaD approach. 34 – 36 If some of the mechanisms for developmental plasticity described in the DOHaD approach are epigenetic, and disease-related outcomes are related to disruptions of epigenetic processes elicited by the fetal environment, then this emerging field may provide explanatory mechanisms that underlie some of the enduring effects of adverse fetal, infant, and childhood environments. In their review of the DOHaD approach, Gluckman et al 17 provided an excellent summary of epigenetic modification of histones or of DNA itself in a figure (p. 66) that we reproduce here ( Fig. 1 ). This figure summarizes the important epigenetic processes of DNA methylation and histone acetylation and methylation that have been discussed and reviewed elsewhere 31 – 34 in detail and thus are not repeated here.

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Regulation of gene expression through epigenetic processes. Epigenetic modification of histones or of DNA itself controls access of transcription factors (TFs) to the DNA sequence, thereby modulating the rate of transcription to messenger RNA (mRNA). Transcriptionally active chromatin (top) characterized by the presence of acetyl groups (Ac) on specific lysine residues of core histones in the nucleosome, which decreases their binding to DNA and results in a more open chromatin structure that permits access of transcription factors. In addition, cytidine-guanosine (CpG) sequences in the promoter regions (P) of actively transcribed genes are generally unmethylated, allowing for the binding of transcription factors. Transcriptionally inactive chromatin (bottom) is characterized by histone deacetylation, promoter CpG methylation (as indicated by methyl groups [Me]), and decreased binding of transcription factors. (For simplicity, other histone modifications [such as methylation] and additional regulatory factors [such as methyl-CpG binding proteins] are not shown.) A further level of epigenetic control is provided by microRNA molecules (19 to 22 nucleotides in length), which bind to complementary sequences in the 3′ end of mRNA and reduce the rate of protein synthesis. From Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med 2008;359(1):61–73.

Two types of genes are modified epigenetically (i.e., are epigenetically liable): imprinted genes and genes with metastable epialleles. Imprinted genes are those in which specifically either the maternally derived or the paternally derived allele is suppressed, thereby rendering them functionally haploid (i.e., with parent-of-origin monoallelic expression). In other nonimprinted genes, one or both alleles are regulated epigenetically, and these metastable epialleles result in varying levels of gene expression. The modification of DNA from conception forward occurs to establish the epigenetic influence of gene expression, which is clearly presented in an article by Reik et al 37 that is often cited in reviews of imprinting. This article provided a figure outlining the processes of methylation reprogramming in the germ line and in preimplantation embryos that is reproduced here ( Fig. 2 ). As Leudi et al 38 discuss, many undiscovered genes are predicted to be imprinted, and this set of genes is likely to have special importance for reproductive medicine and fetal growth. They speculate about the evolutionary benefits of imprinted genes, which likely are the product of positive Darwinian selection despite potential drawbacks associated with a haploid gene compared with a diploid gene that has a backup copy that may protect from “single-hit” effects of DNA damage. Waterland and Michels 35 pointed out that in the DOHaD field “direct evidence of an involvement of epigenetic dysregulation in human cardiovascular disease, type 2 diabetes, and obesity is scant” compared with the field of cancer. They noted that this may be due to temporal and tissue specificity of the relevant tissue that may have epigenetic dysregulation associated diseases that have been the focus of the DOHaD approach, and they concluded that any disease with a genetic basis is also likely to have an epigenetic basis, but the “tissue-specificity of epigenetic regulation (and dysregulation) will be the major obstacle to epigenetic epidemiology of DOHaD” (p. 379). However, as pointed out by Gluckman and Hanson, 15 there is a strong epigenetic basis for the DOHaD model of disease pathogenesis based on animal studies, which (for example) show that minor alterations in the maternal diet during pregnancy can produce lasting changes in the physiology and metabolism of offspring. We summarize here classic findings from two research programs that have provided evidence about possible epigenetic mechanisms involved in this type of phenotypic plasticity in the DOHaD approach. More detailed description and review was provided as background for a 2006 meeting on Genes, Environments and Human Development, Health and Disease. 39

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(A) Methylation reprogramming in the germ line. Primordial germ cells (PGCs) in the mouse become demethylated early in development. Remethylation begins in prospermatogonia on E16 in male germ cells and after birth in growing oocytes. Some stages of germ cell development are shown (modified from 73). (B) Methylation reprogramming in preimplantation embryos. The paternal genome (blue) is demethylated by an active mechanism immediately after fertilization. The maternal genome (red) is demethylated by a passive mechanism that depends on DNA replication. Both are remethylated around the time of implantation to different extents in embryonic (EM) and extraembryonic (EX) lineages. Methylated-imprinted genes and some repeat sequences (dashed line) do not become demethylated. Unmethylated imprinted genes (dashed line) do not become methylated. From Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293(5532):1089–1093.

