Clinical signs of type 1 diabetes are associated with type 2 diabetes marker transcription factor 7-like 2 polymorphism.

AIMS/INTRODUCTION: We aimed to assess the distribution of transcription factor 7-like 2 gene TCF7L2 (rs7903146) polymorphism and to find possible associations between TCF7L2 and the characteristics of type 1 diabetes. MATERIALS AND METHODS: We studied 190 newly diagnosed type 1 diabetes patients (median age 12.7 years, range 2.0-72.5) and 246 controls (median age 23.8 years, range 1.4-81.5) for TCF7L2 single nucleotide polymorphism. We determined anti-islet autoantibodies, random C-peptide levels, diabetes associated HLA DR/DQ haplotypes and genotypes in all patients. RESULTS: There were no differences in the distribution of TCF7L2 single nucleotide polymorphism between patients and controls. However, patients with in type 1 diabetes, after adjusting for age and sex, subjects carrying C allele were at risk for a C-peptide level lower than 0.5 nmol/L (OR 5.65 [95% CI: 1.14-27.92]) and for zinc transporter 8 autoantibody positivity (5.22 [1.34-20.24]). Participants without T allele were associated with a higher level of islet antigen-2 autoantibodies (3.51 [1.49-8.27]) and zinc transporter 8 autoantibodies (2.39 [1.14-4.99]). CONCLUSIONS: The connection of TCF7L2 polymorphism with zinc transporter 8 and islet antigen-2 autoantibodies and C-peptide levels in patients supports the viewpoint that TCF7L2 is associated with the clinical signs and autoimmune characteristics of type 1 diabetes. The mechanisms of the interaction between the TCF7L2 risk genotype and anti-islet autoantibodies need to be studied further.

Circulating Zonulin Correlates with Density of Enteroviruses and Tolerogenic Dendritic Cells in the Small Bowel Mucosa of Celiac Disease Patients.

BACKGROUND: Impaired intestinal integrity, including increased permeability of the small bowel mucosa, has been shown in patients with celiac disease (CD) as well as with type 1 diabetes (T1D). Zonulin (ZO, pre-haptoglobin), a tight junction regulator, plays a particular role in the regulation of intestinal barrier function and in the pathogenesis of the above-mentioned diseases. AIM: To investigate whether enteroviruses (EVs) and immunoregulatory cells are associated with intestinal permeability in patients with CD alone and with coexistent T1D. MATERIALS AND METHODS: Altogether 80 patients (mean age 10.68 ± 6.69 years) who had undergone small bowel biopsy were studied. Forty patients with functional dyspepsia and normal small bowel mucosa formed the control group. The circulating ZO level in sera was evaluated using ELISA. The densities of EV, FOXP3+ regulatory T cells (Tregs), indoleamine 2,3-dioxygenase (IDO+) dendritic cells (DCs) and glutamic acid dexarboxylase (GAD)65+ cells in small bowel mucosa were investigated by immunohistochemistry. The expression analysis of FOXP3, tight junction protein 1 (TJP1), gap junction (GJA1), IDO and CD103 genes was evaluated by real-time PCR. RESULTS: The ZO level was higher in CD patients compared to subjects with a normal small bowel mucosa, particularly in those with Marsh IIIc atrophy (p = 0.01), and correlated with the density of EV (r = 0.63; p = 0.0003) and IDO+ DCs (r = 0.58; p = 0.01) in the small bowel mucosa. The density of GAD65+ epithelial cells was correlated with the density of EV (r = 0.59; p = 0.03) and IDO+ DCs (r = 0.78; p = 0.004) in CD patients. The relative expression of FOXP3 mRNA in the small bowel mucosa tissue was significantly higher in patients with CD, compared to subjects with a normal mucosa, and correlated with the density of EV (r = 0.62; p = 0.017) as well as with the relative expression of IDO mRNA (r = 0.54; p = 0.019). CONCLUSIONS: The CD is associated with elevation of the circulating ZO level, the value of which correlates with the density of EV in CD patients with severe atrophic changes in the small bowel mucosa, particularly in cases of concomitant T1D. The CD is also characterized by the close relationship of the density of GAD65+ epithelial cells with the EV, ZO level and IDO+ DCs.

