In medicine, the hygiene hypothesis is a hypothesis that states that a lack of early childhood exposure to infectious agents, symbiotic microorganisms (e.g., gut flora or probiotics), and parasites increases susceptibility to allergic diseases by suppressing the natural development of the immune system.
The original formulation of the hygiene hypothesis dates from 1989 when Strachan proposed that lower incidence of infection in early childhood could be an explanation for the rapid 20th century rise in allergic diseases such as asthma and hay fever. Over the last 2 decades researchers have recognised that the concept of a link between microbial exposure and allergy is probably correct, but the idea that children who have more infections or are less hygienic are less likely to develop allergies is wide of the mark.
It is now also recognised that the "reduced microbial exposure" concept applies to a much broader range of chronic inflammatory diseases (CIDs) than asthma and hay fever, which includes diseases such as type 1 diabetes and multiple sclerosis, and also some types of depression and cancer.
In 2003 Graham Rook proposed the "Old Friends Hypothesis" which seems to offer a more rational explanation for the link between microbial exposure and inflammatory disorders. He argues that the vital microbial exposures are not colds, influenza, measles and other common childhood infections which have evolved relatively recently over the last 10,000 years, but rather the microbes already present during mammalian and human evolution, that could persist in small hunter gatherer groups as microbiota, tolerated latent infections or carrier states. He proposes that we have become so dependent on these Old Friends that our immune systems neither develop properly nor function properly without them.
Strachan’s original formulation of the hygiene hypothesis also centred around the idea that smaller families provided insufficient microbial exposure partly because of less person to person spread of infections, but also because of "improved household amenities and higher standards of personal cleanliness". It seems likely that this was the reason he named it the "hygiene" hypothesis". Although the "hygiene revolution" of the nineteenth and twentieth centuries may have been a major factor, it now seems more likely that, although public health measures such as sanitation, potable water and garbage collection were instrumental in reducing our exposure to cholera, typhoid and so on, they also deprived us of our exposure to the "old friends" that occupy the same environmental habitats.
Some evidence indicates that autism is correlated to factors (such as certain cytokines) that are indicative of an immune disease. One publication speculated that the lack of early childhood exposure could be a cause of autism.
The risk of CIDs also depends on factors such as diet, pollution, physical activity, obesity, socio-economic factors and stress. Genetic predisposition is also a factor.
Although the idea that exposure to certain infections may decrease the risk of allergy is not new, Strachan was one of the first to formally propose it, in an article published in the British Medical Journal (now the BMJ), in 1989. This article proposed to explain the observation that hay fever and eczema, both allergic diseases, were less common in children from larger families, which were presumably exposed to more infectious agents through their siblings, than in children from families with only one child.
The hypothesis was extensively investigated by immunologists and epidemiologists and has become an important theoretical framework for the study of chronic inflammatory disorders. It explains the increase in allergic diseases that has been seen since industrialization and the higher incidence of allergic diseases in more developed countries. Epidemiological studies continue to confirm the protective effect of large family size and of growing up on a farm. However, exposure to common childhood infections such as chickenpox or measles is not thought to be protective.
The "Old Friends" hypothesis proposed in 2003 may offer a better explanation for the link between microbial exposure and inflammatory diseases. This hypothesis argues that the vital exposures are not common childhood and other recently-evolved infections, over the last 10,000 years, but rather microbes already present in hunter-gatherer times when the human immune system was evolving. Conventional childhood infections are mostly "crowd infections" that kill or immunise and thus cannot persist in isolated hunter-gatherer groups. Crowd infections started to appear after the neolithic agricultural revolution, when human populations increased in size and proximity. The microbes that co-evolved with mammalian immune systems are much more ancient. Humans became so dependent on them that their immune systems can neither develop nor function properly without them.
Rook proposed that these microbes most likely include:
Ambient species that exist in the same environments as humans
Species that inhabit human skin, gut and respiratory tract, and that of the animals we live with
Organisms such as viruses and helminths (worms) that establish chronic infections or carrier states that humans can tolerate and so could co-evolve a specific immunoregulatory relationship with the immune system.
The modified hypothesis later expanded to include exposure to symbiotic bacteria and parasites.
