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The prevalence of stress-associated somatic and psychiatric disorders is increased in environments offering a narrow relative to a wide range of microbial exposure. Moreover, different animal and human studies suggest that an overreactive immune system not only accompanies stress-associated disorders, but might even be causally involved in their pathogenesis. In support of this hypothesis, we recently showed that urban upbringing in the absence of daily contact with pets, compared to rural upbringing in the presence of daily contact with farm animals, is associated with a more pronounced immune activation following acute psychosocial stressor exposure induced by the Trier Social Stress Test (TSST). Here we employed 16S rRNA gene sequencing to test whether this difference in TSST-induced immune activation between urban upbringing in the absence of daily contact with pets ( n = 20) compared with rural upbringing in the presence of daily contact with farm animals ( n = 20) is associated with differences in the composition of the salivary microbiome. Although we did not detect any differences in alpha or beta diversity measures of the salivary microbiome between the two experimental groups, statistical analysis revealed that the salivary microbial beta diversity was significantly higher in participants with absolutely no animal contact ( n = 5, urban participants) until the age of 15 compared to all other participants ( n = 35) reporting either daily contact with farm animals ( n = 20, rural participants) or occasional pet contact ( n = 15, urban participants). Interestingly, when comparing these urban participants with absolutely no pet contact to the remaining urban participants with occasional pet contact, the former also displayed a significantly higher immune, but not hypothalamic-pituitary-adrenal (HPA) axis or sympathetic nervous system (SNS) activation, following TSST exposure. In summary, we conclude that only urban upbringing with absolutely no animal contact had long-lasting effects on the composition of the salivary microbiome and potentiates the negative consequences of urban upbringing on stress-induced immune activation., (Copyright © 2020 Langgartner, Zambrano, Heinze, Stamper, Böbel, Hackl, Jarczok, Rohleder, Rook, Gündel, Waller, Lowry and Reber.)

Urbanization is on the rise, and environments offering a narrow range of microbial exposures are linked to an increased prevalence of both physical and mental disorders. Human and animal studies suggest that an overreactive immune system not only accompanies stress-associated disorders but might even be causally involved in their pathogenesis. Here, we show in young [mean age, years (SD): rural, 25.1 (0.78); urban, 24.5 (0.88)] healthy human volunteers that urban upbringing in the absence of pets ( n = 20), relative to rural upbringing in the presence of farm animals ( n = 20), was associated with a more pronounced increase in the number of peripheral blood mononuclear cells (PBMCs) and plasma interleukin 6 (IL-6) concentrations following acute psychosocial stress induced by the Trier social stress test (TSST). Moreover, ex vivo-cultured PBMCs from urban participants raised in the absence of animals secreted more IL-6 in response to the T cell-specific mitogen Con A. In turn, antiinflammatory IL-10 secretion was suppressed following TSST in urban participants raised in the absence of animals, suggesting immunoregulatory deficits, relative to rural participants raised in the presence of animals. Questionnaires, plasma cortisol, and salivary α-amylase, however, indicated the experimental protocol was more stressful and anxiogenic for rural participants raised in the presence of animals. Together, our findings support the hypothesis that urban vs. rural upbringing in the absence or presence of animals, respectively, increases vulnerability to stress-associated physical and mental disorders by compromising adequate resolution of systemic immune activation following social stress and, in turn, aggravating stress-associated systemic immune activation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The hygiene or "Old Friends" hypothesis proposes that the epidemic of inflammatory disease in modern urban societies stems at least in part from reduced exposure to microbes that normally prime mammalian immunoregulatory circuits and suppress inappropriate inflammation. Such diseases include but are not limited to allergies and asthma; we and others have proposed that the markedly reduced exposure to these Old Friends in modern urban societies may also increase vulnerability to neurodevelopmental disorders and stress-related psychiatric disorders, such as anxiety and affective disorders, where data are emerging in support of inflammation as a risk factor. Here, we review recent advances in our understanding of the potential for Old Friends, including environmental microbial inputs, to modify risk for inflammatory disease, with a focus on neurodevelopmental and psychiatric conditions. We highlight potential mechanisms, involving bacterially derived metabolites, bacterial antigens, and helminthic antigens, through which these inputs promote immunoregulation. Though findings are encouraging, significant human subjects' research is required to evaluate the potential impact of Old Friends, including environmental microbial inputs, on biological signatures and clinically meaningful mental health prevention and intervention outcomes.

Aims: To review the burden of allergic and infectious diseases and the evidence for a link to microbial exposure, the human microbiome and immune system, and to assess whether we could develop lifestyles which reconnect us with exposures which could reduce the risk of allergic disease while also protecting against infectious disease., Methods: Using methodology based on the Delphi technique, six experts in infectious and allergic disease were surveyed to allow for elicitation of group judgement and consensus view on issues pertinent to the aim., Results: Key themes emerged where evidence shows that interaction with microbes that inhabit the natural environment and human microbiome plays an essential role in immune regulation. Changes in lifestyle and environmental exposure, rapid urbanisation, altered diet and antibiotic use have had profound effects on the human microbiome, leading to failure of immunotolerance and increased risk of allergic disease. Although evidence supports the concept of immune regulation driven by microbe-host interactions, the term 'hygiene hypothesis' is a misleading misnomer. There is no good evidence that hygiene, as the public understands, is responsible for the clinically relevant changes to microbial exposures., Conclusion: Evidence suggests a combination of strategies, including natural childbirth, breast feeding, increased social exposure through sport, other outdoor activities, less time spent indoors, diet and appropriate antibiotic use, may help restore the microbiome and perhaps reduce risks of allergic disease. Preventive efforts must focus on early life. The term 'hygiene hypothesis' must be abandoned. Promotion of a risk assessment approach (targeted hygiene) provides a framework for maximising protection against pathogen exposure while allowing spread of essential microbes between family members. To build on these findings, we must change public, public health and professional perceptions about the microbiome and about hygiene. We need to restore public understanding of hygiene as a means to prevent infectious disease., (© Royal Society for Public Health 2016.)

