Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

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.

Plasma cytokines during pregnancy provide insight into the risk of diabetes in the gestational diabetes risk group.

AIMS/INTRODUCTION: Gestational diabetes (GDM) is characterized by low-grade systemic inflammation, which manifests as changes in the levels of cytokines in the blood. We aimed to investigate plasma immune mediators during gestational weeks 23-28 in 213 women at risk for GDM, and to find associations between GDM and its complications. MATERIALS AND METHODS: We quantified the levels of adipokines: adiponectin, leptin, plasminogen activator inhibitor-1 and resistin; chemokines: C-C motif chemokine ligand 2 (CCL2), CCL4, C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10; and cytokines: granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-1ß, soluble (s)IL-1RI, IL-2, sIL-2Ra, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-27, transforming growth factor (TGF)-ß1, TGF-ß2, TGF-ß3, tumor necrosis factor-α and soluble tumor necrosis factor receptor 2 using the Milliplex®MAP Magnetic Bead assay on Luminex®200™, and compared the results with clinical data from pregnancy and post-partum follow up. RESULTS: Lower levels of adiponectin and higher levels of CCL2 (Wilcoxon test, P = 3.4E-03 and P = 0.03, respectively) were found in women with GDM. IL-27 levels were associated with lower odds of GDM (adjusted logistic regression 0.90, P = 2.4E-03), and showed a risk association with glutamic acid decarboxylase autoantibody positivity (adjusted odds ratio 1.13, P = 2.8E-03). Similarly, higher IL-22 levels increased the odds of glutamic acid decarboxylase autoantibody positivity (adjusted odds ratio 4.23, P = 0.04). TGF-ß1 was associated with post-partum fasting glucose levels, and CCL4 with post-partum C-peptide levels (linear regression, P = 0.04 and P = 0.01, respectively). Women who developed pregnancy complications had higher levels of CXCL10 and CCL4 (linear regression, P = 7.0E-04 and P = 0.01, respectively). CONCLUSIONS: Plasma adiponectin and CCL2 concentrations distinguish women with GDM. IL-27 and IL-22 levels might select women with an autoimmune reaction, whereas increased TGF-ß1 and CCL4 are associated with post-partum glucose and insulin metabolism.

Interleukin-7, T helper 1, and regulatory T-cell activity-related cytokines are increased during the second trimester of healthy pregnancy compared to non-pregnant women.

PROBLEM: Healthy pregnancy is associated with a physiologic increase in inflammatory responses. The objective of this study was to assess changes in plasma cytokines associated with uncomplicated pregnancy. METHOD OF STUDY: To examine these changes, plasma levels of immune response mediators from healthy gravidas (N = 115, gestation weeks 23-30) were compared with those from healthy non-pregnant women (N = 42). Comparisons were performed using multiplex analysis for Th1 activity-related cytokines (IFN-γ, IL-2, sIL-2Rα, IL-12[P70], and IL-27), Th2 activity-related cytokines (IL-4, IL-5, and IL-13), other immune response mediators (GM-CSF, IL-1ß, sIL-1RI, IL-6, IL-8, IL-15, IL-17A, IL-17F, IL-21, IL-22, IL-23, TGFß1, TGFß2, TGFß3, and TNFα), regulatory T cell-related cytokines (IL-10 and sTNFRII), adipokines (adiponectin, leptin, PAI-1, and resistin), chemokines (IP-10, MCP-1, and MIP-1ß), and hematopoietic growth factor IL-7. RESULTS: Multivariate linear regression models showed increased levels of IL-7, Th1-, and Treg activity-related cytokines and decreased levels of adipokines and chemokines in healthy gravidas compared with healthy non-pregnant women. Additionally, season of the year, age, pre-pregnancy body mass index, and HLA-DR/DQ genotypes for type 1 diabetes risk showed different and sometimes reciprocal influence on cytokine levels. CONCLUSION: Our study stresses the importance of profiling immune response mediators during pregnancy to better understand the effect of healthy pregnancy on cytokine levels.

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.

Early-life exposure to common virus infections did not differ between coeliac disease patients and controls.