The "microbial diversity" hypothesis, proposed by Paolo Matricardi and developed by von Hertzen, holds that diversity and turnover of bacterial species in the gut mucosa and other sites is a key factor for priming and regulating the immune system, rather than stable colonisation with a particular species. It is not clear whether diversity per se, or that a diverse population will include certain organisms without which the immune system fails to develop. Rook likened the embryonic immune system to a computer that contains programmes but little data. During gestation and infancy exposure to diverse organisms builds a "database" that allows the immune system to identify and respond to harmful agents and normalize once the danger is eliminated.
For allergic disease, the most important times for exposure are: early in development; later during pregnancy; and the first few days or months of infancy. Exposure needs to be maintained over a significant period. This fits with evidence that delivery by Caesarean section may be associated with increased allergies, whilst breastfeeding can be protective. The extent to which exposures need to be maintained after infancy and whether these conditions could be managed by on-going exposure is as yet unknown.
Allergic conditions are caused by inappropriate immunological responses to harmless antigens driven by a TH2-mediated immune response. Many bacteria and viruses elicit a TH1-mediated immune response, which down-regulates TH2 responses. The mechanism of action of the hygiene hypothesis was insufficient stimulation of the TH1 arm, stimulating the cell defence of the immune system and leading to an overactive TH2 arm, stimulating the antibody-mediated immunity of the immune systems, which in turn led to allergic disease.
An alternative explanation is that the developing immune system must receive stimuli (from infectious agents, symbiotic bacteria, or parasites) to adequately develop regulatory T cells. Without that stimuli it becomes more susceptible to autoimmune diseases and allergic diseases, because of insufficiently repressed TH1 and TH2 responses, respectively. For example, all chronic inflammatory disorders show evidence of failed immunoregulation. Secondly, helminths, non-pathogenic ambient pseudocommensal bacteria or certain gut commensals and probiotics) drive immunoregulation. They block or treat models of all chronic inflammatory conditions. Thirdly, some such organisms (or molecules that they secrete), specifically expand populations of regulatory T cells (Treg), or cause dendritic cells (DC) to switch to regulatory forms that preferentially drive immunoregulation. Finally, when multiple sclerosis patients become infected with helminths, the disease stops progressing and circulating myelin-recognising regulatory T cells (Treg) appear in the peripheral blood. This indicates that helminths act as Treg adjuvants. This observation led to clinical trials.
The hygiene hypothesis is supported by epidemiological data. Studies have shown that various immunological and autoimmune diseases are much less common in the developing world than the industrialized world and that immigrants to the industrialized world from the developing world increasingly develop immunological disorders in relation to the length of time since arrival in the industrialized world. This is true for asthma and other chronic inflammatory disorders.
In developed countries where childhood diseases were eliminated, the asthma rate for youth is approximately 10%. In the 19th century, hay-fever, an easily recognisable allergy, was a very rare condition.
Longitudinal studies in Ghana demonstrate an increase in immunological disorders as it grew more affluent and presumably cleaner. The use of antibiotics in the first year of life has been linked to asthma and other allergic diseases. The use of antibacterial cleaning products has also been associated with higher incidence of asthma. Increased asthma rates are associated with birth by Caesarean section. The data supporting links to antibiotic use and caesarean section (but not to antibacterial use) are rapidly strengthening.
Antibiotic usage, which reduces the diversity of gut microbiota, is another cited factor. Although several studies have shown associations between antibiotic use and later development of asthma or allergy, other studies suggest that the effect is due to more frequent antibiotic use in asthmatic children. Trends in vaccine use may also be relevant, but epidemiological studies provide no consistent support for a detrimental effect of vaccination/immunisation on atopy rates.
King et al. 2004 showed that when short lived T cells were replaced during a state of too few long lived T-cells (Memory T cell), because of lack of infections, the risk of developing autoimmune diseases increases. They showed that in a state of too few long lived T-cells, because of lack of infections, not enough short lived T-cells could be produced by long lived T-cells during homeostatic expansion. Therefore, more auto reactive T-cells divide in such a state, causing multiplying auto reactive T-cells with a greater risk of causing autoimmune diseases like type I diabetes or multiple sclerosis.
