Anti‐C1q autoantibodies are linked to autoimmune thyroid disorders in pregnant women

Anti‐C1q antibodies (anti‐C1q) have been implicated in the pathogenesis of autoimmune diseases, including autoimmune thyroid disorders (AITD). The aim of this study was to evaluate the association between anti‐C1q and thyroid function in pregnancy‐associated AITD. In 96 pregnant women screened positive for AITD (thyroid dysfunction and/or antibodies against thyroperoxidase – TPOAb), anti‐C1q were measured during the 9‐11th gestational week and after delivery (median 16 months after delivery), and compared to the corresponding serum levels of thyroid hormones. As controls, 80 healthy pregnant women, 72 non‐pregnant AITD patients and 72 blood donors were included. In the non‐pregnant AITD group, two serum samples ≥ 6 months apart were analysed. Compared to blood donors, anti‐C1q levels were substantially higher in all pregnant women analysed. In pregnancy, anti‐C1q levels were higher in the TPOAb‐positive women than in controls (37 versus 17·5%, P < 0·0001). Anti‐C1q‐positive pregnant women screened positive for AITD had higher thyroid‐stimulating hormone (TSH) levels than anti‐C1q‐negative women (2·41 versus 1·94 mU/l, P = 0·01), and TSH correlated positively with anti‐C1q (r = 0·226, P = 0·045) in the TPOAb‐positive women. After delivery, serum levels of anti‐C1q decreased in the positively screened TPOAb‐negative women (8·8 versus 5·9 U/l, P = 0·002), but not in the TPOAb‐positive ones, and they no longer correlated with TSH. Anti‐C1q antibody levels increase during pregnancy in general and even more in the context of AITD, where they correlate with thyroid stimulating hormone levels.     

Serum cytokine pattern in young children with screening detected coeliac disease

Coeliac disease is an autoimmune disease characterized by inflammation localized to the small bowel, but less is known about systemic signs of inflammation. The aim was to measure cytokines of the T helper 1 (Th1) and T helper 2 (Th2) cell patterns in children with screening‐detected coeliac disease before and after treatment with a gluten‐free diet. Serum samples selected before and after the start of a gluten‐free diet from 26 3‐year‐old children diagnosed with biopsy‐proven coeliac disease and from 52 matched controls were assayed in an multiplex enzyme‐linked immunosorbent assay (ELISA) for the 10 cytokines: interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐8, IL‐10, IL‐12p70, IL‐13 and tumour necrosis factor (TNF)‐α. Among Th1 cytokines, IFN‐γ and IL‐12p70 were elevated significantly in children with coeliac disease compared to controls (P < 0·001 and P = 0·001, respectively). Similar findings were demonstrated for the Th2 cytokines IL‐5 (P < 0·001), IL‐10 (P = 0·001) and IL‐13 (P = 0·002). No difference in cytokine levels between the two groups was found for TNF‐α, IL‐1β, IL‐2, IL‐4 and IL‐8. After gluten‐free diet, levels of IL‐5, IL‐12 and IL‐10 decreased significantly (P < 0·001, P = 0·002 and P = 0·007) and IFN‐γ levels were reduced (P = 0·059). Young children with coeliac disease detected by screening demonstrate elevated levels of serum cytokines at time of diagnosis. A prolonged systemic inflammation may, in turn, contribute to long‐term complications known to be associated with untreated coeliac disease.     

A novel bioassay for anti‐thyrotrophin receptor autoantibodies detects both thyroid‐blocking and stimulating activity

Autoantibodies to the thyrotrophin (TSH) receptor (anti‐TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid‐stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti‐TSHR antibodies block TSH and can cause hypothyroidism. Thyroid‐blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)‐Luc cells was more than five‐fold lower compared with the wild‐type CHO‐Luc cells. We tested the performance of this bioassay using a thyroid‐blocking monoclonal antibody K1‐70, established an assay cut‐off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R² 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20‐fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred‐fold and still exhibit blocking activity above the cut‐off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti‐TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.     