AIM: Our aim was to compare the presence of various common viruses (rhinovirus, enterovirus, adenovirus, Epstein-Barr virus, cytomegalovirus, norovirus, parechovirus) in stool and nasal swab samples as well as virus-specific antibodies in serum samples between children who developed coeliac disease and controls. METHODS: A case-control study was established based on the DIABIMMUNE Study cohorts. During the study, eight Estonian children and 21 Finnish children aged 1.5 years to five years developed coeliac disease and each was matched with a disease-free control. Nasal swabs and stool samples were taken at the age of three to six months and the serum samples at the time of diagnosis. RESULTS: Rhinovirus ribonucleic acid was detected in the nasal swabs from five coeliac disease children, but none of the control children (p = 0.05). There were no statistically significant differences in the level of viral antibodies between cases and controls. Enterovirus immunoglobulin G class antibodies were found more frequently in the Estonian than in the Finnish children (63% versus 23%, p = 0.02). CONCLUSION: This study did not find any marked overall differences in laboratory-confirmed common viral infections between the children who developed coeliac disease and the controls. However, rhinovirus infections were detected slightly more often in those patients who developed coeliac disease.

Salmon fibrinogen and chitosan scaffold for tissue engineering: in vitro and in vivo evaluation.

3D fibrous scaffolds have received much recent attention in regenerative medicine. Use of fibrous scaffolds has shown promising results in tissue engineering and wound healing. Here we report the development and properties of a novel fibrous scaffold that is useful for promoting wound healing. A scaffold made of salmon fibrinogen and chitosan is produced by electrospinning, resulting in a biocompatible material mimicking the structure of the native extracellular matrix (ECM) with suitable biochemical and mechanical properties. The scaffold is produced without the need for enzymes, in particular thrombin, but is fully compatible with their addition if needed. Human dermal fibroblasts cultured on this scaffold showed progressive proliferation for 14 days. Split-thickness experimental skin wounds treated and untreated were compared in a 10-day follow-up period. Wound healing was more effective using the fibrinogen-chitosan scaffold than in untreated wounds. This scaffold could be applicable in various medical purposes including surgery, tissue regeneration, burns, traumatic injuries, and 3D cell culture platforms.

Unstimulated Adult Human B Cells Include an IL-10+ Population with Suppressive Properties and an Activated Phenotype.

B cells with regulatory properties have been recently identified and described in different immune disorders, including autoimmunity, infection, cancer, and allergy. in vitro studies of regulatory B cells are usually performed following prolonged cell culture and stimulation in order to obtain B cells capable of IL-10 secretion. We describe the isolation of viable IL-10-positive B cells directly from ex vivo unstimulated samples using the IL-10 secretion assay from Miltenyi Biotec, which was originally designed for IL-10-positive T cell analysis and isolation. IL-10-positive B cells from unstimulated samples represented approximately 2% of all B cells in healthy individuals, suppressed T cell proliferation and were enriched for surface markers of B cell activation. This tool has a potential to boost functional studies of IL-10-secreting B cells in health and disease. © 2018 International Society for Advancement of Cytometry.

Avoidance of Cow's Milk-Based Formula for At-Risk Infants Does Not Reduce Development of Celiac Disease: A Randomized Controlled Trial.

