We studied the effects of probiotic treatment on the innate immune system during infancy. The study included a subgroup of infants recruited to the pilot study testing the feasibility of probiotics intervention in infants with genetic risk of type 1 diabetes (T1D). A mixture of Lactobacillus rhamnosus GG (5 x 109 cfu), Lactobacillus rhamnosus LC705 (5 x 109 cfu), Bifidobacterium breve Bbi99 (2 x 108 cfu) and Propionibacterium freudenreichii ssp. Shermani JS (2 x 109 cfu) was given to the infants beginning one to three weeks after birth until the age of 6 months. Blood samples were drawn at the age of 6, 12 and 24 months for the analyses of beta-cell autoantibodies and the phenotype and stimulation response of monocytes with flow-cytometry, including surface markers on circulating CD14+ monocytes and expression of co-stimulatory markers on CD14+ monocytes as response to stimulation with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). Also gene expression of toll-like receptor (TLR) signalling molecules was studied in the peripheral blood mononuclear cell (PBMC) population.

In the children who received probiotics the number of circulating CD14+ monocytes expressing CD58 was reduced at the age of 6 months, and a tendency for a decreased induction of CCR5, CD80 and CD58 expressing monocytes as response to LTA was seen when compared to the children who received placebo. At the age of 12 months, the number of monocytes expressing CCR5 was decreased in the probiotic group, and a decreased spontaneous expression of TNFRSF1A and an increased spontaneous expression of TLR9 was observed in the PBMC from the children treated with probiotics. In the whole study group, the numbers of circulating monocytes expressing CD80 increased with age as well as the induction of CCR5, CD80 and CD58 on monocytes as response to stimulation. By the age of 24 months one child in both groups developed multiple autoantibodies.

We demonstrated that probiotics modulated the activation stage and stimulation response of monocytes, and that prolonged effects of the treatment were seen at the age of 12 months. The findings suggest that early microbial exposure may program the function of the innate immune system for later life.

Type 1 diabetes (TlD) is considered to be an autoimmune disease leading to destruction of the insulin producing beta cells in pancreas. Interaction between genetic factors and environmental factors are believed to trigger the autoimmune response finally causing T1D. A number of environmental factors have been suggested to be associated with TlD. Microbiotic colonization of the newborn infant's gut ecosystem by specific bacterial species may be important in the initial regulation of the developing immune system. Development of TlD has been associated with intestinal immune activation and enhanced immunity to food antigens. Probiotics is defined as nonpathogenic cultures of living bacteria with a health-promoting effect. The effect of probiotics is unspecific, likely mediated via the modulation of the innate immune system. The actions of probiotics in the prevention of TID could include reduced occurrence of enteral virus infections, enhanced maturation of the gut immune system, decreased gut permeability, and support for development of oral tolerance and/or induced immune regulation. The aim was to test the safety of probiotics in infants with risk genes for TlD and to study the effect of probiotics on the activation stage of monocytes ex vivo and on their response to in vitro LTA and LPS stimulation. We conclude that probiotics seems to be safe and we demonstrate here to our knowledge the first evidence in humans that probiotics are able to modulate the function of monocytes in vivo and even longstanding effects of the treatment during the early infancy are seen. The findings support the hygiene hypothesis suggesting that early microbial exposure may program the function of the innate immune system for later life. The probiotic induced hyporesponsiveness of the innate immune system to stimuli may protect from over-whelming inflammatory responses that could further contribute to the development of harmful innate and adaptive immune responses seen in allergies and autoimmune diseases. These results explain, at least partly, the beneficial effects of probiotics seen in the prevention and treatment of allergies in children.

The final aim of the PRODIA study is to determine whether the use of probiotics during the first 6 months of life decreases the appearance of type 1 diabetes mellitus (T1DM)-associated autoantibodies in children with genetic risk for T1DM. A pilot study including 200 subjects was planned to show whether the use of probiotics during the first 6 months of life is safe and feasible. The prevalence of autoantibodies among the study subjects at 6, 12, and 24 months of age was at levels close to the expected and the clinical follow-up did not either indicate problems in the feasibility of the study.

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