Increased Blood Levels of Growth Factors, Proinflammatory Cytokines, and Th17 Cytokines in Patients with Newly Diagnosed Type 1 Diabetes.

The production of several cytokines could be dysregulated in type 1 diabetes (T1D). In particular, the activation of T helper (Th) type 1 (Th1) cells has been proposed to underlie the autoimmune pathogenesis of the disease, although roles for inflammatory processes and the Th17 pathway have also been shown. Nevertheless, despite evidence for the role of cytokines before and at the onset of T1D, the corresponding findings are inconsistent across studies. Moreover, conflicting data exist regarding the blood cytokine levels in T1D patients. The current study was performed to investigate genetic and autoantibody markers in association with the peripheral blood cytokine profiles by xMap multiplex technology in newly diagnosed young T1D patients and age-matched healthy controls. The onset of young-age T1D was characterized by the upregulation of growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-7, the proinflammatory cytokine IL-1ß (but not IL-6 or tumor necrosis factor [TNF]-α), Th17 cytokines, and the regulatory cytokines IL-10 and IL-27. Ketoacidosis and autoantibodies (anti-IA-2 and -ZnT8), but not human leukocyte antigen (HLA) genotype, influenced the blood cytokine levels. These findings broaden the current understanding of the dysregulation of systemic levels of several key cytokines at the young-age onset of T1D and provide a further basis for the development of novel immunoregulatory treatments in this disease.

Increased carotid artery intima-media thickness and myeloperoxidase level in children with newly diagnosed juvenile idiopathic arthritis.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is a frequent childhood rheumatic disease characterized by chronic inflammation. The latter has been related to impairment of arterial functional-structural properties, atherogenesis and later cardiovascular events. The objective of this study was to examine intima-media thickness (IMT) and the parameters of arterial stiffness in children with JIA at diagnosis and their correlation with JIA subtype and markers of inflammation and atherosclerosis. METHODS: Thirty-nine newly diagnosed patients with JIA (26 girls; mean age, 13.2 ± 2.6 years) and 27 healthy controls (9 girls; mean age, 13.6 ± 3.4 years) were included in the study. Twelve patients had oligoarthritis, fifteen had extended oligoarthritis and twelve had rheumatoid factor-negative polyarthritis. IMT of the common carotid artery was determined by ultrasonography, carotid-femoral pulse wave velocity (cfPWV) and augmentation index adjusted to a heart rate of 75 beats/min (AIx@75) were determined by applanation tonometry. The serum levels of atherosclerosis-related biomarkers, such as asymmetric dimethylarginine (ADMA), myeloperoxidase (MPO) and adiponectin, were measured by enzyme-linked immunosorbent assay. RESULTS: Mean IMT (0.46 ± 0.04 vs. 0.42 ± 0.04 mm; p = 0.0003) and MPO concentration (115.2 [95% confidence interval {95% CI}, 97.4-136.3] vs. 57.6 [95% CI, 47.1-70.3] ng/ml; p < 0.0001) were higher in the patients with JIA than in the control subjects. The cfPWV, AIx@75 and serum ADMA and adiponectin levels did not significantly differ between the groups and JIA subtypes. Serum adiponectin level correlated negatively with AIx@75 in patients with JIA (r = -0.38; p < 0.05). CONCLUSIONS: Patients with JIA have increased mean IMT and elevated MPO levels at early stages of the disease. AIx@75 was inversely independently associated with adiponectin level in the patients, suggesting that lower adiponectin levels might influence arterial subclinical stiffening in patients with newly diagnosed JIA.

Differences in Gut Microbiota Between Atopic and Healthy Children.