BACKGROUND & AIMS: Feeding during the first months of life might affect risk for celiac disease. Individuals with celiac disease or type 1 diabetes have been reported to have high titers of antibodies against cow's milk proteins. Avoidance of cow's milk-based formula for infants with genetic susceptibility for type 1 diabetes reduced the cumulative incidence of diabetes-associated autoantibodies. We performed a randomized controlled trial in the same population to study whether weaning to an extensively hydrolyzed formula reduced the risk of celiac disease autoimmunity or celiac disease. METHODS: We performed a double-blind controlled trial of 230 infants with HLA-defined predisposition to type 1 diabetes and at least 1 family member with type 1 diabetes. The infants were randomly assigned to groups fed a casein hydrolysate formula (n = 113) or a conventional formula (control, n = 117) whenever breast milk was not available during the first 6-8 months of life. Serum samples were collected over a median time period of 10 years and analyzed for antibodies to tissue transglutaminase (anti-TG2A) using a radiobinding assay, to endomysium using an immunofluorescence assay, and antibodies to a deamidated gliadine peptide using an immunofluorometry assay. Duodenal biopsies were collected if levels of anti-TG2A exceeded 20 relative units. Cow's milk antibodies were measured during the first 2 years of life. RESULTS: Of the 189 participants analyzed for anti-TG2A, 25 (13.2%) tested positive. Of the 230 study participants observed, 10 (4.3%) were diagnosed with celiac disease. We did not find any significant differences at the cumulative incidence of anti-TG2A positivity (hazard ratio, 1.14; 95% confidence interval, 0.51-2.54) or celiac disease (hazard ratio, 4.13; 95% confidence interval, 0.81-21.02) between the casein hydrolysate and cow's milk groups. Children who developed celiac disease had increased titers of cow's milk antibodies before the appearance of anti-TG2A or celiac disease. CONCLUSIONS: In a randomized controlled trial of 230 infants with genetic risk factors for celiac disease, we did not find evidence that weaning to a diet of extensively hydrolyzed formula compared with cow's milk-based formula would decrease the risk for celiac disease later in life. Increased titers of cow's milk antibody before anti-TG2A and celiac disease indicates that subjects with celiac disease might have increased intestinal permeability in early life. ClinicalTrials.gov Number: NCT00570102.

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 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.

Changes in Blood B Cell-Activating Factor (BAFF) Levels in Multiple Sclerosis: A Sign of Treatment Outcome.

Multiple sclerosis (MS) is mediated primarily by autoreactive T cells. However, evidence suggesting the involvement of humoral immunity in brain diseases has increased interest in the role of B cells and their products during MS pathogenesis. The major survival factor for B cells, BAFF has been shown to play a role in several autoimmune conditions. Elevated BAFF levels have been reported in MS animal model and during MS relapse in patients. Moreover, disease-modifying treatments (DMT) reportedly influence blood BAFF levels in MS patients, but the significance of these changes remains unclear. The present study addresses how blood BAFF levels are associated with the clinical course of relapsing-remitting MS and the effectiveness of DMT and short-term steroid treatment. During a prospective longitudinal follow-up of 2.3 years, BAFF was measured in the blood of 170 MS patients in the stable phase and within 186 relapses. BAFF levels were significantly higher in MS patients compared to healthy controls. However, stable MS patients without relapses exhibited significantly higher BAFF levels than relapsing patients. Treatment with interferon-ß and immunosuppressants raised BAFF blood levels. Interestingly, a similar effect was not seen in patients treated with glatiramer acetate. Short-term treatment with high doses of intravenous methylprednisolone did not significantly alter plasma BAFF levels in 65% of relapsing-remitting MS patients. BAFF were correlated weakly but significantly with monocyte and basophil counts, but not with other blood cell types (neutrophils, lymphocytes, or eosinophils) or inflammatory biomarkers. To our knowledge, this is the first report demonstrating that higher blood BAFF levels may reflect a more stable and effective MS treatment outcome. These results challenge hypotheses suggesting that elevated blood BAFF levels are associated with more severe disease presentation and could explain the recent failure of pharmaceutical trials targeting BAFF with soluble receptor for MS treatment.

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.

