FABP4 and I-FABP Levels in Pregnant Women Are Associated with Body Mass Index but Not Gestational Diabetes.

Objectives: Gestational diabetes mellitus (GDM) is glucose intolerance detected initially during pregnancy. GDM poses an increased risk for the development of diabetes later in life. Fatty acid-binding protein 4 (FABP4) is a regulator of lipid metabolism and is associated with obesity, insulin resistance, and type 2 diabetes. Increased level of intestinal fatty acid-binding protein (I-FABP) may indicate impaired intestinal permeability, which may be an important contributor to the pathogenesis of type 1 diabetes and GDM. We aimed to compare FABP4 and I-FABP levels in pregnant women with GDM and in healthy pregnant controls, taking into consideration their prepregnancy body mass index (BMI), past exposures to enteroviruses (EV), and adipokine and cytokine levels, which have been shown to decrease insulin sensitivity. Material and Methods. Forty patients with GDM (median age 30.5) and 40 pregnant healthy controls (median age 31.1) were divided on the basis of their prepregnancy BMI into two groups: normal weight (BMI < 25, n = 20) and overweight (BMI ≥ 25, n = 20). FABP4 and I-FABP were measured from serum samples using commercial ELISA kits. Results: FABP4 and I-FABP levels did not differ between women with GDM and healthy pregnant controls (p > 0.05 for both comparisons). However, both levels were associated with BMI (p < 0.001 for both comparisons). Median I-FABP level was the highest in healthy controls with lower BMI (<25) (p = 0.0009). FABP4 levels correlated with BMI and C-peptide values in both groups (p < 0.001). Anti-EV antibody levels did not correlate with FABP4 or I-FABP levels. FABP4 and adiponectin levels were negatively correlated in controls (r = -0.61, p = 0.0009), while I-FABP correlated positively with adiponectin (r = 0.58, p = 0.04) and resistin (r = 0.67, p = 0.04) levels in the GDM group. Conclusion: FABP4 and I-FABP levels were not dependent on the diagnosis of GDM, but rather on BMI. The correlation of I-FABP with adiponectin and resistin levels in women with GDM may suggests the importance of lipid metabolism in GDM-associated changes in intestinal permeability.

Celiac Disease in Children, Particularly with Accompanying Type 1 Diabetes, Is Characterized by Substantial Changes in the Blood Cytokine Balance, Which May Reflect Inflammatory Processes in the Small Intestinal Mucosa.

Cytokines play a pivotal role in the maintenance of intestinal homeostasis inducing pro- or anti-inflammatory response and mucosal barrier function in celiac disease (CD) and type 1 diabetes (T1D). We aimed to compare the levels of pro- and anti-inflammatory cytokines in CD patients without and with coexisting T1D, as well as to evaluate its association with the presence of enteroviruses (EV), regulatory T cells (Tregs), and dendritic cells (DCs) in small bowel mucosa. Altogether, 72 patients (median age 10.1 years) who had undergone small bowel biopsy were studied. The study group consisted of 24 patients with CD (median age 6.5 years), 9 patients with CD and concomitant T1D (median age 7.0 years), two patients with T1D (median age 8.5 years), and 37 patients (median age 14.0 years) with functional gastrointestinal disorders (FGD) and a normal small bowel mucosa as controls. The levels of 33 cytokines in serum were measured by multiple analysis using the Milliplex® MAP Magnetic Bead assay. The densities of FOXP3+ Tregs, CD11c+ DC, indoleamine 2,3-dioxygenase+ (IDO+) DC, langerin+ (CD207+) DCs, and EV were evaluated by immunohistochemistry as described in our previous studies. Circulating anti-EV IgA and IgG were evaluated using ELISA. The most important finding of the study is the significant increase of the serum levels of IL-5, IL-8, IL-13, IL-15, IL-17F, IL-22, IL-27, IP-10, MIP-1ß, sIL-2Rα, sTNFRII, and TNFα in CD patients compared to controls and its correlation with the degree of small bowel mucosa damage graded according to the Marsh classification. The leptin level was higher in females in all study groups. The levels of IL-2, IL-6, IL-12 (P70), IL-15, IP-10, and IFNγ correlated significantly with the density of FOXP3+ Tregs in lamina propria of the small bowel mucosa, which supports the evidence about the signaling role of these cytokines in the peripheral maintenance of FOXP3+ Tregs. At the same time, a significant negative correlation occurred between the level of IL-4 and density of FOXP3+ Tregs in controls. Another important finding of our study was the correlation of IL-17F, IP-10, sTNFRII, MCP-1, and GM-CSF with the density of EV-positive cells in the lamina propria of the small bowel mucosa. Correlation of MIP-1 (CCL-4) with CD103+ DC and langerin+ DC densities may point to their significance in the recruitment of immune cells into the lamina propria and in driving the inflammatory response in CD patients. Our results suggest the predominance of Th1 and Th17 immune responses over EV VP1 protein in CD and T1D patients. The significant elevation of Th2 cytokines, like IL-5 and IL-13, but not IL-4, in CD patients and its correlation with the degree of small bowel mucosa damage could reflect the role of these cytokines in gut defense and inflammation.