The Agouti Mouse Model

A group at Duke University has used a mouse model to investigate the effects of maternal diet during pregnancy on the phenotype manifested in the offspring ( www.geneimprint.com ). The viable yellow agouti (A vy ) mouse has a mutation that causes yellow hair pigmentation. A vy /a animals manifest a broad range of coat-color phenotypes from brown to mottled to yellow. Waterland and Jirtle 40 investigated methyl supplementation of the diet of mothers during pregnancy and showed when a standard diet is supplemented by methyl donors, methylation of the A vy gene increases and the coat-color distribution shifts toward the brown phenotype. Dolinoy et al 41 extended this work by investigation of effects of soy-rich diets before and during pregnancy in a/a females. The distribution of coat color in A vy offspring was shifted toward brown, and adult weight of the yellow phenotype was ~50% higher than in the brown phenotype. This demonstrated that for the A vy /a genotype of the viable yellow agouti mouse model, a high-soy diet results in epigenetic changes (increased methylation of CpG during fetal development), affects coat color, and also reduces obesity.

The Rat Model of Nurturing

A group at McGill University has used a rodent model to investigate epigenetic effects of maternal care. In rats, an important component of maternal care consists of licking and grooming, which varies widely across individuals. Meaney and Szyf 42 showed that increased licking and grooming increased hippocampal expression of the glucocorticoid receptor (GR) mRNA and protein, decreased hypothalamic corticotrophin-release factor, and reduced hypothalamic-pituitary-adrenal response to stress. This work showed a direct relationship between maternal behavior and DNA methylation in the rat hippocampal GR gene (specifically in the exon 1 7 promoter). Weaver et al 43 demonstrated further that the stress responses in adult rats that are programmed early in life by maternal care can be reversed by central infusion of methionine (a methyl donor), suggesting that the inherently stable epigenomic marks established by behavioral programming at a critical period early in life are potentially reversible later in life. This provides a biological basis for speculations about the effects of poverty on early experience, and how exposure to abuse, family strife, emotional neglect, and harsh discipline may have epigenetic effects that produce individual differences in neural and endocrine response to stress and may increase the susceptibility to common adult disorders such as depression and anxiety, drug abuse, and diabetes, heart disease, and obesity.

Recent Epigenetic Studies in Primates

Aaggard-Tillery et al 44 used a nonhuman primate model to investigate the effects of maternal diet on alternations to the epigenome that may be related to obesity. A high-fat diet (35% fat) was established that produced obesity in pregnant monkeys. In comparison with control animals with a control diet (13% fat), the offspring of the obese monkeys were obese. In the first stage of investigation, Aagaard-Tillery et al 44 identified an epigenetic change in the liver of these offspring (hyperacetylation of fetal hepatic tissue) that was associated with the high-fat diet and the resulting obesity. In the second stage, they altered the fat content of the monkeys’ diets during pregnancy. Even though obesity was maintained, the epigenetic changes in offspring were no longer present. This finding suggests that obesity may, in part, be due to the effects of maternal diet rather than maternal obesity on the fetal environment. This study in primates is important because it showed “ in utero exposure (caloric-dense high-fat maternal diet) induces site-specific alterations in fetal hepatic H3 acetylation” and that this leads “to epigenetically altered fetal chromatin structure in primates via covalent modifications of histones and hence lends a molecular basis to the fetal origins of adult disease hypothesis” (p. 91).