Although gut microbiota has been studied relatively extensively in the context of allergic diseases, there have been several contradictions between these studies. By applying high-throughput sequencing, we aimed to analyze the differences in gut microbiota between atopic and healthy children at 5 and 12 years of age. 51 stool samples were collected from 14 atopic and 15 healthy children and analyzed with 454 pyrosequencing of the 16S rRNA gene. At the ages of 5 and 12 years, Bacteroides, Prevotella, and Dialister dominated gut microbiota in both atopic and healthy groups of children. Children in the atopic group had lower abundance and prevalence of Akkermansia in gut microbiota than their healthy counterparts. Thus, the composition of gut microbiota does not seem to be significantly different between atopic and healthy children, but lower abundance and prevalence of Akkermansia indicate that this bacterium may accompany or play a role in IgE-mediated atopic diseases.

Increased density of tolerogenic dendritic cells in the small bowel mucosa of celiac patients.

AIM: To investigate the densities of dendritic cells (DCs) and FOXP3(+) regulatory T cells (Tregs) and their interrelations in the small bowel mucosa in untreated celiac disease (CD) patients with and without type 1 diabetes (T1D). METHODS: Seventy-four patients (45 female, 29 male, mean age 11.1 ± 6.8 years) who underwent small bowel biopsy were studied. CD without T1D was diagnosed in 18 patients, and CD with T1D was diagnosed in 15 patients. Normal small bowel mucosa was found in two T1D patients. Thirty-nine patients (mean age 12.8 ± 4.9 years) with other diagnoses (functional dyspepsia, duodenal ulcer, erosive gastritis, etc.) formed the control group. All CD patients had partial or subtotal villous atrophy according to the Marsh classification: Marsh grade IIIa in 9, grade IIIb in 21 and grade IIIc in 3 cases. Thirty-nine patients without CD and 2 with T1D had normal small bowel mucosa (Marsh grade 0). The densities of CD11c(+), IDO(+), CD103(+), Langerin (CD207(+)) DCs and FOXP3(+) Tregs were investigated by immunohistochemistry (on paraffin-embedded specimens) and immunofluorescence (on cryostat sections) methods using a combination of mono- and double-staining. Sixty-six serum samples were tested for IgA-tissue transglutaminase (tTG) using a fully automated EliA™ Celikey(®) IgA assay (Pharmacia Diagnostics, Freiburg, Germany). RESULTS: The density of CD11c(+) DCs was significantly increased in CD patients compared with patients with normal mucosa (21.67 ± 2.49 vs 13.58 ± 1.51, P = 0.007). The numbers of FOXP3(+) cells were significantly higher in CD patients (10.66 ± 1.50 vs 1.92 ± 0.37, P = 0.0002) and in patients with CD and coexisting T1D (8.11 ± 1.64 vs 1.92 ± 0.37, P = 0.002) compared with patients with normal mucosa. The density of FOXP3(+) cells significantly correlated with the histological grade of atrophic changes in the small bowel mucosa according to the March classification (r = 0.62; P < 0.0001) and with levels of IgA antibody (r = 0.55; P < 0.0001). The densities of IDO(+) DCs were significantly higher in CD patients (21.6 ± 2.67 vs 6.26 ± 0.84, P = 0.00003) and in patients with CD and coexisting T1D (19.08 ± 3.61 vs 6.26 ± 0.84, P = 0.004) compared with patients with normal mucosa. A significant correlation was identified between the densities of IDO(+) DCs and FOXP3(+) T cells (r = 0.76; P = 0.0001). The mean values of CD103(+) DCs were significantly higher in CD patients (10.66 ± 1.53 vs 6.34 ± 0.61, P = 0.01) and in patients with CD and associated T1D (11.13 ± 0.72 vs 6.34 ± 0.61, P = 0.00002) compared with subjects with normal small bowel mucosa. The mean value of Langerin(+) DCs was higher in CD patients compared with persons with normal mucosa (7.4 ± 0.92 vs 5.64 ± 0.46, P = 0.04). CONCLUSION: The participation of diverse DC subsets in the pathological processes of CD and the possible involvement of tolerogenic DCs in Tregs development to maintain intestinal immunological tolerance in CD patients are revealed.