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.

The impact of langerin (CD207)+ dendritic cells and FOXP3+ Treg cells in the small bowel mucosa of children with celiac disease and atopic dermatitis in comparison to children with functional gastrointestinal disorders.

In the present study we aimed to evaluate the impact of langerin (CD207)+ dendritic cells (DCs) and FOXP3+ Treg cells in the intestinal mucosa of children with celiac disease (CD) and atopic dermatitis (AD) in comparison to children with functional gastrointestinal disorders (FGD). Seventy-five children (37 male, mean age 8.4 ± 4.8 years), who randomly underwent small bowel biopsy, were studied. The CD was diagnosed in 14 children, including five persons with concomitant AD (all positive for anti-tissue transglutaminase IgA antibodies and with small bowel atrophy). Normal small bowel mucosa was found in eight patients with AD and in 53 patients with FGD. The sera of all patients were tested for total and specific IgE antibodies to food allergen panels. Staining for CD11c+, langerin (CD207+) DCs, CD4+, and FOXP3+ Treg cells was performed on paraffin-embedded sections of bioptates using immunohistochemistry. The density of CD11c+ DCs, CD4+, and FOXP3+ Treg cells was higher in the CD patients compared to the AD and FGD patients (p = 0.02; p = 0.001). In AD, significantly higher density of CD11c+ DCs was detected in patients positive for specific IgE to food allergen panels (p = 0.02). The FGD patients with elevated total IgE had increased density of langerin (CD207)+ DCs compared to the patients with normal total IgE levels (p = 0.01). The increased density of FOXP3+ Treg cells, CD4+, cells and CD11c+ DCs was associated with CD but not with AD. The elevated level of total IgE or specific IgE to food allergens was associated with more pronounced expression of DCs, indicating a possible link between the presence of these cells in small bowel mucosa with elevated level of serum IgE.

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.

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.

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.

Helicobacter pylori immunology and vaccines.

Helicobacter pylori infection causes chronic gastritis, peptic ulcer, and gastric cancer. Colonization of H. pylori in the stomach activates Toll-like and Nod-like receptors to induce not only innate immunity but also adaptive Th1 responses against this organism. Adaptive Th1 response is not sufficient to clear this organism and, as a result, the infection persists. Insufficient adaptive immunity can be explained by poor activation of Toll-like receptors, suppressive effects of bacterial factors, and induction of regulatory T-cell responses. Significant progress in the understanding of innate and adaptive immunity against H. pylori was made during the past year. Recent findings in the fields of vaccines for H. pylori are also reviewed.

Immune response to Helicobacter pylori and its association with the dynamics of chronic gastritis in the antrum and corpus.

The aim of the study was to establish possible factors which play a role in progression of gastritis to atrophic gastritis in long-term follow-up among the Estonian population, to assess the association between the host immune response and different Helicobacter pylori antigens and autoantigens in relation to the histological parameters of gastritis in the antrum and corpus. ELISA and immunoblot were used for detection of IgG to H. pylori acid glycine-extracted cell surface proteins, CagA protein, and H. pylori HSP60. Anticanalicular autoantibodies (ACAB) in the serum were evaluated according to Faller et al. (1996). Apoptosis was evaluated using the TUNEL method. Study subjects were 1958 persons from an unselected Estonian population, and 70 persons from a sample from Saaremaa Island, who had been investigated over a period of 18 years. Seropositivity for CagA was a sign of gastritis activity [OR=14.8 (4.5-50.3)] and atrophy [OR=7.0 (2.1-23.1)] and might predict development of atrophy, particularly in the corpus [OR=7.1 (1.8-27.7)]. The prevalence of ACAB increased significantly with duration of H. pylori gastritis from 22% in 1985 to 46% in 1997 (p=0.004). Immune response to H. pylori HSP60 indicates chronic inflammation in the antrum (p=0.003). Apoptosis of gastric epithelial cells is largely dependent on grade of activity of gastritis, and, particularly in the antrum, on grade of H. pylori colonization (p=0.01; p=0.02), but is not associated with development of atrophy. Seropositivity for different H. pylori antigens (CagA, HSP 60) serves as a marker of different histological manifestations in the antrum and corpus mucosa.