Cooke showed that NOD mice (which spontaneously develop type 1 diabetes) had significantly reduced incidence of this disease when infected with the helminth parasite Schistosoma mansoni. Other animal models also showed that helminth infections block or treat various chronic inflammatory disorders were comprehensively reviewed recently.
A double blind study performed on 2500 pregnant women in Uganda showed that infants of the women treated with anthelminthic medication for worm infections had double the rate of doctor-diagnosed infantile eczema.
Early life exposure to specific microbe-enriched environments decreases susceptibility to diseases such as IBD and asthma, whereas its absence, as in antibiotic treatment during childhood, may have the opposite effect. Olszak and colleagues, compared germ free mice (GF) and pathogen specific free mice (SPF), challenged with aerosol ovoalbumin to promote allergen-induced airway inflammation. They found that early exposure to conventional microbiota protected animals from developing asthma.
Public health trends
Since allergies and other CIDs are largely diseases of the last 100 years or so, the "hygiene" revolution of the last 200 years came under scrutiny as a possible cause. During the 1800s radical improvements to sanitation and water quality occurred in Europe and North America. The introduction of toilets and sewer systems and the clean up of city streets, and cleaner food were part of this program. This in turn led to a rapid decline in infectious diseases, particularly during the period 1900-1950, through reduced exposure to infectious agents.
Public health activities have also played a part in affecting diet and lifestyle, such as physical activity levels and locations.
While no hygiene-related treatments are part of the standard of care, various approaches are under investigation. Helminth therapy is one alternative.Probiotic (drinks or foods) can reintroduce microbes to the gut. As yet, therapeutically relevant microbes have not been specifically identified.
Lifestyle changes could increase microbial exposure, but whether this on balance improves the balance of risks remains the subject of research. Proposals include natural childbirth, sustained breast feeding and physical interaction between siblings, and encouraging children to spend more time in "uncleaned" outdoor environments.
Should these therapies become accepted, public policy implications include providing green spaces in urban areas or even providing access to agricultural environments for children.
The anti-inflammatory effects of helminth infection are prompting interest and research into diseases that involve inflammation but that are not currently considered to include autoimmunity or immune dysregulation as a causative factor. Heart disease and arteriosclerosis both have similar epidemiological profiles as autoimmune diseases and both involve inflammation. Their increased incidence cannot be solely attributed to environmental factors. Recent research explored the eradication of helminths as contributing to this discrepancy.
Helminthic therapy emerged from the search for reasons why the incidence of immunological disorders and autoimmune diseases correlates with the level of industrial development.
Related therapies include use other types of infectious organisms, such as protozoa.
No evidence supports the idea that reducing modern practices of cleanliness and hygiene would have any impact on rates of chronic inflammatory and allergic disorders, but a significant amount of evidence that it would increase the risks of infectious diseases.
If home and personal cleanliness contributes to reduced exposure to vital microbes, its role is likely to be small. The idea that homes can be made “sterile” through excessive cleanliness is implausible. The evidence shows that, as fast as they are removed by cleaning, microbes are replaced, via dust and air from outdoors, by shedding from the body and other living things as well as from food. The key point may be that the microbial content of urban housing has altered, not because of home and personal hygiene habits, but because they are part of urban environments. Diet and lifestyle changes also affects the gut, skin and respiratory microbiota.
At the same time that concerns about allergies and other CIDs have been increasing, so also have concerns about infectious disease. Infectious diseases continue to exert a heavy health toll. Preventing pandemics and reducing antibiotic resistance are global priorities. Hygiene is a cornerstone of containing these threats.
Infection risk management
The International Scientific Forum on Home Hygiene has developed a risk management approach to reducing home infection risks. This approach uses microbiological and epidemiological evidence to identify the key routes of infection transmission in the home. These data indicate that the critical routes involve the hands, hand and food contact surfaces and cleaning utensils. Clothing and household linens involve somewhat lower risks. Surfaces that contact the body, such as baths and hand basins, can act as infection vehicles, as can surfaces associated with toilets. Airborne transmission can be important for some pathogens. A key aspect of this approach is that it maximises protection against pathogens and infection, but is more relaxed about visible cleanliness in order to sustain normal exposure to other human, animal and environmental microbes.
