Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.

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.

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.

Plants, animals and humans, are colonized by microorganisms (microbiota) and transiently exposed to countless others. The microbiota affects the development and function of essentially all organ systems, and contributes to adaptation and evolution, while protecting against pathogenic microorganisms and toxins. Genetics and lifestyle factors, including diet, antibiotics and other drugs, and exposure to the natural environment, affect the composition of the microbiota, which influences host health through modulation of interrelated physiological systems. These include immune system development and regulation, metabolic and endocrine pathways, brain function and epigenetic modification of the genome. Importantly, parental microbiotas have transgenerational impacts on the health of progeny. Humans, animals and plants share similar relationships with microbes. Research paradigms from humans and other mammals, amphibians, insects, planktonic crustaceans and plants demonstrate the influence of environmental microbial ecosystems on the microbiota and health of organisms, and indicate links between environmental and internal microbial diversity and good health. Therefore, overlapping compositions, and interconnected roles of microbes in human, animal and plant health should be considered within the broader context of terrestrial and aquatic microbial ecosystems that are challenged by the human lifestyle and by agricultural and industrial activities. Here, we propose research priorities and organizational, educational and administrative measures that will help to identify safe microbe-associated health-promoting modalities and practices. In the spirit of an expanding version of "One health" that includes environmental health and its relation to human cultures and habits (EcoHealth), we urge that the lifestyle-microbiota-human health nexus be taken into account in societal decision making.

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.anxiety | chronic psychosocial stress | fear | microbiota | posttraumatic stress disorder