Association of a CTLA-4 3′ untranslated region (CT60) single nucleotide polymorphism with autoimmune thyroid disease in the Japanese population

The etiology of the autoimmune thyroid diseases (AITDs), Graves' disease (GD) and Hashimoto's thyroiditis is largely unknown. However, genetic susceptibility is believed to play a major role. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene, encoding a negative regulator of the T-lymphocyte immune response, had been reported to be associated and/or linked to AITD. Recently, AITD susceptibility in the Caucasians was mapped to the 6.1-kb 3′UTR of the CTLA-4 gene, in which the single-nucleotide polymorphism (SNP), CT60, was most strongly associated with AITD. In order to determine the association of the CTLA-4 gene with AITD in the Japanese, case-control association analysis for the CT60 of the CTLA-4 gene using 264 AITD patients and 179 healthy controls was done. The frequency of the disease-susceptible G allele of the CT60 of the Japanese control was higher than that of the Caucasians (72.6 vs. 52.3%). However, the G allele of the CT60 was associated with GD (84.0 vs. 72.6%, P=0.0008) and AITD (80.1 vs. 72.6%, P=0.009) in the Japanese. Furthermore, the G allele of the CT60 was associated with the increased risk for GD [P=0.004, odds ratio (OR)=2.0] and AITD (P=0.03, OR=1.6) in a recessive model. These results suggested that the CTLA-4 gene is involved in the susceptibility for GD and AITD in the Japanese.     

The functional polymorphisms of VDR,GC and CYP2R1 are involved in the pathogenesis of autoimmune thyroid diseases

Vitamin D is a multi-functional immune regulator, and a low serum concentration of vitamin D promotes autoimmune inflammation. In this study, we evaluate the association between the prognosis of autoimmune thyroid disease (AITD) and the functional polymorphisms of genes that regulate vitamin D metabolism. For 139 Graves’ disease (GD) patients, 116 Hashimoto''s disease (HD) patients and 76 control subjects, we genotyped the following polymorphisms using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP): vitamin D receptor (VDR): rs731236, rs7975232, rs2228570 and rs1544410; group-specific component (GC): rs7041 and rs4588; and CYP2R1: rs10741657. The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The frequency of the C allele for the rs7975232 polymorphism was higher in GD patients than in control subjects (P = 0·0349). The proportion of GD patients whose anti-thyrotrophin receptor antibody (TRAb) level was >51% was higher in those with the CC genotype than in those with the CA+AA genotypes (P = 0·0065). The frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in control subjects (P = 0·0174) and GD patients (P = 0·0149). The frequency of the Gc1Gc1 genotype for the GC polymorphism and the AG genotype for the CYP2R1 polymorphism were lower in intractable GD than in GD in remission (P = 0·0093 and 0·0268, respectively). In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved with the intractability of GD.     

Prevalence and clinical relevance of thyroid stimulating hormone receptor‐blocking antibodies in autoimmune thyroid disease

The prevalence and clinical relevance of thyroid stimulating hormone (TSH) receptor (TSHR) blocking antibodies (TBAb) in patients with autoimmune thyroid disease (AITD) was investigated. Serum TBAb were measured with a reporter gene bioassay using Chinese hamster ovary cells. Blocking activity was defined as percentage inhibition of luciferase expression relative to induction with bovine TSH alone (cut‐off 40% inhibition). All samples were measured for TSHR stimulatory antibody (TSAb) and TSHR binding inhibiting immunoglobulins (TBII). A total of 1079 unselected, consecutive patients with AITD and 302 healthy controls were included. All unselected controls were negative for TBAb and TSAb. In contrast, the prevalence of TBAb‐positive patients with Hashimoto's thyroiditis and Graves' disease was 67 of 722 (9·3%) and 15 of 357 (4·2%). Of the 82 TBAb‐positive patients, thirty‐nine (48%), 33 (40%) and 10 (12%) were hypothyroid, euthyroid and hyperthyroid, respectively. Ten patients were both TBAb‐ and TSAb‐positive (four hypothyroid, two euthyroid and four hyperthyroid). Thyroid‐associated orbitopathy was present in four of 82 (4·9%) TBAb‐positive patients, with dual TSHR antibody positivity being observed in three. TBAb correlated positively with TBII (r = 0·67, P < 0·001) and negatively with TSAb (r = –0·86, P < 0·05). The percentage of TBII‐positive patients was higher the higher the level of inhibition in the TBAb assay. Of the TBAb‐positive samples with  > 70% inhibition, 87% were TBII‐positive. Functional TSHR antibodies impact thyroid status. TBAb determination is helpful in the evaluation and management of patients with AITD. The TBAb assay is a relevant and important tool to identify potentially reversible hypothyroidism.     