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

It is increasingly evident that inflammation is an important determinant of cognitive function and emotional behaviors that are dysregulated in stress-related psychiatric disorders, such as anxiety and affective disorders. Inflammatory responses to physical or psychological stressors are dependent on immunoregulation, which is indicated by a balanced expansion of effector T-cell populations and regulatory T cells. This balance is in part driven by microbial signals. The hygiene or "old friends" hypothesis posits that exposure to immunoregulation-inducing microorganisms is reduced in modern urban societies, leading to an epidemic of inflammatory disease and increased vulnerability to stress-related psychiatric disorders. With the global trend toward urbanization, humans are progressively spending more time in built environments, thereby, experiencing limited exposures to these immunoregulatory "old friends." Here, we evaluate the implications of the global trend toward urbanization, and how this transition may affect human microbial exposures and human behavior., (© 2016 Elsevier Inc. All rights reserved.)

The immune system influences brain development and function. Hygiene and other early childhood influences impact the subsequent function of the immune system during adulthood, with consequences for vulnerability to neurodevelopmental and psychiatric disorders. Inflammatory events during pregnancy can act directly to cause developmental problems in the central nervous system (CNS) that have been implicated in schizophrenia and autism. The immune system also acts indirectly by "farming" the intestinal microbiota, which then influences brain development and function via the multiple pathways that constitute the gut-brain axis. The gut microbiota also regulates the immune system. Regulation of the immune system is crucial because inflammatory states in pregnancy need to be limited, and throughout life inflammation needs to be terminated completely when not required; for example, persistently raised levels of background inflammation during adulthood (in the presence or absence of a clinically apparent inflammatory stimulus) correlate with an increased risk of depression. A number of factors in the perinatal period, notably immigration from rural low-income to rich developed settings, caesarean delivery, breastfeeding and antibiotic abuse have profound effects on the microbiota and on immunoregulation during early life that persist into adulthood. Many aspects of the modern western environment deprive the infant of the immunoregulatory organisms with which humans co-evolved, while encouraging exposure to non-immunoregulatory organisms, associated with more recently evolved "crowd" infections. Finally, there are complex interactions between perinatal psychosocial stressors, the microbiota, and the immune system that have significant additional effects on both physical and psychiatric wellbeing in subsequent adulthood. This article is part of a Special Issue entitled Neuroimmunology in Health And Disease., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

The immune system evolved to require input from at least three sources that we collectively term the 'old friends': (i) the commensal microbiotas transmitted by mothers and other family members; (ii) organisms from the natural environment that modulate and diversify the commensal microbiotas; and (iii) the 'old' infections that could persist in small isolated hunter-gatherer groups as relatively harmless subclinical infections or carrier states. These categories of organism had to be tolerated and co-evolved roles in the development and regulation of the immune system. By contrast, the 'crowd infections' (such as childhood virus infections) evolved later, when urbanization led to large communities. They did not evolve immunoregulatory roles because they either killed the host or induced solid immunity, and could not persist in hunter-gatherer groups. Because the western lifestyle and medical practice deplete the 'old' infections (for example helminths), immunoregulatory disorders have increased, and the immune system has become more dependent upon microbiotas and the natural environment. However, urbanization maintains exposure to the crowd infections that lack immunoregulatory roles, while accelerating loss of exposure to the natural environment. This effect is most pronounced in individuals of low socioeconomic status (SES) who lack rural second homes and rural holidays. Interestingly, large epidemiological studies indicate that the health benefits of living close to green spaces are most pronounced for individuals of low SES. Here we discuss the immunoregulatory role of the natural environment, and how this may interact with, and modulate, the proinflammatory effects of psychosocial stressors in low SES individuals., (© 2014 The Authors. Clinical and Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society. for Immunology.)

Regulation of the immune system is an important function of the gut microbiota. Increasing evidence suggests that modern living conditions cause the gut microbiota to deviate from the form it took during human evolution. Contributing factors include loss of helminth infections, encountering less microbial biodiversity, and modulation of the microbiota composition by diet and antibiotic use. Thus the gut microbiota is a major mediator of the hygiene hypothesis (or as we prefer, "Old Friends" mechanism), which describes the role of organisms with which we co-evolved, and that needed to be tolerated, as crucial inducers of immunoregulation. At least partly as a consequence of reduced exposure to immunoregulatory Old Friends, many but not all of which resided in the gut, high-income countries are undergoing large increases in a wide range of chronic inflammatory disorders including allergies, autoimmunity and inflammatory bowel diseases. Depression, anxiety and reduced stress resilience are comorbid with these conditions, or can occur in individuals with persistently raised circulating levels of biomarkers of inflammation in the absence of clinically apparent peripheral inflammatory disease. Moreover poorly regulated inflammation during pregnancy might contribute to brain developmental abnormalities that underlie some cases of autism spectrum disorders and schizophrenia. In this chapter we explain how the gut microbiota drives immunoregulation, how faulty immunoregulation and inflammation predispose to psychiatric disease, and how psychological stress drives further inflammation via pathways that involve the gut and microbiota. We also outline how this two-way relationship between the brain and inflammation implicates the microbiota, Old Friends and immunoregulation in the control of stress resilience.

Epidemiological studies suggest that living close to the natural environment is associated with long-term health benefits including reduced death rates, reduced cardiovascular disease, and reduced psychiatric problems. This is often attributed to psychological mechanisms, boosted by exercise, social interactions, and sunlight. Compared with urban environments, exposure to green spaces does indeed trigger rapid psychological, physiological, and endocrinological effects. However, there is little evidence that these rapid transient effects cause long-term health benefits or even that they are a specific property of natural environments. Meanwhile, the illnesses that are increasing in high-income countries are associated with failing immunoregulation and poorly regulated inflammatory responses, manifested as chronically raised C-reactive protein and proinflammatory cytokines. This failure of immunoregulation is partly attributable to a lack of exposure to organisms ("Old Friends") from mankind's evolutionary past that needed to be tolerated and therefore evolved roles in driving immunoregulatory mechanisms. Some Old Friends (such as helminths and infections picked up at birth that established carrier states) are almost eliminated from the urban environment. This increases our dependence on Old Friends derived from our mothers, other people, animals, and the environment. It is suggested that the requirement for microbial input from the environment to drive immunoregulation is a major component of the beneficial effect of green space, and a neglected ecosystem service that is essential for our well-being. This insight will allow green spaces to be designed to optimize health benefits and will provide impetus from health systems for the preservation of ecosystem biodiversity.