Low prevalence of IgA anti-transglutaminase 1, 2, and 3 autoantibodies in children with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a multifactorial chronic inflammatory skin disease presenting with a relapsing clinical pattern similar to chronic autoimmune disease. Several human transglutaminases have been defined and keratinocyte transglutaminase (TG1) and epidermal transglutaminase (TG3) expressed in the epidermis are associated with epidermal barrier dysfunction. Since impairments to the epidermal barrier represent an important factor in AD, we hypothesized that IgA autoantibodies specific for TG1 (IgA-anti-TG1) and TG3 (IgA-anti-TG3) may affect AD development during childhood. METHODS: Active AD patients (n = 304), 28 patients with biopsy-confirmed coeliac disease (CD), 5 patients with active AD and CD, and 55 control patients without CD and skin diseases were enrolled into the study. IgA-anti-TG1 and IgA-anti-TG3 reactivity was determined using an enzyme-linked immunosorbent assay. IgA-anti-TG2 were defined using a fluoroenzyme immunoassay. RESULTS: IgA-anti-TG1 antibodies were found in 2% and IgA-anti-TG3 antibodies in 3% of patients with active AD. Two out of the 5 patients with AD and concomitant CD had IgA-anti-TG1 and IgA-anti-TG2 antibodies. In CD patients, 36% of individuals presented with elevated IgA-anti-TG1 antibodies and 18% presented with elevated IgA-anti-TG3 antibodies and all CD patients presented with IgA-anti-TG2 antibodies (significantly different from AD patients and controls, p < 0.05). In CD patients, IgA-anti-TG1 and/or IgA-anti-TG3 seropositivity tended to appear concurrently, whereas only one patient with AD had both types of autoantibodies. CONCLUSIONS: IgA-anti-TG1 and IgA-anti-TG3 seropositivity was rare in active AD but frequent in CD patients. The level of circulating antibodies related to skin lesions could be studied by determining the levels of IgA-anti-TG1 and IgA-anti-TG3 in skin biopsies of AD patients.

Autoimmune activation toward embryo implantation is rare in immune-privileged human endometrium.

Human embryo implantation represents embryo apposition, adhesion to the endometrial epithelium, and invasion into the stromal extracellular matrix within 1 to 2 days during days 6 to 9 after ovulation. The major molecular mechanisms mediating implantation include adhesion molecules, including mucins, selectins, integrins, and cadherins; extracellular matrix components, such as laminins and collagens and their degrading enzymes; phospholipids and immune regulatory molecules, including prostaglandins, cytokines; and immunosuppressive molecules expressed by invasive trophoblasts and endometrial cells. Many of these molecules are the targets for autoimmune reactions in autoimmune diseases and cancer; however, the relevance of those in immune-mediated implantation failure has not been defined. In this review, we will describe the molecules involved in 2-day event of human embryo implantation, which may also be involved in immune system activation and subsequently cause immune-mediated implantation failure. We speculate that the data in the literature are limited concerning antiendometrial antibodies because the endometrium might be taken as an immune-privileged site that avoids autoimmune activation that might harm the implantation process. Antibodies affecting human fertility in ways other than impairing implantation are outside the scope of the current article and will not be discussed.

Celiac disease in children with atopic dermatitis.

Celiac disease (CD) is an autoimmune disorder of the small intestine with highly variable clinical presentation and frequently associated with various immune-mediated diseases. Among these immune-mediated diseases, atopy has been found frequently in individuals with CD. We aimed to study the prevalence of CD in Estonian children with atopic dermatitis (AD), a common multifactorial chronic inflammatory skin disease. We recruited 351 consecutive children with active AD (mean age 5.8 yrs, 57.6% boys) at Tallinn Children's Hospital, Estonia. Sera of all patients were tested for total serum immunoglobulin (Ig) A, for IgA- and IgG-type autoantibodies to tissue transglutaminase (IgA-anti-TG2, IgG-anti-TG2) and to deamidated gliadin peptides (IgA-anti-DGP, IgG-anti-DGP). The diagnosis of CD was confirmed histologically by small intestine biopsy according to the European Society of Paediatric Gastroenterology, Hepatology and Nutrition diagnostic criteria. IgA deficiency was detected in nine patients with AD (2.6%), none of whom had IgG-anti-TG2 or IgG-anti-DGP seropositivity. IgA-anti-TG2 positivity was found in 4 (1.1%), IgG-anti-TG2 positivity in 2 (0.6%), IgA-anti-DGP positivity in 11 (3.1%), and IgG-anti-DGP in 10 (2.8%) patients. Celiac disease was confirmed in five (1.4%) patients with AD (95% confidence interval 0.46, 3.32) and all were histologically characterized as Marsh IIIa-IIIc stages and two presented with silent CD. In AD patients, CD prevalence was more than four times as high as in previously studied randomly selected schoolchildren in Estonia. Two patients with AD diagnosed with CD had no symptoms indicative of CD, in spite of extensive histologic changes in the small intestine mucosa. Therefore our study emphasizes the need for evaluating the cost-effectiveness of screening individuals with AD for CD in time to prevent long-term complications.