Response of IgG1 and IgG2 subclasses to Helicobacter pylori in subjects with chronic inflammation of the gastric mucosa, atrophy and gastric cancer in a country with high Helicobacter pylori infection prevalence.

Polarized immune response to Helicobacter pylori and induction of chronic inflammation may increase the risk of gastric atrophy and adenocarcinoma. We studied the association of the response of IgG1 and IgG2 antibodies to H. pylori with grade of gastric chronic inflammation and atrophy in a population with a high prevalence of H. pylori, and compared these data with the data obtained from the study of gastric cancer patients, as well as with the data for CagA positivity. Altogether, 114 persons from two adult population samples from Estonia and 45 consecutive gastric cancer patients were studied. All patients were positive for the H. pylori antibody determined by ELISA. Adenocarcinoma was classified histologically according to the Laurén's system. The response of the IgG subclasses to H. pylori (acid glycine-extracted whole cell proteins) was determined by ELISA and the results were compared with the ELISA results for the recombinant fragment of the CagA protein. Helicobacter pylori IgG level was lower in atrophic gastritis compared with nonatrophic gastritis (chronic inflammation) (p=0.001). In the group of cancer patients, the response of IgG and IgG1 was lower compared with both gastritis groups (p=0.01 and p=0.0002 for IgG, and p=0.001 and p=0.0005 for IgG1). IgG2 was lower for gastric cancer localized in the corpus (p=0.03). In conclusion, atrophic gastritis and gastric cancer were associated with a significant decline in IgG and IgG1 response to H. pylori compared with nonatrophic gastritis. Higher value of CagA antibodies was seen in gastric cancer and in gastric atrophy compared with nonatrophic gastritis; in gastric cancer patients, IgG1 response to H. pylori was correlated with CagA status.

Helicobacter pylori infection in children in Estonia: decreasing seroprevalence during the 11-year period of profound socioeconomic changes.

BACKGROUND: The prevalence of Helicobacter pylori infection is inversely associated with socioeconomic conditions in childhood. In Estonia, a high prevalence of H. pylori infection has been observed among children born in 1987 and earlier. Since 1991, after the dissolution of the USSR, profound social and economic changes have taken place in the country. The aim of the study was to evaluate changes in the seroprevalence of H. pylori infection among children in the period 1991-2002. MATERIALS AND METHODS: The hospital-based study population consisted of two groups of children enrolled in 1991 (n = 425) and 2002 (n = 296) according to the same inclusion criteria. The immunoglobulin G antibodies to the cell surface proteins of H. pylori were determined by enzyme-linked immunosorbent assay, and the sera with the borderline results were analyzed by immunoblot analysis. Multiple regression analysis was used to determine the associations between H. pylori seropositivity and different variables such as demographic characteristics, diagnoses and year of enrollment. RESULTS: The only two variables linked independently to H. pylori serostatus were age and year of enrollment: the adjusted odds of being H. pylori seropositive were 1.92 [95% confidence interval (CI) 1.33-2.76] times higher for the children enrolled in 1991 compared with the children enrolled in 2002. The age-standardized seroprevalence rate was 42.2% (95% CI 37.4-47.0%) for the group of 1991 and 28.1% (95% CI 23.1-33.6%) for the group of 2002. CONCLUSION: The prevalence of H. pylori infection among children has significantly decreased during the 11-year period of profound socioeconomic changes in Estonia.

Immune responses to bile-tolerant helicobacter species in patients with chronic liver diseases, a randomized population group, and healthy blood donors.

Bile-tolerant Helicobacter species such as Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD) in humans. Antibody responses to cell surface proteins of H. pullorum, H. bilis, and H. hepaticus in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to Helicobacter pylori. For analysis of a possible cross-reactivity between bile-tolerant Helicobacter species and H. pylori, sera from a subpopulation of each group were absorbed with a whole-cell extract of H. pylori and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for H. pullorum was significantly higher in patients with CLD than in healthy blood donors (P = 0.01). Antibody reactivity to cell surface protein of H. hepaticus was also significantly higher in the CLD patients than in the healthy blood donors and the population group (P = 0.005 and P = 0.002, respectively). Following the absorption, antibody responses to H. pullorum decreased significantly in all three groups (P = 0.0001 for CLD patients, P = 0.0005 for the population group, and P < 0.0001 for the blood donors), indicating that cross-reactivity between H. pylori and other Helicobacter spp. occurs. The antibody responses to H. hepaticus and H. bilis in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance of this finding requires further investigations.