Chronic inflammatory diseases (autoimmunity, allergy and inflammatory bowel diseases) are increasing in prevalence in urban communities in high-income countries. One important factor is reduced exposure to immunoregulation-inducing macro- and microorganisms and microbiota that accompanied mammalian evolution (the hygiene hypothesis or 'Old Friends' mechanism). Reduced exposure to these organisms predisposes to poor regulation of inflammation. But inflammation is equally relevant to psychiatric disorders. Inflammatory mediators modulate brain development, cognition and mood, and accompany low socioeconomic status and some cases of depression in developed countries. The risk of all these conditions (chronic inflammatory and psychiatric) is increased in urban versus rural communities, and increased in immigrants, particularly if they move from a low- to a high-income country during infancy, and often the prevalence increases further in second generation immigrants, suggesting that critical exposures modulating disease risk occur during pregnancy and infancy. Diminished exposure to immunoregulation-inducing Old Friends in the perinatal period may enhance the consequences of psychosocial stressors, which induce increased levels of inflammatory mediators, modulate the microbiota and increase the risk for developing all known psychiatric conditions. In later life, the detrimental effects of psychosocial stressors may be exaggerated when the stress occurs against a background of reduced immunoregulation, so that more inflammation (and therefore more psychiatric symptoms) result from any given level of psychosocial stress. This interaction between immunoregulatory deficits and psychosocial stressors may lead to reduced stress resilience in modern urban communities. This concept suggests novel interpretations of recent epidemiology, and novel approaches to the increasing burden of psychiatric disease.

Studies of IL-4 have revealed a wealth of information on the diverse roles of this cytokine in homeostatic regulation and disease pathogenesis. Recent data suggest that instead of simple linear regulatory pathways, IL-4 drives regulation that is full of alternatives. In addition to the well-known dichotomous regulation of Th cell differentiation by IL-4, this cytokine is engaged in several other alternative pathways. Its own production involves alternative mRNA splicing, yielding at least two functional isoforms: full-length IL-4, encoded by the IL-4 gene exons 1-4, and IL-4δ2, encoded by exons 1, 3, and 4. The functional effects of these two isoforms are in some ways similar but in other ways quite distinct. When binding to the surface of target cells, IL-4 may differentially engage two different types of receptors. By acting on macrophages, a cell type critically involved in inflammation, IL-4 induces the so-called alternative macrophage activation. In this review, recent advances in understanding these three IL-4-related branch points--alternative splicing of IL-4, differential receptor engagement by IL-4, and differential regulation of macrophage activation by IL-4--are summarized in light of their contributions to inflammation.

Major depression is common in the context of autoimmune and inflammatory diseases and is frequently associated with persistently raised levels of proinflammatory cytokines and other markers of inflammation, even in the absence of another diagnosable immune pathology to account for these findings. Therefore immunoregulation-inducing vaccines or manipulations of the gut microbiota might prevent or treat depression. These strategies are already undergoing clinical trials for chronic inflammatory disorders, such as allergies, autoimmunity and inflammatory bowel disease. In this article, we summarize data suggesting that this approach might be effective in depression and encourage the initiation of clinical vaccination trials in this disorder., (Copyright © 2012. Published by Elsevier Ltd.)

Throughout the twentieth century, there were striking increases in the incidences of many chronic inflammatory disorders in the rich developed countries. These included autoimmune disorders such as Type 1 diabetes and multiple sclerosis. Although genetics and specific triggering mechanisms such as molecular mimicry and viruses are likely to be involved, the increases have been so rapid that any explanation that omits environmental change is incomplete. This chapter suggests that a series of environmental factors, most of them microbial, have led to a decrease in the efficiency of our immunoregulatory mechanisms because we are in a state of evolved dependence on organisms with which we co-evolved (and that had to be tolerated) as inducers of immunoregulatory circuits. These organisms ("Old Friends") are depleted from the modern urban environment. Rather than considering fetal programming by maternal microbial exposures, neonatal programming, the hygiene hypothesis, gut microbiota, and diet as separate and competing hypotheses, I attempt here to integrate these ideas under a single umbrella concept that can provide the missing immunoregulatory environmental factor that is needed to explain the recent increases in autoimmune disease.

Bronchopulmonary inflammation, such as that associated with asthma, activates afferent neural pathways. We recently demonstrated that localized inflammation in the lungs, induced by intratracheal administration of ovalbumin in ovalbumin-preimmunized mice (an animal model of asthma) results in activation of the dorsolateral part of the nucleus of the solitary tract, a major target of vagal afferent fibers innervating the lungs and airways. Activation of the nucleus of the solitary tract was evident in the absence of activation of the area postrema, a circumventricular organ, consistent with the hypothesis that localized inflammation in the bronchopulmonary system can signal to the central nervous system via specific neural pathways, in the absence of circulating proinflammatory mediators. The pattern of brain activation in ovalbumin-challenged mice differs from the pattern of activation in mice challenged with heat-killed Mycobacterium vaccae, suggesting that qualitative aspects of bronchopulmonary inflammation determine the overall pattern of brain activation. The mechanisms through which localized bronchopulmonary inflammation signals to the central nervous system is poorly understood, but appears to involve both vagal and spinal afferent pathways. In this chapter, we review our current understanding of the anatomical pathways through which localized inflammation in the bronchopulmonary system influences central nervous system function., (Copyright © 2012 S. Karger AG, Basel.)

