Objective. To study T-helper (Th)1--Th2--Th3 gene activation profile in the small intestine and peripheral blood of children with celiac disease (CD) with special interest in the response to the gluten-free diet (GFD) treatment in order to elucidate an immune dysregulation not triggered by gluten. Material and methods. Small intestinal biopsies and venous blood were taken from seven children with CD (mean age: 8 years, four girls) at presentation and after 1 year of strict GFD. The Th1--Th2--Th3 gene expression profile was examined by real-time PCR arrays. The findings were compared with the corresponding expressions in peripheral blood and small intestinal biopsies from six reference children without CD (mean age: 6 years, four girls). Results. The Th1 gene expression profile including interferon (IFN)-gamma gamma, signal transducer and activator of transcription (STAT) 1 and interferon regulatory factor (IRF) 1 together with reduced interleukin (IL)-2 expression was pronounced in small intestinal biopsies from children with untreated CD. A downregulation of IFN-gamma gamma transcripts was seen after 1 year of GFD, but there was still increased expression of STAT1 and IRF1 in association with low IL-2 expression in spite of eliminated exposure to wheat gluten. By contrast, the decreased intestinal expression of Th2 gene markers observed at presentation was normalized with GFD. The alterations in the mucosal gene expression profile were not reflected in peripheral blood. Conclusion. The GFD did not correct the increased activation of the IFN-gamma gamma signaling pathway related markers and reduced IL-2 expression, suggesting that they represent an immune dysregulation not dependent on gluten exposure.

Up-regulation of interleukin (IL)-17 in small intestinal mucosa has been reported in coeliac disease (CD) and in peripheral blood in type 1 diabetes (T1D). We explored mucosal IL-17 immunity in different stages of CD, including transglutaminase antibody (TGA)-positive children with potential CD, children with untreated and gluten-free diet-treated CD and in children with T1D. Immunohistochemistry was used for identification of IL-17 and forkhead box protein 3 (FoxP3)-positive cells and quantitative polymerase chain reaction (qPCR) for IL-17, FoxP3, retinoic acid-related orphan receptor (ROR)c and interferon (IFN)-γ transcripts. IL-1β, IL-6 and IL-17 were studied in supernatants from biopsy cultures. Expression of the apoptotic markers BAX and bcl-2 was evaluated in IL-17-stimulated CaCo-2 cells. The mucosal expression of IL-17 and FoxP3 transcripts were elevated in individuals with untreated CD when compared with the TGA-negative reference children, children with potential CD or gluten-free diet-treated children with CD (P andlt; 0·005 for all IL-17 comparisons and P andlt; 0·01 for all FoxP3 comparisons). The numbers of IL-17-positive cells were higher in lamina propria in children with CD than in children with T1D (P andlt; 0·05). In biopsy specimens from patients with untreated CD, enhanced spontaneous secretion of IL-1β, IL-6 and IL-17 was seen. Activation of anti-apoptotic bcl-2 in IL-17-treated CaCo-2 epithelial cells suggests that IL-17 might be involved in mucosal protection. Up-regulation of IL-17 could, however, serve as a biomarker for the development of villous atrophy and active CD.

Objective. The aim was to investigate the mucosal activation of a broad range of genes associated with the T-helper 17 cell (Th17) signaling pathway in children at different stages of celiac disease (CD), including children with increased risk for CD and children with untreated and gluten-free diet (GFD)-treated CD. Material and methods. Small intestinal biopsies were taken from children with untreated and GFD-treated CD, transglutaminase antibody (TGA)-positive children with potential CD, and reference children. Real-time polymerase chain reaction (PCR) arrays were used to study the gene expression pattern of Th17-related genes, and quantitative PCR was used to study the interleukin (IL)-17A expression. Results. The mucosal expression of CD8A was elevated at all stages of CD. Children with untreated CD had diminished levels of IL-17RE, IL-23R, RORc, STAT6, CCL22, NFATC2, IL-18, CD4, CD247, and matrix metalloproteinase (MMP)9 but had elevated levels of MMP3, IL-17, interferon-gamma (IFN-gamma) and CD8A, compared to references. The majority of the aforementioned genes, being differentially expressed in untreated CD, displayed similar expression in GFD-treated children and references. Children with untreated and GFD-treated CD had elevated expression of IFN-gamma but had reduced expression of CD247. Interestingly, children with potential CD displayed reduced FOXP3, IL-21, and IL-17A levels. Conclusion. Mucosal upregulation of Th17 immunity occurs at the late stage of disease and is downregulated with dietary treatment, thus indicating that IL-17 immunity is not a fundamental feature of CD as Th1 immunity, which is not fully downregulated by GFD.

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.