Expression of B7 and CD28 family genes in newly diagnosed type 1 diabetes.

Type-1 diabetes (T1D) is caused by T-cell mediated autoimmune reaction directed against the insulin-secreting beta cells. We hypothesized that in addition to antigen/MHC recognition the co-stimulatory B7 and CD28 pathway is also strongly affected in T1D. CD80, CD86, CD28, CTLA4, ICOS and CD25 mRNA expression was characterized in 49 newly diagnosed young T1D patients (mean age 11 ± 5 years, 25 male/24 women) and 31 controls (mean age 14 ± 7 years, 14 male/17 women). In addition, polymorphisms in CTLA4 (rs231806, rs231775, rs3087243) were genotyped and soluble CTLA4 plasma levels measured by ELISA. T1D patients presented with higher peripheral blood expression levels of CD80 (Mann-Whitney U-test, p=0.0001) and lower ICOS levels compared to healthy controls (Glm adjusted for age, p=0.0004). CD80 expression in T1D patients correlated with expression of two CTLA4 splice variants (Spearman's rank correlation, rho=0.56, p=0.0002 for sCTLA4; rho=0.61, p<0.0001 for flCTLA4). In controls, CD80 expression correlated with CD25 expression (Spearman's rank correlation, rho=0.57, p=0.002). A strong correlation was observed between sCTLA4 and flCTLA4 (Spearman's rank correlation, rho=0.94, p<0.0001). We also found a tendency that the CTLA4 +49A/G polymorphism influenced sCTLA4 mRNA expression in T1D individuals and was lowest in individuals with the GG genotype (Mann-Whitney U-test, p=0.039). However, we could not identify associations between gene expression and plasma levels of sCTLA4. To summarize, we expect that newly diagnosed T1D among children and adolescents is associated with activation of CD80 and CTLA4 in peripheral blood. Additional studies will be needed to elucidate the role of CD80/CTLA4 signaling in T1D.

Lower expression of tight junction protein 1 gene and increased FOXP3 expression in the small bowel mucosa in coeliac disease and associated type 1 diabetes mellitus.

BACKGROUND: The role of regulatory T cells expressing FOXP3 in the pathogenesis of coeliac disease (CD) and type 1 diabetes (T1D) has been reported. Recent data have placed special focus on the interplay between the intestinal barrier and immunoregulatory processes. We aimed to determine whether the expression of tight junction protein 1 (TJP1), which reflects small bowel mucosa permeability, is changed in CD and T1D. METHODS: Transcription levels of TJP1 and FOXP3 genes were evaluated in the small bowel biopsies of 14 children with CD, 12 with CD and coexisting T1D and 40 controls using real-time PCR. Serum IgA and IgG to deamidated gliadin, bovine ß-lactoglobulin, bovine α-casein and human tissue transglutaminase (tTG) were determined by ELISA. RESULTS: The highest expression of FOXP3 mRNA was seen in patients with CD and T1D compared to patients with CD alone and controls (p = 0.02). In contrast, the lowest level of TJP1 mRNA expression was found in patients with CD and T1D (p = 0.01). The levels of IgA to deamidated gliadin and tTG were highest in patients with CD and T1D (p = 0.0001 and 0.01, respectively). The expression of FOXP3 mRNA correlated highly with the level of anti-gliadin IgA (p = 0.02) and anti-tTG IgA antibodies (p = 0.004). CONCLUSION: The significant decline in TJP1 expression in CD patients, particularly in those with coexisting T1D, was accompanied by an increase in FOXP3 expression. This might reflect an attempt to maintain immune tolerance to counterbalance the loss of mucosal integrity in the small intestine in CD associated with T1D.

Celiac disease in patients with type 1 diabetes: a condition with distinct changes in intestinal immunity?