Clinical, epidemiological and therapeutic studies indicate that some human depression is associated with proinflammatory cytokines, chronic inflammatory disorders, and inflammation-inducing lifestyle factors. Moreover depression can be induced by administration of proinflammatory cytokines, including IL-2 or IFN-α. However, recent studies in specific pathogen-free (SPF) rodents suggest a different--and potentially contradictory--relationship between immune processes and mental health. These studies suggest that effector T cells specific for central nervous system (CNS) antigens can assist recovery from an array of environmental insults ranging from nerve injury to psychological stress, while in contrast, regulatory T cells (Treg) oppose such recovery. Indeed, some reported effects of this so-called "protective autoimmunity" seem of direct relevance to depressive disorders. These findings pose a dilemma for those intending to manipulate inflammatory pathways as a treatment for depression. Should we administer anti-inflammatory treatments, or should we induce self-reactive T cells? We re-examine the rodent findings and outline immunological peculiarities of SPF rodents, the abnormal properties of their regulatory T cells, and the impact of gut microbiota. We find that "protective autoimmunity" is likely to be relevant only to very clean SPF animals that lack normal levels of activated T cells, CNS T cell traffic and mature Treg. The data indicate that even in SPF models the effectors of beneficial effects are not the proinflammatory autoimmune cells themselves, but rather unidentified regulatory cells. This reinterpretation of findings relevant to "protective autoimmunity" suggests that ongoing, and planned, clinical trials of anti-inflammatory strategies to treat depressive disorders are justified., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The role of Th2 cytokines and Th2-associated chemokines in tuberculosis (TB) remains controversial, though in Mexico a polymorphism causing increased production of CCL2 is a risk factor. We studied levels of the Th2-associated chemokines CCL2 and CCL18, circulating soluble IL-4 receptors (sIL-4R), IL-4 and the inhibitory splice variant of IL-4 (IL-4δ2) in a cohort of patients with pulmonary TB and their healthy contacts. These were followed for 2 years during which time 10 contacts developed pulmonary TB. Results were compared with measurements made in renal and meningeal TB, and in disease controls with bacterial pneumonias or Dengue fever that have large Th2 components. In these disease controls both chemokines were significantly raised. They were also very significantly raised in all forms of TB, irrespective of age or disease site. Levels of CCL18 were raised least in meningeal TB, and most in pulmonary patients with long histories, when levels were similar to those in disease controls. Levels of CCL2, although also raised in all three forms of TB, were negatively correlated with CCL18. We found that levels of sIL-4R were strikingly reduced in all forms of TB, particularly meningeal. Contacts who progressed could not be distinguished from contacts who remained healthy at 2 years in terms of IL-4, sIL-4R, CCL2 or CCL18. However contacts had raised expression of IL-4δ2 as previously found. These results indicate vigorous and previously unrecorded activity within the Th2 axis, and further investigation is warranted., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As man has moved rapidly from the hunter-gatherer environment to the living conditions of the industrialized countries, the incidences of some cancers have increased alarmingly. Recent increases are usually attributed to dietary changes or to altered exposures to putative carcinogens associated with the modern lifestyle. However, the changes in cancer incidence parallel similar increases in non-neoplastic chronic inflammatory disorders (inflammatory bowel disease, allergies, and autoimmunity), and the epidemiology is often strikingly similar. This parallel is worth exploring, because the increases in chronic inflammatory disorders are at least partly explained by immunoregulatory defects resulting from diminished exposure to microorganisms that co-evolved with mammals and developed a role in driving immunoregulatory circuits (the hygiene hypothesis). Dysregulated chronic inflammation can drive oncogenesis and also provides growth and angiogenic factors that enhance the subsequent proliferation and spread of tumor cells. Thus, a modern failure to downregulate inappropriate inflammation could underlie increases in some cancers in parallel with the increases in chronic inflammatory disorders. This possibility is supported by recent work showing that in some circumstances regulatory T cells protect against cancer, rather than aggravating it, as previously assumed. A greater understanding of these interactions might pave the way to improved microbe-based immunotherapies., (© 2011 John Wiley & Sons A/S.)

The current 'Darwinian' synthesis of the hygiene (or 'Old Friends') hypothesis suggests that the increase in chronic inflammatory disorders that started in Europe in the mid-19th century and progressed until the late 20th century is at least partly attributable to immunodysregulation resulting from lack of exposure to microorganisms that were tasked by co-evolutionary processes with establishing the 'normal' background levels of immunoregulation, a role that they perform in concert with the normal microbiota. This is an example of 'evolved dependence'. The relevant organisms co-evolved with mammals, already accompanied early hominids in the Paleolithic era and are associated with animals, mud and faeces. These organisms often establish stable carrier states, or are encountered continuously in primitive environments as 'pseudocommensals' from mud and water. These organisms were not lost during the first epidemiological transition, which might even have resulted in increased exposure to them. However, the crucial organisms are lost progressively as populations undergo the second epidemiological transition (modern urban environment). Recently evolved sporadic 'childhood infections' are not likely to have evolved immunoregulatory roles, and epidemiology supports this contention. The consequences of the loss of the Old Friends and distortion of the microbiota are aggravated by other modern environmental changes that also lead to enhanced inflammatory responses (obesity, vitamin D deficiency, pollution (dioxins), etc.). The range of chronic inflammatory disorders affected may be larger than had been assumed (allergies, autoimmunity, inflammatory bowel disease, but also coeliac disease, food allergy, vascular disease, some cancers, and depression/anxiety when accompanied by raised inflammatory cytokines)., (Copyright © 2011 S. Karger AG, Basel.)

Context: Inflammation is increasingly recognized as contributing to the pathogenesis of major depressive disorder (MDD), even in individuals who are otherwise medically healthy. Most studies in search of sources for this increased inflammation have focused on factors such as psychosocial stress and obesity that are known to activate inflammatory processes and increase the risk for depression. However, MDD may be so prevalent in the modern world not just because proinflammatory factors are widespread, but also because we have lost contact with previously available sources of anti-inflammatory, immunoregulatory signaling., Objective: To examine evidence that disruptions in coevolved relationships with a variety of tolerogenic microorganisms that were previously ubiquitous in soil, food, and the gut, but that are largely missing from industrialized societies, may contribute to increasing rates of MDD in the modern world., Data Sources: Relevant studies were identified using PubMed and Ovid MEDLINE., Study Selection: Included were laboratory animal and human studies relevant to immune functioning, the hygiene hypothesis, and major depressive disorder identified via PubMed and Ovid MEDLINE searches., Data Extraction: Studies were reviewed by all authors, and data considered to be potentially relevant to the contribution of hygiene-related immune variables to major depressive disorder were extracted., Data Synthesis: Significant data suggest that a variety of microorganisms (frequently referred to as the "old friends") were tasked by coevolutionary processes with training the human immune system to tolerate a wide array of non-threatening but potentially proinflammatory stimuli. Lacking such immune training, vulnerable individuals in the modern world are at significantly increased risk of mounting inappropriate inflammatory attacks on harmless environmental antigens (leading to asthma), benign food contents and commensals in the gut (leading to inflammatory bowel disease), or self-antigens (leading to any of a host of autoimmune diseases). Loss of exposure to the old friends may promote MDD by increasing background levels of depressogenic cytokines and may predispose vulnerable individuals in industrialized societies to mount inappropriately aggressive inflammatory responses to psychosocial stressors, again leading to increased rates of depression., Conclusion: Measured exposure to the old friends or their antigens may offer promise for the prevention and treatment of MDD in modern industrialized societies.