Tyckol 45 addressed the special nature of imprinted genes in placental growth. The placenta is the principal metabolic, respiratory, excretory, and endocrine organ of the fetus, which has substantial molecular variation across the fetal and maternal compartment (see Sood et al 46 ) and affects birthweight even after adjustment for placental weight (see Salafia et al 47 ). There are ~30 known imprinted genes, and a large percentage are expressed in trophoblasts and regulate placental growth. 48 In this review, placental phenotypes controlled by imprinted genes were discussed, as well as the role of oppositely imprinted genes (e.g., Igf2 and Igf2r) related to the evolutionary theory of genetic conflict described by Haig 49 that proposes different maternal and paternal self-interest in fetal growth. Some genes expressed in the placenta normally are maternally silenced/paternally expressed genes that promote growth (e.g., MEST) and others normally are maternally expressed/paternally silenced that limit growth (e.g., PHLDA2). In a study of intrauterine growth restriction (IUGR) and non-IUGR placenta, an altered expression of imprinted genes in placental response to maternal vascular underperfusion was investigated by McMinn et al. 50 In this study, IUGR was characterized by increased expression of PHLDA2 and decreased expression of MEST in placenta tissue. This suggested unbalanced expression of these two oppositely imprinted genes was one component of the adaptive response of placental tissue to chronic maternal vascular underperfusion associated with IUGR, which provides some support for the conflict hypothesis. New methods are emerging for assessing epigenetic marks, such as the MSNP approach for determining allele-specific methylation and allele-specific expression patterns that may be dependent or independent of genome sequence (see Kerkel et al 51 ). In the study of IUGR, there was no evidence of altered DNA methylation in imprinting centers of the PHLDA2 and MEST genes, which led McNinn et al 50 to conclude that “the high PHLDA2/MEST mRNA ratios in this subset of IUGR may reflect altered DNA methylation in as yet uncharacterized cis -acting regulatory sequences, but more likely reflects conventional transcriptional dysregulation by trans acting factors in placental cytotrophoblasts” (p. 543).

RELEVANCE OF EPIGENETIC AND THE DOHaD HYPOTHESIS TO REPRODUCTIVE MEDICINE

Niemtz and Feinberg 52 provide an example of early epigenetic research in reproductive medicine. 52 They investigated a possible a link between assisted reproductive therapy (ART) and Beckwith-Wiedemann syndrome (BWS). 53 This syndrome is characterized by genetic heterogeneity, but about half the cases are associated with loss of imprinting in genes related to growth (e.g., the LIT1 gene located on the tip of chromosome 11). Niemitz and Feinberg 52 suggested that “epigenetic alterations could arise from some aspect of ART” (e.g., in the in vitro culture itself or the media used) or that “epigenetic alteration could be a significant cause for infertility, rather than a consequence of the procedures used to treat it” (p. 605). Chang et al 54 tested that hypothesis that culture media would be implicated as a common factor among children with BWS conceived after ART, but in a small sample of 19 they reported that in vitro fertilization (IVF) was a common factor but otherwise “no common factor was identified among reproductive endocrine records,” and they concluded “larger prospective studies are needed to systematically assess the potential risk factors associated with BWS and ART” (p. 353).

A summary of recent studies relevant to reproductive medicine is facilitated by two recent reviews in the series of Seminars in Reproductive Medicine, 55 , 56 which provide detailed background for the application of the DOHaD approach to reproductive medicine and epigenetic processes ( Fig. 1 ) that may operate at the time of conception ( Fig. 2 ).

Rinaudo and Lamb 55 summarized the literature on in utero stress related to deficient maternal-placental nutrient supply and some adverse childhood and adult outcomes (i.e., cardiovascular disease, hypertension, diabetes, and dysregulation of the hypothalamic-pituitary-adrenal axis) associated with stressful fetal environments during the postimplantation period of fetal growth. They also provided a brief review of perinatal morbidity associated with stress during the preimplantation period related to in vitro culture in assisted reproduction. They concluded that the evidence linking stress in utero to risk for adult disorders is convincing, and that the preimplantation embryo development is particularly sensitive to epigenetic regulation and dysregulation.

Kalra and Molinaro 56 summarized the literature on association of in vitro fertilization with perinatal morbidity and the risk for congenital abnormalities, preterm birth, low birthweight, and other pregnancy-related complications. They concluded that children “conceived after IVF do seem to be at an increased risk for congenital and chromosomal anomalies compared with that of natural conceptions, particularly if ICSI [intracytoplasmic sperm injection] is used” (p. 432), but that effects have not yet been determined for long-term outcomes related to the DOHaD approach and physical, emotional, or cognitive development.

Other reviews of reproductive outcomes after IVF 57 and ICSI 58 also are available and add to the growing recognition that alteration of biochemical and biophysical conditions at conception and during early embryonic life associated with ARTs may result in changes in epigenetic processes and produce short- and long-term effects on development and health.