Two common chronic childhood diseases-celiac disease (CD) and type 1 diabetes (T1D)-result from complex pathological mechanisms where genetic susceptibility, environmental exposure, alterations in intestinal permeability and immune responses play central roles. In this study, we investigated whether these characteristics were universal for CD independently of T1D association. For this purpose, we studied 36 children with normal small-bowel mucosa and 26 children with active CD, including 12 patients with T1D. In samples from the small-bowel mucosa, we detected the lowest expression of tight junction protein 1 (TJP1) mRNA in CD patients with T1D, indicating an increase in intestinal permeability. Furthermore, these samples displayed the highest expression of forkhead box P3 (FoxP3) mRNA, a marker for regulatory T cells, as compared with other patient groups. At the same time, serum levels of IgA antibodies specific for the CD-related antigens deamidated gliadin and tissue transglutaminase (tTG) were the highest in CD patients with T1D. In contrast, no significant differences were found in IgA or IgG antibodies specific for bovine beta-lactoglobulin or Bifidobacterium adolescentis DSM 20083-derived proteins. There were also no differences in the transamidating activity of serum autoantibodies between patients and control individuals. Our results show that patients with T1D and newly detected CD exhibit severely altered intestinal permeability, strong local immune activation and increased immunoregulatory mechanisms in the small bowel. Further study is required to determine whether these extreme changes in this CD subgroup are due to some specific environmental factors (virus infections), unknown genetic effects or autoimmune reactions to antigenic targets in intracellular tight junctions.

Increasing incidence of childhood-onset type 1 diabetes mellitus among Estonian children in 1999-2006. Time trend analysis 1983-2006.

BACKGROUND: The incidence of childhood-onset type 1 diabetes mellitus (T1DM) among Estonian children under 15 years of age was 10.1 per 100,000 per year in 1983-1990 and 12.2 per 100,000 per year in 1991-1998 with the highest incidence in age-group 10.0-14.9 years in both periods. From 1983 to 1998, the incidence increased most rapidly in age-group 0-4.9 years. OBJECTIVE: To determine the incidence of T1DM among Estonian children in 1999-2006 and to compare the results with the data from 1983 to 1998. SUBJECTS AND METHODS: In 1999-2006, population-based incidence data were collected from two centers where all children with T1DM are seen after the diagnosis. Data for earlier periods were obtained from previously published data. Subjects were divided into three age-groups: 0-4.9 years, 5.0-9.9 years and 10.0-14.9 years. RESULTS: Between 1999 and 2006, 310 new cases of T1DM were diagnosed in Estonian children aged 0-14.9 years. The age-standardized incidence rate for that period was 17.2 [95% confidence interval (CI) 13.1-21.2]. The incidence was the highest, 21.2 (95% CI 17.7-25.3) in age-group 5.0-9.9 years. Over the time period 1983-2006, the incidence of childhood-onset T1DM in Estonian children under 15 years of age increased annually by an average 3.3% with the most rapid annual increase-9.3%-occurring in the youngest age-group. CONCLUSIONS: The incidence of childhood-onset T1DM in Estonia continues to rise and the age of onset of the disease becomes younger.

Symptomless celiac disease in type 1 diabetes: 12-year experience in Estonia.

BACKGROUND: We aimed to determine the prevalence and characteristics of celiac disease in children with type 1 diabetes in Estonia, a country with a formerly low frequency of both diseases. METHODS: Altogether, 271 patients with diabetes were studied over 12 years (1995-2006): 122 at diagnosis and 149 patients 0.1-14.8 years after diagnosis. In addition, 73 patients were followed up over 1-6 years. Immunoglobulin A type endomysium and tissue transglutaminase antibodies were determined. Patients with antibodies and/or with celiac-disease-related symptoms were invited for a small-intestinal biopsy. RESULTS: At the primary screening, celiac disease was histologically confirmed in nine patients (all without symptoms), that is, in 3.3% (95% confidence interval: 1.63-6.42) of type 1 diabetes cases. At follow up, celiac disease was additionally detected in two (2.7%) of 73 diabetic patients, that is, in 0.016 (95% confidence interval: 0-0.072) celiac disease cases per follow-up year. CONCLUSION: The prevalence of celiac disease among type 1 diabetes patients in Estonia is similar to that in countries with a high incidence of celiac disease and type 1 diabetes. As celiac disease is mostly symptomless, all children with type 1 diabetes, irrespective of their geographic origin, should be regularly screened for celiac disease.