The current synthesis of the 'hygiene hypothesis' suggests that the recent increase in chronic inflammatory disorders is at least partly attributable to immunodysregulation resulting from lack of exposure to microorganisms that have evolved an essential role in the establishment of the immune system. This document provides a background for discussion of the following propositions. 1. The essential role of these organisms is an example of 'evolved dependence'. 2. The most relevant organisms are those that co-evolved with mammals, and already accompanied early hominids in the Paleolithic. 3. More recently evolved 'childhood infections' are not likely to have evolved this role, and recent epidemiology supports this contention. 4. This mechanism is interacting with other modern environmental changes that also lead to enhanced inflammatory responses [inappropriate diet, obesity, psychological stress, vitamin D deficiency, pollution (dioxins), etc.]. 5. The range of chronic inflammatory disorders that is affected is potentially larger than usually assumed [allergies, autoimmunity, inflammatory bowel disease, but also vascular disease, some cancers, depression/anxiety (when accompanied by raised inflammatory cytokines), and perhaps neurodegenerative disorders and type 2 diabetes].

Immunotherapies have the potential to improve the outcome in all patients with tuberculosis (TB) including those with multidrug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB. Immunotherapy for TB may shorten duration of treatment and reduce pathology in individuals cured by chemotherapy, potentially preventing recurrence. Currently none of the available candidate agents have proof of efficacy for use in MDR-TB or XDR-TB. Further development and evaluation of existing immunotherapeutic agents is required to identify an effective agent that can be used adjunctively with chemotherapy to improve treatment outcomes for drug-susceptible TB, MDR-TB, and XDR-TB. With a range of potential immunotherapeutics, some of which have been produced to good manufacturing practice (GMP) standards and are registered for other indications in humans, the immunotherapy option should no longer be ignored.

Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.

The currently available vaccine for tuberculosis (TB) is ineffective in developing countries. We need to understand the pathogenesis of TB in those countries and how it differs from the pathogenesis of TB in wealthy countries, to facilitate the design and interpretation of clinical trials of new vaccine candidates that are now available. We show here that these geographical differences parallel the strikingly different immunology and bacterial growth curves seen in animal models after high-dose and low-dose challenge with M. tuberculosis (Mtb). We consider this point in the light of recent insights into the multiple pathways used by the immune response to control M. tuberculosis and the susceptibilities of these pathways to regulation and suppression. There are important implications for the screening, testing, and likely success of vaccine candidates.

Man has moved rapidly from the hunter-gatherer environment to the living conditions of the rich industrialized countries. The hygiene hypothesis suggests that the resulting changed and reduced pattern of exposure to microorganisms has led to disordered regulation of the immune system, and hence to increases in certain inflammatory disorders. The concept began with the allergic disorders, but there are now good reasons for extending it to autoimmunity, inflammatory bowel disease, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers. This review discusses these possibilities in the context of Darwinian medicine, which uses knowledge of evolution to cast light on human diseases. The Darwinian approach enables one to correctly identify some of the organisms that are important for the 'Hygiene' or 'Old Friends' hypothesis, and to point to the potential exploitation of these organisms or their components in novel types of prophylaxis with applications in several branches of medicine.

The environmental saprophyte Mycobacterium vaccae induces a Th1 response and cytotoxic T cells that recognize M. tuberculosis, and by subcutaneous injection, it is therapeutic for pulmonary tuberculosis (TB) induced by high-dose challenge in BALB/c mice. However, M. vaccae also drives regulatory T cells that inhibit Th2 responses, and this is seen in allergy models, not only following subcutaneous injection but also after oral administration. An oral immunotherapeutic for TB would be clinically useful, so we investigated M. vaccae given orally by gavage at 28-day intervals in the TB model. We used two different protocols: starting the oral M. vaccae either 1 day before or 32 days after infection with M. tuberculosis. Throughout the infection (until 120 days), we monitored outcome (CFU), molecules involved in the development of immunoregulation (Foxp3, hemoxygenase 1, idoleamine 2,3-dioxygenase, and transforming growth factor beta [TGF-beta]), and indicators of cytokine balance (tumor necrosis factor, inducible nitric oxide synthase, interleukin-4 [IL-4], and IL-4delta2; an inhibitory splice variant of IL-4 associated with improved outcome in human TB). Oral M. vaccae had a significant effect on CFU and led to increased expression of Th1 markers and of IL-4delta2, while suppressing IL-4, Foxp3, and TGF-beta. When administered 1 day before infection, oral M. vaccae induced a striking peak of expression of hemoxygenase 1. In conclusion, we show novel information about the expression in TB of murine IL-4delta2 and molecules involved in immunoregulation and show that these can be modulated by oral administration of a saprophytic mycobacterium. A clinical trial of oral M. vaccae in extensively drug-resistant TB might be justified.

: There is much to be gained from examining human diseases within the expanding framework of Darwinian medicine. This is particularly true of those conditions that change in frequency as populations develop from the human "environment of evolutionary adaptedness" to the living conditions of the rich industrialized countries. This development entails major changes in lifestyle, leading to reductions in contact with environmental microorganisms and helminths that have evolved a physiologic role as drivers of immunoregulatory circuits. It is suggested that a deficit in immunoregulation in rich countries is contributing not only to increases in the incidence of allergic disorders but also to increases in other chronic inflammatory conditions that are exacerbated by a failure to terminate inappropriate inflammatory reponses. These include autoimmunity, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers.

