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.

Increased levels of anti-BSA antibodies in children with Down syndrome.

Introduction: Autoimmune diabetes occurs more often in the first 2 years of life in children with Down syndrome (DS) compared with the general population. We previously observed increased frequencies of islet autoantibodies, including insulin autoantibodies (IAA), in children with DS. Assays for IAA using 125I-labelled insulin require competition to overcome cross reactivity with antibodies to the cow's milk protein, bovine serum albumin (BSA). 125I-IAA assay results suggested that levels of antibodies to BSA may also be increased in children with DS. The aim of this study therefore was to determine whether the levels of anti-BSA antibodies differed in children with DS compared with controls. Methods: Samples were available from two populations with DS: one from the UK, (UK DS cohort n=106, 58 male, median age 12.5 years) and one from Estonia (Estonian DS cohort: n=121, 65 male, median age 9.75 years). A UK control population was provided by sex and age-matched healthy siblings of probands participating in the Bart's Oxford (BOX) family study of type 1 diabetes. A competitive-displacement radiobinding assay (RBA) and a Dissociation Enhanced Lanthanide Fluoroimmunoassay (DELFIA) were developed to measure and confirm anti-BSA antibody levels. HLA class II genotype was analysed by PCR using sequence specific primers (PCR-SSP). Results: Overall, levels of anti-BSA antibodies were increased in those with DS compared with controls (p<0.0001) but this was not HLA associated. Conclusion: Increased levels of anti-BSA antibodies may reflect a defect in immune maturation or increased gut permeability in children with DS, increasing their risk of developing autoimmunity.

Profiling of peripheral blood B-cell transcriptome in children who developed coeliac disease in a prospective study.

Background: In coeliac disease (CoD), the role of B-cells has mainly been considered to be production of antibodies. The functional role of B-cells has not been analysed extensively in CoD. Methods: We conducted a study to characterize gene expression in B-cells from children developing CoD early in life using samples collected before and at the diagnosis of the disease. Blood samples were collected from children at risk at 12, 18, 24 and 36 months of age. RNA from peripheral blood CD19+ cells was sequenced and differential gene expression was analysed using R package Limma. Findings: Overall, we found one gene, HNRNPL, modestly downregulated in all patients (logFC -0·7; q = 0·09), and several others downregulated in those diagnosed with CoD already by the age of 2 years. Interpretation: The data highlight the role of B-cells in CoD development. The role of HNRPL in suppressing enteroviral replication suggests that the predisposing factor for both CoD and enteroviral infections is the low level of HNRNPL expression. Funding: EU FP7 grant no. 202063, EU Regional Developmental Fund and research grant PRG712, The Academy of Finland Centre of Excellence in Molecular Systems Immunology and Physiology Research (SyMMyS) 2012-2017, grant no. 250114) and, AoF Personalized Medicine Program (grant no. 292482), AoF grants 292335, 294337, 319280, 31444, 319280, 329277, 331790) and grants from the Sigrid Jusélius Foundation (SJF).

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.

IgA-Type Enterovirus Antibodies Are Increased among Adults and Children with Recently Diagnosed Type 1 Diabetes.

Enteroviruses (EV) are among the leading environmental triggers of childhood-onset type 1 diabetes (T1D). Our aim was to determine the prevalence of antibodies against EV and their association with T1D in different age groups (n = 62), including young adults, and to compare these data with results from HLA-matched control participants (n = 62). IgA, IgG, and IgM antibodies against EV were detected. IgA EV antibodies were present in 46.8% of participants with T1D (median level 10.9 EIU) and in 11.3% of controls (median level 3.4 EIU). IgA EV positivity and higher level of IgA EV antibodies were both significant risk factors for T1D (odds ratio (OR) 8.33; 95% confidence interval (CI) 2.52-27.6; p = 0.0005 and OR 1.04; 95% CI 1.01-1.06; p = 0.0105, respectively). Importantly, the prevalence of IgA EV antibodies in the subgroups of both children and young adults was also significantly different between participants with T1D and their matched controls (p = 0.0089 and p = 0.0055, respectively). Such differences were not seen for IgG and IgM EV antibodies. However, IgG EV antibodies were associated with 65 kDa glutamic acid decarboxylase antibodies, but not with zinc transporter 8 and protein tyrosine phosphatase IA2 antibodies. The genotype frequency of PTPN22 (rs2476601) and IFIH1 (rs1990760) was not associated with EV positivity. This study showed that EV infections may be an important disease-promoting factor of T1D not only in childhood-onset but also in adult-onset T1D. However, to further confirm this association, direct virological studies are needed in the latter T1D group.

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.