Survivin promoter polymorphisms and autoantibodies in endometriosis.

Expression of survivin, an inhibitor of apoptosis, is increased in endometriotic lesions and probably favors the survival of endometrial fragments in the peritoneal cavity. The aim of this study was to evaluate associations between survivin promoter polymorphisms and the risk of endometriosis, as well as to compare the immunoreactivity to survivin in sera of patients with and without endometriosis. We studied 149 women with endometriosis, 196 fertile women from the general population (control group A) and 47 women who had undergone diagnostic laparoscopy and had no evidence of endometriosis (control group B). There were no significant differences in the genotypic distribution of the survivin gene promoter region -241C/T, -235G/A and -31G/C single nucleotide polymorphisms (SNP) between endometriosis patients and the two control groups. In addition, also median anti-survivin autoantibody levels were similar among patients and controls (group B). However, anti-survivin antibody concentrations seemed to be influenced by cigarette smoking, being significantly lower in sera of actively smoking women compared to non-smokers (median OD: 0.019 vs. 0.155, respectively, P<0.001), and by the -235G/A SNP, as A allele carriers were significantly more frequent among women with a high antibody level (OD≥2.0) compared to those with lower concentrations (OD<2.0) (23.1% vs. 4.1%, respectively, P=0.008). Based on these results, we conclude that survivin promoter polymorphisms are not associated with susceptibility to endometriosis in the Estonian population, and though the expression of survivin is increased in endometriotic lesions, autoimmune reactivity against it is similar in women with and without the disease.

Kinetic and functional characterisation of the heparin-binding peptides from human transglutaminase 2.

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin-binding regions of human TG2 by studying binding kinetics of the predicted heparin-binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high-affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202-215) and P2 (261-274), were found to bind heparin. The amino acid sequences corresponding to the heparin-binding peptides were located close to each other on the surface of the TG2 molecule as part of the α-helical structures. The heparin-binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco-2 intestinal epithelial cell attachment to the FN and FN-TG2 coated surfaces. We propose that TG2 amino acid sequences 202-215 and 261-274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin-binding peptides of TG2 with CD patient's IgA supports the previously described role of anti-TG2 autoantibodies interfering with this interaction.

A 10-year serological follow-up of celiac disease in an Estonian population.

BACKGROUND: Celiac disease (CD) is induced by wheat gluten and related prolamines. Its prevalence may be underestimated in many geographic regions and populations, and has recently increased in several countries. In 1998 and 1999, a random sample of Estonian schoolchildren was screened with IgA-type tissue transglutaminase antibodies (IgA-tTG) for CD. The results revealed a CD prevalence of 0.34%, which is lower compared with many other European countries. OBJECTIVE: We rescreened the same population for CD using IgA-tTG after a 10-year interval. MATERIALS AND METHODS: A total of 891 patients from the initial sample were rescreened using the IgA-tTG assay for a participation rate of 76.8% (median age, 24.3 years). As in the initial study, the IgA-tTG results were evaluated by ImmunoCAP EliA Celikey using an IgG-tTG and deamidated gliadin antibody assay for IgA-deficient cases. RESULTS: No new cases of CD were found in this follow-up study. Of note, 75% of patients with initial IgA-tTG-positive results and biopsy-proven CD remained seropositive. One patient with a negative seroconversion at the time of rescreening followed a strict gluten-free diet during the follow-up years. CONCLUSION: In a 10-year follow-up period, no new cases of CD were found in this Estonian population of school-children and young adults. Therefore, we presume no increase in CD during the last decade among this age group in Estonia. Additional studies are needed to determine whether similar results would be obtained in other age groups, because of differences in the CD prevalence between Estonian and other European populations.