The hygiene hypothesis proposes that several chronic inflammatory disorders (allergies, autoimmunity, inflammatory bowel disease) are increasing in prevalence in developed countries because a changing microbial environment has perturbed immunoregulatory circuits which normally terminate inflammatory responses. Some stress-related psychiatric disorders, particularly depression and anxiety, are associated with markers of ongoing inflammation, even without any accompanying inflammatory disorder. Moreover, pro-inflammatory cytokines can induce depression, which is commonly seen in patients treated with interleukin-2 or interferon-alpha. Therefore, some psychiatric disorders in developed countries might be attributable to failure of immunoregulatory circuits to terminate ongoing inflammatory responses. This is discussed in relation to the effects of the immune system on a specific group of brain serotonergic neurons involved in the pathophysiology of mood disorders.

The 'Hygiene' or 'Old Friends' hypothesis suggests that increases in chronic inflammatory disorders (allergies, inflammatory bowel disease and autoimmunity) in developed countries are partly attributable to diminishing exposure to organisms that were part of mammalian evolutionary history. Crucial organisms, including helminths and saprophytic mycobacteria, are recognised by the innate immune system as harmless or, in the case of helminths, as organisms that once established must be tolerated. This recognition then triggers development of regulatory dendritic cells that drive regulatory T-cell responses to the 'Old Friends' themselves and to simultaneously processed 'forbidden' target antigens of the chronic inflammatory disorders.

Background: NOD2, an intracellular pathogen recognition sensor, modulates innate defences to muropeptides derived from various bacterial species, including Mycobacterium tuberculosis (MTB). Experimentally, NOD2 attenuates two key putative mycobactericidal mechanisms. TNF-alpha synthesis is markedly reduced in MTB-antigen stimulated-mononuclear cells expressing mutant NOD2 proteins. NOD2 agonists also induce resistance to apoptosis, and may thus facilitate the survival of MTB in infected macrophages. To further define a role for NOD2 in disease pathogenesis, we analysed NOD2 transcriptional responses in pulmonary leucocytes and mononuclear cells harvested from patients with pulmonary tuberculosis (PTB)., Methods: We analysed NOD2 mRNA expression by real-time polymerase chain-reaction in alveolar lavage cells obtained from 15 patients with pulmonary tuberculosis and their matched controls. We compared NOD2 transcriptional responses, in peripheral leucocytes, before and after anti-tuberculous treatment in 10 patients. In vitro, we measured NOD2 mRNA levels in MTB-antigen stimulated-mononuclear cells., Results: No significant differences in NOD2 transcriptional responses were detected in patients and controls. In some patients, however, NOD2 expression was markedly increased and correlated with toll-like-receptor 2 and 4 expression. In whole blood, NOD2 mRNA levels increased significantly after completion of anti-tuberculosis treatment. NOD2 expression levels did not change significantly in mononuclear cells stimulated with mycobacterial antigens in vitro., Conclusion: There are no characteristic NOD2 transcriptional responses in PTB. Nonetheless, the increased levels of NOD2 expression in some patients with severe tuberculosis, and the increases in expression levels within peripheral leucocytes following treatment merit further studies in selected patient and control populations.

Background: Data about T cell antigen-specific (ESAT-6 and CFP-10) IFN-gamma release assays (IGRAs) during and after completion of anti-tuberculous (TB) treatment are limited and highly discordant. Thus, the utility of IGRAs as a surrogate marker of mycobacterial burden remain unclear., Methods: To investigate factors that modulate IGRA responses during anti-TB treatment we used a standardised assay (T-SPOT.TB) in 33 patients with culture positive tuberculosis., Results: Significantly more patients in the early (< or = 4 months of anti-TB treatment) rather than the late phase (> 4 months or completed anti-TB treatment) had positive IGRA responses [10/12 (83%) vs 4/21 (19%); p < or = 0.01]. Thus, 17/21 (81%) in the late phase or who had completed treatment (mean duration of treatment = 8.7 months) were IGRA negative, despite having robust antigen-specific recall proliferative responses. In these 17 patients prolonged incubation (5 days vs overnight), use of different antigen preparations (protein vs peptide) and addition of endotoxin, failed to elicit positive responses., Conclusions: In treated TB patients the discordant IGRA data remain unexplained by variation in laboratory protocols and are more likely due to host or environmental factors. In a low burden setting IGRAs may be a promising surrogate marker of mycobacterial disease burden.

The increasing threat posed by drug-resistant strains of M. tuberculosis is leading to a reappraisal of the possibility of treating tuberculosis (TB) by immunotherapy. We analyze 6 strategies that have been shown to be therapeutic in animal models of TB and identify a common pathway underlying the activity of the superficially different immunotherapeutic protocols. This pathway involves enhanced induction of CD8(+) cytotoxic T lymphocytes (CTLs) and down-regulation of interleukin-4 and transforming growth factor- beta , leading to further enhancement of the activity of CD8(+) CTLs and of other microbicidal pathways. This unifying analysis strengthens the rationale for future trials of immunotherapy in humans and points to surrogate markers that could be studied in such trials.

Regulatory T lymphocytes (T(regs)) that express FOXP3 are involved in the beneficial attenuation of immunopathology, but are also implicated in down-regulation of protective responses to infection. Their role in tuberculosis (TB) is unknown. We classified 1272 healthy TB contacts according to their tuberculin skin test (TST) and interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) results and 128 TB cases, and studied the expression of FOXP3 and interleukin (IL)-10 in blood samples. Compared to the uninfected contact group (TST(-), ELISPOT(-)), we observed higher levels of FOXP3 mRNA in blood from TB patients (< 0.001), but IL-10 expression was slightly lower (P = 0.04). In contrast, FOXP3 expression levels were significantly lower (P = 0.001) in the recently infected contacts (TST(+), ELISPOT(+)) but there was no difference for IL-10 (P = 0.74). We hypothesize that during early/subclinical TB, most of which will become latent, FOXP3(+) T(regs) may be sequestered in the lungs, but when TB becomes progressive, FOXP3 reappears at increased levels in the periphery. While these findings do not reveal the role, beneficial or harmful, of T(regs) in TB, they emphasize the probable importance of these cells.