NEXT STEPS: LARGE HUMAN COHORT STUDIES

There are many recent developments in the field of epigenetics. The NIH Roadmap program on the Epigenomics of Human Health and Disease ( www.nih.gov ) was initiated to accelerate new developments, including the characterization of reference epigenomes in non-disease states and the perturbation of these epigenomes in disease states that may be temporally and tissue specific. Recent workshops (Epigenomics of Addiction) and conferences (Epigenomics of Human Health and Disease) have summarized these new developments in the understanding of mechanisms and methods that are being applied in the current investigations. The basic processes that describe how cells with the same molecular instructions (the DNA code) become differentiated during development through differential expression of genes have been reviewed in detail, with information available on the NIH Web site (see www.nida.nih.gov ).

The next phase of research to address epigenetic mechanisms in humans would benefit substantially from large birth cohort studies with prospective measures of broad domains of exposures and outcomes. 35 , 59 There are good examples in the literature. Recently, the Avon Longitudinal Study of Parents and Children (ALSPAC) study, 60 the Danish National Birth Cohort, 61 and the SWS 11 have taken initial steps to identify critical developmental processes that underlie fetal growth and development 13 , 62 , 63 and later outcomes in childhood related to the overnutrition hypothesis, 64 preterm birth, 61 and body composition. 13

A prospective birth cohort, the National Children’s Study (NCS), is now underway in the United States. The NCS will recruit a nationally representative birth cohort at 105 sites with ~100,000 children. The recruitment will start with randomly designated neighborhoods and a survey of households within them to identify ~750,000 women between the 18 and 40 years of age who are not pregnant or within the first trimester of pregnancy. These women will be evaluated and followed until ~1000 births occur at each of the 105 locations (for an expected total of ~105,000). Broad exposures and outcome domains will be assessed at multiple times across stages of development (before conception, during pregnancy, and at birth; in infancy, childhood, adolescence; and into adulthood). A recent summary of the background, controversies, and status of the NCS was provided by Landrigan et al. 65 The NCS will provide a prospective evaluation of a large birth cohort with early and frequent direct observation of the same individuals over time, which will provide rich information about health and disease that will be stored in clinical databases as well as biological and environmental samples that will be stored in biospecimen repositories. This information will be used initially to evaluate 29 “priority” hypotheses that were developed during the early consensus-development phase of the project in 2002 and then updated by the Vanguard Centers of the NCS in 2007 (see www.nationalchildrensstudy.gov ). However, with such a large and representative birth cohort characterized by broad exposure and outcome domains, many more hypotheses will be tested and will emerge as the NCS progresses.

Although the NCS does not provide specific funding for genetic or epigenetic analyses, it does have a goal to provide the NCS clinical databases and biospecimen repositories for use in approved and separately funded adjunct studies designed to take advantage of the extensive infrastructure of the NCS. Swanson and Wadhwa 32 presented and discussed three critical issues that highlight the extraordinary value of the birth cohort design used by the NCS, and here we note some significant issues and problems associated with each one:

If the nature of the combined effect of multiple genetic and environmental risk factors on complex common disorders is nonadditive, as assumed by the DOHaD approach, then the best way to elucidate these interactive effects is to assess the various risk factors simultaneously in the same cohort rather than in separate cohorts as proposed by Willet et al. 66 However, the cost and subject burden required to conduct a birth cohort study are limiting factors that are now being considered in the NCS. 67
If vulnerability to a particular risk factor or disease phenotype is determined not only by the genome acquired at conception but by the nature of its interplay with the environments during successive critical periods of development, a key feature of the DOHaD approach, then a longitudinal assessment of the environment from before conception through pregnancy, fetal life, birth, and infancy may be necessary, instead of relying on assessment of the environment in adult life as proposed by Collins and Manolio, 68 to better understand disease susceptibilities that may have fetal or developmental origins. However, the investment in time, the retention of the sample, and the continuity of funding are issues that are facing the NCS. 69
If the interplay between genes and environment is characterized by dynamic modifications to the genome, as assumed by the DOHaD approach, then not only the stable DNA sequence but also the epigenetic modifications to nuclear DNA and chromatin structure must be investigated serially during critical periods in early development over time and under various environmental conditions to assess gene–environment interactions adequately. However, the issues of temporal and tissue specificity must be addressed. 35 The need for multiple collections of environmental exposures and biological specimens and the limitation inherent in human studies of the availability of many specific tissue types create truly daunting tasks that must be addressed before the full study can be initiated. 70 The Vanguard Centers (7 of the 105 locations) are now conducting a field test of the NCS draft protocol that is intended to provide an empirical basis for decisions about feasibility and costs, which will be used to establish the final protocol for implementation in all 105 locations. 71