Growth of MIN-6 Cells on Salmon Fibrinogen Scaffold Improves Insulin Secretion.

The incidence of type I diabetes has been increasing worldwide at an annual rate of approximately 3%. One of the strategies to treat type I diabetes is islet transplantation, in which damaged ß-cells are replaced with new islets. To improve ß-cells' expansion and pseudoislet formation, studies are focusing on using extracellular-matrix-resembling substrates. We evaluated the potential of salmon fibrinogen and chitosan electrospun scaffold as cell substrate for cultivating MIN-6 cells. The morphology of cells, insulin secretion and gene expression was evaluated and compared with other substrates (nanofibrous scaffold, microporous scaffold and tissue culture polystyrene). We found that all tested 3D conditions favored the pseudoislet formation of MIN-6 cells. The insulin secretion of MIN-6 cells after stimulation with high-glucose media shows approximately a 9-fold increase compared to the control group when a fibrinogen/chitosan-based electrospun scaffold was used for cultivation. The differences in insulin secretion were corroborated by differences in gene expression. The differences in insulin secretion could probably be attributed to the differences in the mechanical and/or chemical nature of the tested substrates.

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.

Maternal breast milk microbiota and immune markers in relation to subsequent development of celiac disease in offspring.

The potential impact of the composition of maternal breast milk is poorly known in children who develop celiac disease (CD). The aim of our study was to compare the microbiota composition and the concentrations of immune markers in breast milk from mothers whose offspring carried the genetic predisposition to CD, and whether they did or did not develop CD during follow-up for the first 3 years of life. Maternal breast milk samples [CD children (n = 6) and healthy children (n = 18)] were collected 3 months after delivery. Enzyme-linked immunosorbent assays were used to measure TGF-ß1, TGF-ß2, sIgA, MFG-E8 and sCD14. For microbiota analysis, next generation (Illumina) sequencing, real-time PCR and denaturing gradient gel electrophoresis were used. Phylotype abundance and the Shannon 'H' diversity index were significantly higher in breast milk samples in the CD group. There was higher prevalence of the phyla Bacteroidetes and Fusobacteria, the classes Clostridia and Fusobacteriia, and the genera Leptotrichia, Anaerococcus, Sphingomonas, Actynomyces and Akkermansia in the CD group. The immunological markers were differently associated with some Gram-negative bacterial genera and species (Chryseobacterium, Sphingobium) as well as Gram-positive species (Lactobacillus reuteri, Bifidobacterium animalis). In conclusion, the microbiota in breast milk from mothers of genetically predisposed offspring who presented CD showed a higher bacterial phylotype abundance and diversity, as well as a different bacterial composition, as compared with the mothers of unaffected offspring. These immune markers showed some associations with bacterial composition and may influence the risk for development of CD beyond early childhood.

Epigenetic quantification of immunosenescent CD8+ TEMRA cells in human blood.

Age-related changes in human T-cell populations are important contributors to immunosenescence. In particular, terminally differentiated CD8+ effector memory CD45RA+ TEMRA cells and their subsets have characteristics of cellular senescence, accumulate in older individuals, and are increased in age-related chronic inflammatory diseases. In a detailed T-cell profiling among individuals over 65 years of age, we found a high interindividual variation among CD8+ TEMRA populations. CD8+ TEMRA proportions correlated positively with cytomegalovirus (CMV) antibody levels, however, not with the chronological age. In the analysis of over 90 inflammation proteins, we identified plasma TRANCE/RANKL levels to associate with several differentiated T-cell populations, including CD8+ TEMRA and its CD28- subsets. Given the strong potential of CD8+ TEMRA cells as a biomarker for immunosenescence, we used deep-amplicon bisulfite sequencing to match their frequencies in flow cytometry with CpG site methylation levels and developed a computational model to predict CD8+ TEMRA cell proportions from whole blood genomic DNA. Our findings confirm the association of CD8+ TEMRA and its subsets with CMV infection and provide a novel tool for their high throughput epigenetic quantification as a biomarker of immunosenescence.

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes.

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 × 10-8) with GDM, mapping to/near MTNR1B (P = 4.3 × 10-54), TCF7L2 (P = 4.0 × 10-16), CDKAL1 (P = 1.6 × 10-14), CDKN2A-CDKN2B (P = 4.1 × 10-9) and HKDC1 (P = 2.9 × 10-8). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.

Higher circulating EGF levels associate with a decreased risk of IgE sensitization in young children.