Lower bone mineral density in children with type 1 diabetes is associated with poor glycemic control and higher serum ICAM-1 and urinary isoprostane levels.

The purpose of the study was to investigate bone mineral density (BMD) in children with type 1 diabetes (DM1) and to establish the relationships between BMD, physical activity, glycemic control, and markers of systemic oxidative stress and inflammation. We studied 30 children with DM1, aged 4.7-18.6 years, and 30 healthy subjects, matched by sex, age, and body mass index (BMI). Mean duration of DM1 was 5.4 +/- 3.4 years and mean glycosylated hemoglobin (HbA(1c)) level over 12 months was 9.8 +/- 1.5%. Lumbar and total bone mineral density (BMD, g/cm(2)) were measured by dual-energy X-ray absorptiometry (DXA). We calculated the apparent volumetric lumbar BMD (BMDvol, g/cm(3)) and total mineral content adjusted for age and height (BMCadj), and measured plasma intercellular adhesion molecule-1 (ICAM-1), high sensitivity C-reactive protein (hs-CRP), and urinary 8-iso-prostaglandin F(2a) (F(2)-IsoPs). Calcium (Ca) intake was assessed by questionnaire and physical activity by questionnaire and accelerometer (ActiGraph, count/h). Total BMCadj and lumbar BMDvol were significantly lower in children with DM1 than in controls (101.8 +/- 7.7 vs. 107 +/- 5.7%, P = 0.005; 0.32 +/- 0.08 vs. 0.36 +/- 0.09 g/cm(3), P = 0.05, respectively). These differences were mostly caused by the differences in boys. Plasma ICAM-1 and hs-CRP levels were significantly higher in the DM1 group compared to the controls. Ca intake and urine F(2)-IsoPs levels were similar between the groups. Diabetic boys were less active than controls (18231 +/- 6613 vs. 24145 +/- 7449 count/h, P = 0.04). In the DM1 group, lumbar BMDvol correlated inversely with urinary F(2)-IsoPs (r = -0.5; P = 0.005) and plasma ICAM-1 levels (r = -0.4; P = 0.02), and also with HbA(1c) levels after adjustment for age (r = -0.45; P < 0.05). Total BMCadj correlated inversely with HbA(1c) levels (r = -0.4; P = 0.02). We conclude that children with DM1, particularly boys, have lower BMD. Poor glycemic control, elevated markers of oxidative stress, and inflammation are associated with lower BMD.

Arterial stiffness, carotid artery intima-media thickness and plasma myeloperoxidase level in children with type 1 diabetes.

OBJECTIVE: The present study investigated the functional-structural changes of the arteries along with a new biochemical marker of atherosclerosis, plasma myeloperoxidase level, in children with type 1 diabetes (T1DM). METHODS: We studied 30 children with T1DM, aged 4.7-18.6 years (mean T1DM duration 5.4+/-3.4 years, mean HbA(1c) 9.8%) and 30 healthy subjects, matched by sex, age and body mass index. Fasting blood samples were obtained for myeloperoxidase (MPO). Ultrasonography and pulse wave analysis were used to measure carotid intima-media thickness (IMT), augmentation index corrected to a heart rate of 75 (AIx@75) and pulse wave velocity (PWV). RESULTS: Children with diabetes had significantly higher plasma MPO levels (p=0.006), increased AIx@75 (p=0.02), IMT (p=0.005) and IMT standard deviation scores (IMT SDS) (p=0.02), compared to the control group. IMT SDS correlated positively with HbA(1c) (r=0.39, p=0.05). PWV, adjusted for age and mean arterial blood pressure, correlated with diabetes duration (r=0.49, p=0.02). CONCLUSIONS: Children with T1DM have substantially elevated plasma levels of myeloperoxidase as well as atherosclerosis-related structural and functional changes of the arterial wall.