The NCS provides an extraordinary opportunity to develop genetic and epigenetic projects and make use of the DOHaD approach to address specific mechanisms related to common disorders (e.g., obesity 72 ) that are increasing by epidemic proportions around the world and define critical issues for consideration in the area of reproductive medicine.

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Schematic representation linking hypothesis of disease predisposition
| Illustration of the different hypotheses of Alzheimer's Disease (AD
Hypothesis for Pathophysiology of Alzheimer's disease
The amyloid hypothesis of Alzheimer's disease at 25 years
Pathophysiology of the main hypotheses of Alzheimer's disease. Created
Schematic expression of the hypothesis: Disease specific expression

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The hygiene hypothesis in autoimmunity: the role of pathogens and
The hypothesis was confirmed by epidemiological studies and was extended to other allergic diseases 3. The term hygiene hypothesis was coined in 2000 and is understood not as a problem of ...
Alzheimer's Disease: An Overview of Major Hypotheses and Therapeutic
2.1. The Amyloid-Beta Hypothesis. This hypothesis is the most recognized one amongst researchers, owing to its explanation for the senile plaque formation and the accumulation of Aβ oligomers as the major highlight of the disease [].The proteolysis of transmembrane protein APP by beta and gamma secretases forms single units of Aβ, which further undergo certain structural modifications to ...
Is the Hygiene Hypothesis True?
You mentioned the hygiene hypothesis, which was postulated back in the '80s. German scientists noticed that families with fewer children tended to have more allergic disease. This was interpreted [to mean] that allergic disease was linked to experiencing fewer infections. I have explored this idea in my research for a couple of decades now.
The 'hygiene hypothesis' for autoimmune and allergic diseases: an
The hygiene hypothesis is based upon epidemiological data, particularly migration studies, showing that subjects migrating from a low-incidence to a high-incidence country acquire the immune disorders with a high incidence at the first generation. However, these data and others showing a correlation between high disease incidence and high socio ...
Hygiene hypothesis
The hygiene hypothesis has difficulty explaining why allergic diseases also occur in less affluent regions. [9] Additionally, exposure to some microbial species actually increases future susceptibility to disease instead, as in the case of infection with rhinovirus (the main source of the common cold ) which increases the risk of asthma.
The hygiene hypothesis: current perspectives and future therapies
In 1989, Strachan proposed the hygiene hypothesis of allergic disease after observing that hay fever was less common among children with older siblings. 8 He reasoned that children growing up in larger families may experience increased exposure to microbes in early childhood due to inevitable unhygienic contact with older siblings or prenatal exposure from the mother infected by similar ...
The Effect of Infections on Susceptibility to Autoimmune and Allergic
The hygiene hypothesis postulates that an environment with a high incidence of infectious diseases protects against allergic and autoimmune diseases, whereas hygienic surroundings increase the inci...
The Hygiene Hypothesis
Last year we celebrated the 30th anniversary of the Hygiene Hypothesis. Since Strachan framed the Hygiene Hypothesis in 1989 his fundamental idea to explain the origins of allergic diseases development has survived the test of time.The basic idea of how humans, their microbiota, and a continuously modernizing environment have interacted to drive immune dysregulation has persisted and become ...
The germless theory of allergic disease: revisiting the hygiene hypothesis
The hypothesis that has gained the most attention is derived from the observation of an inverse relationship between the risk of hay fever and family size 4.This 'hygiene hypothesis' argues that ...
Frontiers
Currently recognized epigenetic pathways overlapping between chronic inflammatory diseases and other disorders such as psychiatric conditions or cancer might extend the Hygiene Hypothesis toward a model explaining in a broader sense the rise of health burdens in westernized societies . Finally, the world-wide challenge caused by the climate ...
Hygiene Hypothesis
The expression " hygiene hypothesis " was cited for the first time by Strachan in 1989 and was based on the fact that microbiota modulates our immune systems [65]. Initially, this hypothesis was investigated associated to allergic disorders; however, actually, it can be applied to most human diseases [66].