There are other hypotheses that try to explain the increase in allergies in developed nations. Major areas of focus include infant feeding, over-exposure and exposure to certain pollutants.
Infant feeding topics includes breastfeeding, when babies begin to eat solid foods and the type of these foods, cow's milk vs other milks and variations in milk processing.
The rising incidence of metabolic disease, including obesity, diabetes and hypertension has been hypothesized to be exacerbated by the absence of certain infections. Inflammation has long been recognized as a major etiological factor for metabolic diseases (MD). Chronic inflammation leading to insulin resistance (IR) is a major etiological factor for metabolic conditions apart from obesity and type-2 diabetes. Insulin resistance typically starts as an organ-specific inflammation affecting the major organs of insulin action namely adipose tissue, skeletal muscles and liver. With disease progression, the inflammation becomes more systemic and starts affecting blood vessels leading to endothelial dysfunction, a pre-stage for vasculopathies. In fact the inflammation associated with macrovasculopathies like cardio-vascular disease, cerebro-vascular disease and perivascular disease seems to be very different from microvascular complications like diabetic retinopathy, nephropathy and neuropathy. The exact cause of inflammation in IR is not known even though dietary, genetic and a variety of environmental factors have been implicated.
The reduced prevalence of LF among diabetic subjects compared to non-diabetic and pre-diabetic subjects was noted, and supported the influence of childhood helminth infestations. Within the diabetic subjects, those who were filarial positive had reduced levels of pro-inflammatory markers compared to those who were filarial negative.
In mice, animals infected with helminths had lesser disease severity compared to uninfected subjects in a diet-induced obesity mice model that is well-recognized for MD. In light of these findings, the decreasing incidence of filarial infection due to mass drug administration could potentially have an unexpected adverse impact on the prevalence of diabetes in developing countries. Infections serve as an important source of inflammation and inflammation itself can serve as a link between infections and metabolic diseases. In general, infections that promote inflammation are thought to augment metabolic diseases while those that dampen inflammation by immunomodulation can confer protection against metabolic diseases. A much higher level of complexity is brought about by the recently identified helminth induced immunomodulation in conferring protection against inflammation/IR.
^ abStrachan, D. (2000). "Family size, infection and atopy: The first decade of the 'hygiene hypothesis'". Thorax55 (90001): 2S. doi:10.1136/thorax.55.suppl_1.S2.edit
^Stene LC, Nafstad P. Relation between occurrence of type 1 diabetes and asthma" Lancet 2001;357:607
^ abRaison CL, Lowry CA, Rook GAW. Inflammation, sanitation and consternation: loss of contact with co-evolved, tolerogenic micro-organisms and the pathophysiology and treatment of major depression" Arch Gen Psychiatry 2010;67(12) 1211-24
^ abcRook GAW, Lowry CA, Raison CL. Microbial Old Friends, immunoregulation and stress resilience. Evolution, Medicine and Pubic Health. 2013;EMPH (2013)(1) 46-64 Rook, G. A. W.; Lowry, C. A.; Raison, C. L. (2013). "Microbial 'Old Friends', immunoregulation and stress resilience". Evolution, Medicine, and Public Health2013: 46. doi:10.1093/emph/eot004.edit
^Rook GAW, Dalgleish A. Infection, immunoregulation and cancer. Immunological Reviews. 2011;240:141-59
^ abcRook GA, Martinelli R, Brunet LR. Innate immune responses to mycobacteria and the downregulation of atopic responses" Curr Opin Allergy Clin Immunol 2003 Oct;3(5) 337-42.