Peripheral immune activation can have profound physiological and behavioral effects including induction of fever and sickness behavior. One mechanism through which immune activation or immunomodulation may affect physiology and behavior is via actions on brainstem neuromodulatory systems, such as serotonergic systems. We have found that peripheral immune activation with antigens derived from the nonpathogenic, saprophytic bacterium, Mycobacterium vaccae, activated a specific subset of serotonergic neurons in the interfascicular part of the dorsal raphe nucleus (DRI) of mice, as measured by quantification of c-Fos expression following intratracheal (12 h) or s.c. (6 h) administration of heat-killed, ultrasonically disrupted M. vaccae, or heat-killed, intact M. vaccae, respectively. These effects were apparent after immune activation by M. vaccae or its components but not by ovalbumin, which induces a qualitatively different immune response. The effects of immune activation were associated with increases in serotonin metabolism within the ventromedial prefrontal cortex, consistent with an effect of immune activation on mesolimbocortical serotonergic systems. The effects of M. vaccae administration on serotonergic systems were temporally associated with reductions in immobility in the forced swim test, consistent with the hypothesis that the stimulation of mesolimbocortical serotonergic systems by peripheral immune activation alters stress-related emotional behavior. These findings suggest that the immune-responsive subpopulation of serotonergic neurons in the DRI is likely to play an important role in the neural mechanisms underlying regulation of the physiological and pathophysiological responses to both acute and chronic immune activation, including regulation of mood during health and disease states. Together with previous studies, these findings also raise the possibility that immune stimulation activates a functionally and anatomically distinct subset of serotonergic neurons, different from the subset of serotonergic neurons activated by anxiogenic stimuli or uncontrollable stressors. Consequently, selective activation of specific subsets of serotonergic neurons may have distinct behavioral outcomes.

The prototype Th2 cytokine IL-4, and its competitive antagonist IL-4delta2, may be important determinants of outcome in human tuberculosis (TB). However, there are no data on how gene expression of these cytokines is regulated. To evaluate this the stability of IL-4 and IL-4delta2 mRNA after the addition of actinomycin-D, was evaluated in whole blood from subjects with pulmonary TB and uninfected healthy volunteers. The Th2/Th1 (IL-4/IFN-gamma) mRNA ratio in unstimulated cells in whole blood was significantly greater in TB subjects than in controls (p<0.05). The mRNA half-life of the agonist (IL-4), but not the antagonist (IL-4delta2), was significantly prolonged in subjects with TB compared to healthy volunteers ( approximately 5-fold, p=0.0016), and the IL-4/IL-4delta2 ratio was higher in TB patients compared to controls (p<0.05). The differential stability of the Th2 agonist, IL-4, compared to the antagonist IL-4delta2, represents a hitherto undescribed post-transcriptional regulatory mechanism that may modulate the polarisation of Th1/Th2 responses in human TB.

We need to understand what is different about susceptibility to tuberculosis (TB) in developing countries where most TB occurs, and where the current vaccine, Bacillus Calmette et Guérin (BCG) usually fails to protect. The presence of a background mixed IFN-gamma and Th2 response to mycobacterial antigens before infection with M. tuberculosis (Mtb), and the development of a large IL-4 response during progressive TB, are characteristics of individuals in the locations where BCG fails, which are also seen in animal models in the same countries. Recent data suggest that the background Th1 component in developing countries protects from low dose challenge with Mtb in mouse and man, but that following high dose challenge the pre-existing IL-4 component increases and blocks immunity unless the individual's immune system releases IL-4delta2, an antagonist of IL-4, which is raised in the blood of donors with stable latent TB. We outline how IL-4 (and IL-13) can undermine Th1-mediated immunity and drive inappropriate alternative activation of macrophages. The mechanisms of the effects of IL-4 include impaired antimicrobial activity due to reduced TNF-alpha-mediated apoptosis of infected cells, reduced activity of iNOS, increased availability of iron to intracellular Mtb, and increased proliferation of antigen-specific FOXP-3+ regulatory T cells. IL-4 also increases the toxicity of TNF-alpha and drives pulmonary fibrosis, thus enhancing immunopathology. The conclusion is that a vaccine that will work in developing countries might need to do more than enhance the existing Th1 response. In these environments it might be more important to block the Th2 component.

Exposure to mycobacteria was inevitable throughout mammalian evolution. Most mycobacteria are saprophytic environmental organisms that are enormously abundant in soil and untreated water and evoke immune responses in the residents of developing countries. A few species are pathogens. For example Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 1/3 of the world's population. Many individuals also receive vaccination with the Bacille Calmette Guérin (BCG), which is an attenuated form of the organism causing bovine TB. In order to understand the possible role that mycobacteria might have in the increases in allergic disorders over the last decades, it is necessary to dissect out these different mycobacterial influences. Above all it is essential, when analysing tuberculin test results, to distinguish between individuals who have latent TB and those who do not. Only then can probable effects of diverse types of exposure emerge. There is no doubt that in animal models mycobacteria can both prevent and treat allergic responses either by boosting Th1 or by driving allergen-specific regulatory T cells (RegT). Clinical trials in man remain inconclusive.

Background: The hygiene hypothesis is often proposed to explain the high prevalence of atopy in the western world. Dysregulation of the immune system may result from inadequate exposure to micro-organisms such as mycobacteria. A small trial suggested that a killed extract of Mycobacterium vaccae ameliorates atopic dermatitis (AD)., Objectives: To confirm in a large clinical trial whether killed M. vaccae ameliorates AD in 5-16-year-old children., Methods: This was a randomized, placebo-controlled, double-blind, multi-centre study of the effect of intradermal injection of killed M. vaccae (0.1 or 1 mg) on patients, aged 5-16, with moderate-to-severe AD. Patients were followed up for 24 weeks. The primary end point was the change in severity of AD at 12 weeks, assessed using the six area, six-sign, atopic dermatitis (SASSAD) score. Secondary end points included changes in disease extent, patient's global assessment and children's dermatology life quality index., Results: There were 166 patients randomized. The mean SASSAD score fell to a similar degree at week 12 in all treatment arms: from 33 to 24, (26%) in the high-dose group, from 30 to 23 (25%) in the low-dose group and from 36 to 27 (24%) in the placebo group (P>0.05). Secondary end points followed the same trend. Adverse events were generally those expected to occur in this population. Injection site reactions occurred in 32 patients at week 4., Conclusions: M. vaccae was no more effective than the placebo in ameliorating the severity of AD.