Revisiting the Hygiene Hypothesis in the Context of Autoimmunity
The hygiene hypothesis in its dynamic aspect is based on the negative correlation observed between the decrease in the frequency of infectious diseases and the increase in that of allergic and autoimmune diseases. The question arises as to whether the trends reported twenty years ago persist today (3).
Hygiene hypothesis and autoimmune diseases: A narrative review of
Evidence about the impact of Hygiene Hypothesis in thyroid autoimmune diseases are few and limited. Epidemiological studies showed that Graves' disease, an organ-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor, is relatively uncommon in developing countries [ 216 ], compatibly with the Hygiene Hypothesis.
Asthma: The Hygiene Hypothesis
The "hygiene hypothesis" is supported by epidemiologic studies demonstrating that allergic diseases and asthma are more likely to occur when the incidence and levels of endotoxin (bacterial ...
The Hygiene Hypothesis and New Perspectives—Current Challenges Meeting
Keywords: hygiene hypothesis, allergy, asthma, non-communicable inflammatory diseases, chronic inflammation. Go to: Throughout its history, the Hygiene Hypothesis has shown itself to be adaptable and flexible whenever it has been challenged by innovation in science (1). A number of new findings need to be considered in this ongoing revisiting ...
The hygiene hypothesis at a glance: Early exposures, immune mechanism
The hygiene hypothesis was proposed almost three decades ago. Nevertheless, its mechanism still remains with relevant controversies. Some studies defend that early exposures during childhood to microbes and parasites are key determinants to prevent allergies and autoimmune diseases; however, other studies demonstrated that these early exposures can even potentiate the clinical scenario of the ...
The Hygiene Hypothesis and Autoimmune Disorders
The hygiene hypothesis is a hypothesis that suggests that the increased incidence of allergic and autoimmune disorders are linked to the tremendous changes in sanitation standards and practices ...
The hygiene hypothesis in autoimmunity: the role of pathogens and
This hypothesis is supported by robust epidemiological data, but the underlying mechanisms are unclear. Pathogens are known to be important, as autoimmune disease is prevented in various experimental models by infection with different bacteria, viruses and parasites. Gut commensal bacteria also play an important role: dysbiosis of the gut flora ...
History and progress of hypotheses and clinical trials for Alzheimer's
Percentage of clinical trials in which each of the various hypotheses for AD were tested up to 2019. The amyloid hypothesis was the most heavily tested (22.3% of trials); the neurotransmitter ...
Cleaning up the hygiene hypothesis
They decided the name has to go (15). "The trouble is, as soon as you use the words 'hygiene hypothesis,' the word hygiene prejudges what the cause is," says Bloomfield. To the public, "hygiene" is interpreted as personal cleanliness: washing hands, keeping food clean and fresh, sanitizing the home.
Disease stages and therapeutic hypotheses in two decades of ...
First, experimental drugs are often broadly categorized as "disease-modifying" if the hypothesis is one of disease modification, regardless of the quality of that hypothesis.
Scientific Hypotheses: Writing, Promoting, and Predicting Implications
The misunderstanding of the hygiene hypothesis that primarily aimed to shed light on the role of the microbiome in allergic and autoimmune diseases resulted in decline of public confidence in hygiene with dire societal implications, forcing some experts to abandon the original idea.27,28 Although that hypothesis is unrelated to the issue of ...
Developmental Origins of Health and Disease: Brief History of the
FROM FETAL ORIGINS OF ADULT DISEASE TO DEVELOPMENTAL ORIGINS OF HEALTH AND DISEASE. Barker's hypothesis stimulated a great deal of worldwide interest and activity in the area of developmental plasticity, Gillman et al 5 summarized in a report of the meetings of the World Congress on Fetal Origins of Adult Disease that were convened in 2001 (Mumbai, India) and 2003 (Brighton, United Kingdom ...

Is the Hygiene Hypothesis True?

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Introduction

Interest and Limitations of Epidemiology

Causal Relationship

Hygiene Hypothesis and Evolution

Intestinal Dysbiosis, Cause, Consequence, or Modulation

Multiple and Complex Underlying Mechanisms

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Asthma: The Hygiene Hypothesis

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