Background: According to the biodiversity hypothesis of immune-mediated diseases, lack of microbiological di-versity in the everyday living environment is a core reason for dysregulation of immune tolerance and - even-tually - the epidemic of immune-mediated diseases in western urban populations. Despite years of intense research, the hypothesis was never tested in a double-blinded and placebo-controlled intervention trial.Objective: We aimed to perform the first placebo-controlled double-blinded test that investigates the effect of biodiversity on immune tolerance. Methods: In the intervention group, children aged 3-5 years were exposed to playground sand enriched with microbially diverse soil, or in the placebo group, visually similar, but microbially poor sand colored with peat (13 participants per treatment group). Children played twice a day for 20 min in the sandbox for 14 days. Sand, skin and gut bacterial, and blood samples were taken at baseline and after 14 days. Bacterial changes were followed for 28 days. Sand, skin and gut metagenome was determined by high throughput sequencing of bacterial 16 S rRNA gene. Cytokines were measured from plasma and the frequency of blood regulatory T cells was defined as a percentage of total CD3 +CD4 + T cells. Results: Bacterial richness (P < 0.001) and diversity (P < 0.05) were higher in the intervention than placebo sand. Skin bacterial community, including Gammaproteobacteria, shifted only in the intervention treatment to resemble the bacterial community in the enriched sand (P < 0.01). Mean change in plasma interleukin-10 (IL-10) concentration and IL-10 to IL-17A ratio supported immunoregulation in the intervention treatment compared to the placebo treatment (P = 0.02). IL-10 levels (P = 0.001) and IL-10 to IL-17A ratio (P = 0.02) were associated with Gammaproteobacterial community on the skin. The change in Treg frequencies was associated with the relative abundance of skin Thermoactinomycetaceae 1 (P = 0.002) and unclassified Alphaproteobacteria (P < 0.001). After 28 days, skin bacterial community still differed in the intervention treatment compared to baseline (P < 0.02). Conclusions: This is the first double-blinded placebo-controlled study to show that daily exposure to microbial biodiversity is associated with immune modulation in humans. The findings support the biodiversity hypothesis of immune-mediated diseases. We conclude that environmental microbiota may contribute to child health, and that adding microbiological diversity to everyday living environment may support immunoregulation.

Howick et al. have reported the findings of a survey that addressed the use of placebos among primary care practitioners in the United Kingdom. They adopted methodology similar to that used in previous studies performed in other countries; however, the use of this approach also means that they repeated the conceptual confusion of the previous surveys. Therefore the findings are not useful. ... The paper’s main finding “placebos are commonly used in UK primary care” is not correct. Only 0.9% of the responding general practitioners reported using pure placebos frequently. The frequency with which impure placebos are used is irrelevant because the concept is useless, as described above. Misleading a patient by administering inert substances without the explicit consent of the patient is unethical. The authors' proposal to “develop ethical and cost-effective placebos” is not possible because saving money by misleading patients is unethical. There is substantial conceptual confusion in the area of placebo and placebo-effect research, and the paper by Howick et al. does not help to reduce this confusion.

The metagenomic data presented in this article are related to the published research of "A Placebo-controlled doubleblinded test of the biodiversity hypothesis of immunemediated diseases: Environmental microbial diversity elicits changes in cytokines and increase in T regulatory cells in young children"This database contains 16S ribosomal RNA (rRNA) metagenomics of sandbox sand and skin and gut microbiota of children in the intervention and placebo daycares. In inter-vention daycares, children aged 3-5 years were exposed to playground sand enriched with microbially diverse soil. In placebo daycares, children were exposed to visually similar as in intervention daycares, but microbially poor sand col-ored with peat. Sand, skin and gut metagenomics were an-alyzed at baseline and after 14 and 28 days of interven-tion by high throughput sequencing of bacterial 16S rRNA gene on the Illumina MiSeq platform. This dataset shows how skin bacterial community composition, including classes Gammaproteobacteria and Bacilli, changed, and how the rel-ative abundance of over 30 bacterial genera shifted on the skin of children in the intervention treatment, while no shifts occurred in the placebo group.(c) 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)