^Ashwood, P.; Wakefield, A. J. (2006). "Immune activation of peripheral blood and mucosal CD3+ lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms". Journal of Neuroimmunology173 (1–2): 126–134. doi:10.1016/j.jneuroim.2005.12.007. PMID16494951.edit
^Zimmerman, A. W.; Jyonouchi, H.; Comi, A. M.; Connors, S. L.; Milstien, S.; Varsou, A.; Heyes, M. P. (2005). "Cerebrospinal Fluid and Serum Markers of Inflammation in Autism". Pediatric Neurology33 (3): 195–201. doi:10.1016/j.pediatrneurol.2005.03.014. PMID16139734.edit
^Molloy, C.; Morrow, A.; Meinzen-Derr, J.; Schleifer, K.; Dienger, K.; Manning-Courtney, P.; Altaye, M.; Wills-Karp, M. (2006). "Elevated cytokine levels in children with autism spectrum disorder". Journal of Neuroimmunology172 (1–2): 198–205. doi:10.1016/j.jneuroim.2005.11.007. PMID16360218.edit
^ abRook GAW. 99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: darwinian medicine and the 'hygiene' or 'old friends' hypothesis. Clin Exp Immunol. 2010 Apr;160(1) 70-9.
^ abFilippi CM, von Herrath MG (2008) Viral trigger for type 1 diabetes: pros and cons" Diabetes 57(11) 2863-2871.
^ abRook GA (2012) Hygiene hypothesis and autoimmune diseases. Clin Rev Allergy Immunol 42(1) 5-15.
^ abRound JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, Mazmanian SK. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science. 2011 May 20;332(6032) 974-7
^ abOsada Y, Kanazawa T. Parasitic helminths: new weapons against immunological disorders. J Biomed Biotechnol. 2010;2010:743-58.
^Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y, Filbey KJ, Finney CAM, Greenwood EJD, Knox DP, Wilson MS, Belkaid Y, Rudensky AY, Maizels RM. Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway" J Exp Med 2010;207(11) 2331-41.
^Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TM, Zaat BA, Yazdanbakhsh M, Wierenga EA, van Kooyk Y, Kapsenberg ML. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol. 2005 Jun;115(6) 1260-7
^Correale J, Farez M. Association between parasite infection and immune responses in multiple sclerosis" Ann Neurol 2007 Feb;61(2) 97-108
^ abFleming J, Isaak A, Lee J, Luzzio C, Carrithers M, Cook T, Field A, Boland J, Fabry Z. Probiotic helminth administration in relapsing-remitting multiple sclerosis: a phase 1 study. Mult Scler. 2011 Mar 3;17(6) 743-54
^Gibson, Peter G.; Henry, Richard L.; Shah, Smita; Powell, Heather; Wang, He (2003). "Migration to a western country increases asthma symptoms but not eosinophilic airway inflammation". Pediatric Pulmonology36 (3): 209–15. doi:10.1002/ppul.10323. PMID12910582.
^Magen, Eli; Bychkov, Vitaly; Ginovker, Alexander; Kashuba, Eduard (2013). "Chronic Opisthorchis felineus infection attenuates atherosclerosis – an autopsy study". International Journal for Parasitology 43 (10) 819–24. doi:10.1016/j.ijpara.2013.04.008. PMID 23792298
^Serreze DV, Ottendorfer EW, Ellis TM, Gauntt CJ, Atkinson MA (2000) Acceleration of type 1 diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive T-cells in pancreatic islets" Diabetes 49(5) 708-711.
^Harrison LC, et al. (2008) Type 1 diabetes: lessons for other autoimmune diseases? J Autoimmun 31(3) 306-310.
^Blackley CH (1873) Experimental Researches on the Causes and Nature of Catarrhus Aestivus (Hay-fever and Hay-asthma) (Baillière Tindall and Cox, London).
^Marra, F.; Lynd, L; Coombes, M; Richardson, K; Legal, M; Fitzgerald, JM; Marra, CA (2006). "Does Antibiotic Exposure During Infancy Lead to Development of Asthma?: A Systematic Review and Metaanalysis". Chest129 (3): 610–8. doi:10.1378/chest.129.3.610. PMID16537858.
^Thavagnanam, S.; Fleming, J.; Bromley, A.; Shields, M. D.; Cardwell, C. R. (2008). "A meta-analysis of the association between Caesarean section and childhood asthma". Clinical & Experimental Allergy38 (4): 629–33. doi:10.1111/j.1365-2222.2007.02780.x. PMID18352976.