BACKGROUND: Decreased exposure to microbial agents in industrialized countries and urban living areas is considered as a risk factor of developing immune-mediated diseases, such as allergies and asthma. Epithelial surfaces in the gastrointestinal and respiratory tracts and in the skin constitute the primary areas in contact with the environmental microbial load. METHODS: We analyzed the levels of 30 cytokines and growth factors in serum or plasma as markers of the immune maturation in the participants in the DIABIMMUNE study from Russian Karelia (n = 60), Estonia (n = 83) and Finland (n = 89), three neighboring countries with remarkable differences in the incidences of allergies, asthma and autoimmune diseases. RESULTS: We observed an upregulation of T helper cell signature cytokines during the first 12 months of life, reflecting natural development of adaptive immune responses. During the first years of life, circulating concentrations of epidermal growth factor (EGF) were significantly higher, especially in Russian children compared with Finnish children. The children who developed IgE sensitization showed lower levels of EGF than those without such responses. CONCLUSION: Our results suggest that low circulating EGF levels associate with the risk of allergies possibly via the effects on the epithelial integrity and mucosal homeostasis.

Human CD4+ and CD8+ T lymphocyte subpopulations have significantly different surface expression patterns of CD226 and TIGIT molecules.

CD226 and the inhibitory T-cell immunoglobulin and ITIM domain (TIGIT) belong to a co-stimulatory receptor system found in both T and natural killer cells. Although data from genome-wide studies have suggested a strong association between the CD226 locus and multiple autoimmune diseases, the understanding of the balance of CD226/TIGIT axis during the activation of human T-cell subpopulation remains incomplete. In this study, we aimed to compare pre- and post-stimulation expression profiles of CD226 and TIGIT with those of CD28 in human CD4+ and CD8+ T-cell subpopulations using flow cytometry. The impact of the CD226 single nucleotide polymorphism, rs763361, on cell surface CD226 expression and effector cytokine secretion was also examined. Peripheral blood mononuclear cells from healthy blood donors (n = 65) were studied. Most naïve CD4+ and CD8+ T-cells did not express CD226 and TIGIT, predominantly upregulating activating receptors following stimulation. Memory CD4+ T-cells exhibited a balanced expression of activating and inhibitory receptors, pre- and post-stimulation. In contrast, memory CD8+ T-cells predominantly expressed TIGIT. The rs763361 TT genotype was associated with both a reduction in CD226 expression on the cell surface of CD4+ memory T-cells (P = .004) and increased interleukin-17A secretion from activated T-cells (P = .036). Description of different expression patterns on T lymphocyte subpopulations provided in this work will lead to a more comprehensive understanding of the role of the CD226/TIGIT axis in control over T-cell activation and suppression.

Quality and best practice in medical laboratories: specific requests for autoimmunity testing.

Special conditions associated with laboratory autoimmune testing are not well compatible with recent developments in regulatory frameworks such as EN/ISO 15189 accreditation or in vitro diagnostic medical device regulation (IVD-R). In addition, international recommendations, guidelines and disease criteria are poorly defined with respect to requirements on autoantibody testing. Laboratory specialists from Austria, Belgium, Croatia, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Norway, Poland, Portugal, South Africa, Spain, Sweden, Switzerland, and The Netherlands collected information, reported national experience, and identified quality issues in relation to autoantibody testing that require consensus on interpretation of the regulatory frameworks and guidelines. This process has been organized by the European Autoimmunity Standardisation Initiative (EASI). By identifying the critical items and looking for a consensus, our objective was to define a framework for, in particular, EN/ISO accreditation purposes. Here, we present a review of current publications and guidelines in this field to unify national guidelines and deliver in this way a European handout on quality control and accreditation for laboratories involved in autoantibody testing. We focus on quality items that can be checked during accreditation visits. Despite various local varieties, we encountered an overwhelming dedication to quality assurance in all contributing countries.

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.

Patients with down syndrome have increased prevalence of rheumatoid factor but not autoantibodies to anti-cyclic citrullinated peptide.

The association between Down syndrome (DS), a genetic disorder resulting from trisomy of the 21st chromosome, and the autoantibodies of rheumatoid arthritis (RA) has been proposed but not unequivocally proven. The aim of this study was to determine whether adult patients with DS present higher levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies and/or rheumatoid factor (RF) than the general population. Our results showed that none of the 68 patients with DS had anti-CCP antibodies, whereas among 204 age- and sex-matched controls these autoantibodies were present in one person. However, DS patients presented a higher number of RF positive cases than controls (11.7% to 3.2% respectively; Fisher's exact test, p = .027). The higher number of RF positive cases in the DS group without increase of anti-CCP antibodies may be indicative of immune disturbances in general rather than RA in these patients. Our study supports the view that RA does not occur with higher frequency in patients with DS than in the general population.

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.