The metabolism of the amino acid L-tryptophan is a highly regulated physiological process leading to the generation of several neuroactive compounds within the central nervous system. These include the aminergic neurotransmitter serotonin (5-hydroxytryptamine, 5-HT), products of the kynurenine pathway of tryptophan metabolism (including 3-hydroxykynurenine, 3-hydroxyanthranilic acid, quinolinic acid and kynurenic acid), the neurohormone melatonin, several neuroactive kynuramine metabolites of melatonin, and the trace amine tryptamine. The integral role of central serotonergic systems in the modulation of physiology and behaviour has been well documented since the first description of serotonergic neurons in the brain some 40 years ago. However, while the significance of the peripheral kynurenine pathway of tryptophan metabolism has also been recognised for several decades, it has only recently been appreciated that the synthesis of kynurenines within the central nervous system has important consequences for physiology and behaviour. Altered kynurenine metabolism has been implicated in the pathophysiology of conditions such as acquired immunodeficiency syndrome (AIDS)-related dementia, Huntington's disease and Alzheimer's disease. In this review we discuss the molecular mechanisms involved in regulating the metabolism of tryptophan and consider the medical implications associated with dysregulation of both serotonergic and kynurenine pathways of tryptophan metabolism.

When IL-4 mRNA was distinguished from mRNA encoding its antagonist, the splice variant IL-4delta2, it was found to correlate directly with expression of TLR2 in fresh peripheral blood mononuclear cells (PBMCs) from normal donors (p=0.0013). Similarly IL-4 mRNA was high when TLR2 mRNA was abundant compared to levels of mRNA encoding its heterodimerisation partners TLR1 (p=0.0007) or TLR6 (p=0.0007). IL-4delta2 tended to show the reverse effect; IL-4delta2 mRNA was high when TLR2 was low relative to TLR1 (p=0.001). When subpopulations of the PBMCs were examined these relationships were found to be restricted to the CD3+ cells. The CD3+ cell population from 5 of 10 donors had detectable TLR2 mRNA. When levels of TLR1, IL-4 and IFN-gamma mRNA were assayed in the TLR2(low) and TLR2(high) CD3+ cells, it was found that IL-4 mRNA was restricted to the TLR2(high) T cells (p=0.007) while TLR1 was higher in the TLR2(low) T cells (p=0.015). IFN-gamma was also somewhat increased in the TLR2(low) (ns). None of these correlations with TLR mRNA expression levels were found in similar samples from tuberculosis patients, or when similar analyses were performed with data for IL-10 mRNA in cells from the same donors. We conclude that in T cell populations from normal donors, expression of IL-4 (but not of its antagonist, IL-4delta2, or of IL-10) is associated with high TLR2 and low TLR1.

In industrialized countries the incidence of diseases caused by immune dysregulation has risen. Epidemiologic studies initially suggested this was connected to a reduction in the incidence of infectious diseases; however, an association with defects in immunoregulation is now being recognized. Effector T(H)1 and T(H)2 cells are controlled by specialized subsets of regulatory T cells. Some pathogens can induce regulatory cells to evade immune elimination, but regulatory pathways are homeostatic and mainly triggered by harmless microorganisms. Helminths, saprophytic mycobacteria, bifidobacteria and lactobacilli, which induce immunoregulatory mechanisms in the host, ameliorate aberrant immune responses in the setting of allergy and inflammatory bowel disease. These organisms cause little, if any, harm, and have been part of human microecology for millennia; however, they are now less frequent or even absent in the human environment of westernized societies. Deficient exposure to these 'old friends' might explain the increase in immunodysregulatory disorders. The use of probiotics, prebiotics, helminths or microbe-derived immunoregulatory vaccines might, therefore, become a valuable approach to disease prevention.

New vaccine candidates for tuberculosis are beginning to enter clinical trials. In this review we discuss issues surrounding the design of these candidates, and the way they were screened in animal models. First, screening vaccines for their ability to attenuate inevitably fatal tuberculosis in immunologically naïve mice might be leading to the selection of inappropriate candidates. We need to screen vaccines for their ability to stop the development of progressive disease, since this is what they must achieve in man. A solution to this problem is proposed. Secondly, we point out that some mouse models of tuberculosis in laboratories in developing countries, where exposure to environmental mycobacteria is large, mimic neglected aspects of human disease more closely than do low-dose infections in hyper-susceptible immunologically naïve mice in the USA or Europe. We need to think more about geographical differences in immunological experience, and these mouse models can help us. Thirdly, we conclude that in developing countries where BCG fails this is not because there is too little Th1 response, but rather because the Th1 response is rendered ineffective and immunopathological by other subversive mechanisms, including IL-4 responses and inappropriate regulatory T cell function. Therefore, we suggest that vaccines that will work in those countries might need to have immunoregulatory properties that can switch off pre-existing subversive mechanisms, and block their development in the future. The development of such vaccines, that might work where BCG does not, will require a greater understanding of the roles of the many types of regulatory T cell in tuberculosis.

There is increasing evidence of a link between tuberculosis and smoking. This paper reviews the epidemiological evidence from the UK, China, India and the USA, summarizing some of the main papers which indicate an association. Where an association has been found there seems to be an increase in tuberculosis case rates of between two- and four-fold for those smoking in excess of 20 cigarettes a day, but it may be difficult to control for other factors, particularly alcohol consumption. The final part of the paper reviews possible mechanisms. A likely possibility is that nicotine turns off the production of TNF-alpha by the macrophages in the lungs, rendering the patient more susceptible to the development of progressive disease from latent Mycobacterium tuberculosis infection.