^Magnus MC, Haberg SE, Stigum H, Nafstad P, London SJ, Vangen S, Nystad W. Delivery by Cesarean section and early childhood respiratory symptoms and disorders: the Norwegian mother and child cohort study. Am J Epidemiol. 2011 Dec 1;174(11) 1275-85.
^Metsala J, Lundqvist A, Virta LJ, Kaila M, Gissler M, Virtanen SM. Mother's and offspring's use of antibiotics and infant allergy to cow's milk" Epidemiology 2013 Mar;24(2) 303-309
^King, Cecile; Ilic, Alex; Koelsch, Kersten; Sarvetnick, Nora (2004). "Homeostatic Expansion of T Cells during Immune Insufficiency Generates Autoimmunity". Cell117 (2): 265–77. doi:10.1016/S0092-8674(04)00335-6. PMID15084263.
^Summers, Robert W.; Elliott, David E.; Qadir, Khurram; Urban Jr, Joseph F.; Thompson, Robin; Weinstock, Joel V. (2003). "Trichuris suis seems to be safe and possibly effective in the treatment of inflammatory bowel disease". The American Journal of Gastroenterology98 (9): 2034–41. doi:10.1111/j.1572-0241.2003.07660.x. PMID14499784.
^Correale, Jorge; Farez, Mauricio (2007). "Association between parasite infection and immune responses in multiple sclerosis". Annals of Neurology61 (2): 97–108. doi:10.1002/ana.21067. PMID17230481.
^ abLeonardi-Bee, Jo; Pritchard, David; Britton, John (2006). "Asthma and Current Intestinal Parasite Infection". American Journal of Respiratory and Critical Care Medicine174 (5): 514–23. doi:10.1164/rccm.200603-331OC. PMID16778161.
^Summers, Robert W.; Elliott, David E.; Urban Jr, Joseph F.; Thompson, Robin A.; Weinstock, Joel V. (2005). "Trichuris suis therapy for active ulcerative colitis: A randomized controlled trial". Gastroenterology128 (4): 825–32. doi:10.1053/j.gastro.2005.01.005. PMID15825065.
^Aoyama, H; Hirata, T; Sakugawa, H; Watanabe, T; Miyagi, S; Maeshiro, T; Chinen, T; Kawane, M; Zaha, O; Nakayoshi, T; Kinjo, F; Fujita, J (2007). "An inverse relationship between autoimmune liver diseases and Strongyloides stercoralis infection". The American journal of tropical medicine and hygiene76 (5): 972–6. PMID17488925.
^Magen, Eli; Borkow, Gadi; Bentwich, Zvi; Mishal, Joseph; Scharf, Shimon (2005). "Can worms defend our hearts? Chronic helminthic infections may attenuate the development of cardiovascular diseases". Medical Hypotheses64 (5): 904–9. doi:10.1016/j.mehy.2004.09.028. PMID15780483.
^Juckett, David A.; Aylsworth, Charles F.; Quensen, Janet Murphy (2008). "Intestinal protozoa are hypothesized to stimulate immunosurveillance against colon cancer". Medical Hypotheses71 (1): 104–10. doi:10.1016/j.mehy.2008.01.024. PMID18343044.
^Bloomfield SF, Scott EA A risk assessment approach to use of antimicrobials in the home to prevent spread of infection American Journal of Infection Control 2013; 41: (5, Suppl) A1-A10, In Disinfection, Sterilization and Antisepsis: Current Issues, New Research and New Technologies. Edited by William A. Rutala and David J. Weber
^Ricardo-Gonzalez, R. R.; Red Eagle, A.; Odegaard, J. I.; Jouihan, H.; Morel, C. R.; Heredia, J. E.; Mukundan, L.; Wu, D.; Locksley, R. M.; Chawla, A. (2010). "IL-4/STAT6 immune axis regulates peripheral nutrient metabolism and insulin sensitivity". Proceedings of the National Academy of Sciences107 (52): 22617. doi:10.1073/pnas.1009152108.edit
Rook, G. A. W.; Adams, V.; Hunt, R.; Palmer, L. R.; Martinelli, J.; Brunet, R. (2004). "Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders". Springer Seminars in Immunopathology25 (3–4): 237–255. doi:10.1007/s00281-003-0148-9. PMID15007629.edit