Elevated plasma adiponectin and decreased plasma homocysteine and asymmetric dimethylarginine in children with type 1 diabetes.

OBJECTIVE: Type 1 diabetes has a bad prognosis concerning the pathogenesis of cardiovascular diseases (CVD). The purpose of this study was to evaluate different possible new risk indices for CVD in children with type 1 diabetes. MATERIAL AND METHODS: The present study included 30 children with diabetes (mean HbA1C 9.8%), aged between 4.7 and 18.6 years and with no clinical evidence of vascular complications, and 30 healthy subjects matched by sex, age and body mass index. Blood pressure was measured and blood samples were obtained for lipid profile, creatinine, glucose, high sensitive C-reactive protein (hsCRP), intercellular adhesion molecule-1 (ICAM-1), asymmetric dimethylarginine (ADMA), adiponectin and homocysteine. RESULTS: Children with diabetes had significantly higher blood pressure, plasma hsCRP, ICAM-1, adiponectin levels and lower homocysteine, ADMA concentrations than their control subjects. In multivariate regression analysis, the best predictors for systolic blood pressure were diabetes group, plasma homocysteine concentration and BMI (Adj R(2) = 0.38, p<0.0001), and for diastolic blood pressure diabetes group and triglycerides level (Adj R(2) = 0.27, p<0.0001). CONCLUSIONS: Children with diabetes, in view of their higher future risk of CVD, are characterized by a higher concentration of protective adiponectin and paradoxically lower blood concentrations of some other possible risk markers of atherosclerosis, i.e. ADMA and homocysteine compared to healthy children.

Increased FOXP3 expression in small-bowel mucosa of children with coeliac disease and type I diabetes mellitus.

OBJECTIVE: To determine whether the expression of FOXP3 is changed in small-bowel mucosa in coeliac disease (CD). MATERIAL AND METHODS: The study comprised 52 patients (mean age 8.01+/-6.14 years) who had undergone small-bowel biopsies. CD only was diagnosed in 16 patients, and CD with type I diabetes mellitus (T1D) in 7. These 23 patients and 4 others without CD had partial or subtotal villous atrophy (PVA, SVA). Twenty-five persons without CD had normal mucosa. The transcription level of the FOXP3 gene (Hs00203958_m1) was evaluated in biopsy samples (small bowel) using TaqMan gene expression assays. FOXP3 protein in mucosal cells was evaluated with mouse anti-human FOXP3 antibodies and CD25(+), and CD4(+) T cells were evaluated by mouse monoclonal antibodies. RESULTS: Expression of FOXP3 mRNA was higher in both PVA and SVA compared to normal mucosa (p=0.007). Patients with CD and T1D had higher expression of FOXP3 mRNA than patients with CD alone (p=0.02). The number of FOXP3(+) cells in intestinal mucosa was higher in patients with CD, especially those with coexisting T1D, than in those with normal mucosa (p=0.01). The results of double staining showed that, among all positive cells, FOXP3 expression alone was revealed in 25.6% of the cells, CD25 positivity in 18% and both markers simultaneously were found in 56.5% of lymphocytes (p=0.03). Double staining for CD4 and FOXP3 showed that 87.5% of cells were CD4(+), 2.8% were FOXP3(+) and 9.7% of cells simultaneously expressed the CD4 and FOXP3 markers. CONCLUSIONS: A more pronounced expression of FOXP3 mRNA and also the number of FOXP3(+) cells (with simultaneous expression of CD25 and CD4 markers) were found in the small-bowel biopsy specimens obtained from children with CD, particularly those with coexisting T1D, compared with the FOXP3 expression in normal mucosa.