Association of CT60 cytotoxic T lymphocyte antigen-4 gene polymorphism with thyroid autoantibody production in patients with Hashimoto's and postpartum thyroiditis

Strong genetic contribution has been demonstrated to influence the development of autoimmune thyroid disease (AITD) as well as thyroid autoantibody production. In order to assess the relation between CT60 cytotoxic T lymphocyte antigen‐4 (CTLA‐4) gene polymorphism and thyroid autoantibody production, we investigated 180 consecutive newly diagnosed patients with two forms of AITD, 105 with Hashimoto's thyroiditis (HT) and 75 with postpartum thyroiditis (PPT). We evaluated thyroid function, measured antibodies against thyroid peroxidase (TPO) and thyroglobulin (Tg), and determined CT60 CTLA‐4 gene polymorphism. In HT, TPO antibody median value was significantly lower in the AA compared to the AG and GG genotypes (65, 122 and 319 U/ml, P < 0·005), while the Tg antibody median value was lower in the AA compared to the AG genotype (91 and 189 U/ml, P < 0·02). In PPT, the frequency of thyroid autoantibody‐positive patients was higher among G‐allele‐carrying genotypes (P < 0·04). Similar to HT, the TPO antibody median value was lower in the AA compared to the AG and GG genotypes (12, 130 and 423 U/ml, P < 0·006). Hypothyroid PPT patients were more often thyroid autoantibody‐positive (P < 0·005) and the TPO antibody median value was higher compared to hyperthyroid PPT patients (500 and 32 U/ml, P < 0·0001). The frequency of the G‐allele was significantly higher among hypothyroid patients (P < 0·05). Our data suggest that in both HT and PPT, the CT60 CTLA‐4 gene polymorphism contributes importantly to thyroid autoantibody production. In PPT, the genotype also seems to influence thyroid function, as patients with the polymorphous allele are more prone to develop hypothyroid form of PPT.     

Activation of cytokines corroborate with development of inflammation and autoimmunity in thromboangiitis obliterans patients

Thromboangiitis obliterans (TAO) is a segmental inflammatory occlusive disorder that affects the arm and leg arteries of young smokers. The immune system seems to play a critical role in the aetiology of TAO; however, knowledge of the aspects involved in the progression of vascular tissue inflammation and, consequently, the evolution of this disease is still limited. This study was carried out to investigate the cytokine levels of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐4, IL‐17 and IL‐23 in the plasma of TAO patients presenting with acute clinical manifestations. The study included 20 TAO patients (n = 10 women; n = 10 men) aged 38–59 years under clinical follow‐up, classified into two groups: (i) TAO former smokers (n = 11) and (ii) TAO active smokers (n = 9); the control groups included normal volunteer non‐smokers (n = 10, active smokers (n = 10) and former smokers (n = 10). Patients' plasma samples were measured using the sandwich enzyme‐linked immunosorbent assay. Statistical analyses were performed using the non‐parametric Mann–Whitney U‐test, with parameters significant at P < 0·05. The activities of all cytokines were different in groups of TAO patients when compared with normal controls, and decreased for control smokers. Increased levels of TNF‐α, IL‐1β, IL‐4, IL‐17 and IL‐23 were significant in patients with TAO when compared to the controls (P < 0·005, all parameters). The results presented here indicate an increased production of cytokines in TAO, possibly contributing to the inflammatory response observed in the patients' vascular levels. In addition, the increased levels of IL‐17 and IL‐23 suggest that the disturbance of TAO is involved with mechanisms of autoimmunity. Thus, the discovery of IL‐17 and its association with inflammation and autoimmune pathology has reshaped our viewpoint regarding the pathogenesis of TAO, which was based previously on the T helper type 1 (Th1)–Th2 paradigm.     

Polymorphisms of HLA Class II Predispose Children and Adolescents with Type 1 Diabetes Mellitus to Autoimmune Thyroid Disease

To determine the prevalence of autoimmune thyroid disease (AITD) in children and adolescents with type 1 diabetes mellitus (DM), and assess whether the development of AITD is correlated with specific DQ-A and DQ-B loci of the HLA class II antigens, we analyzed thyroid function using anti-thyroid antibodies and HLA-DQ-A and -DQ-B polymorphisms in 69 patients with type 1 DM, in 75 normal healthy controls, and in 21 patients with AITD but without type 1 DM. Eighteen patients (26%) in the diabetic patients had AITD. In the diabetic patients, DQA1*0301 and DQB1*0302 occurred more frequently than in controls [DQA1*0301: OR=1.939, 95% CI=1.210–3.109 (P=0.008, P^c (corrected P) <0.05); DQB1*0302: OR=2.558, 95% CI=1.354–4.832 (P=0.005, P_c>0.05)]. Compared with controls, non-diabetic subjects with AITD showed higher frequency of DQA1*0301 (P_c<0.05) and DQB1*0601 (P_c>0.05), but these alleles were not contributing factors in the development of AITD in diabetic patients. In diabetic patients, DQB1*0201, known as susceptible allele of type 1 DM was not a contributing factor in the development of AITD in diabetic patients. Unlike DQB1*0201, DQB1*0401 was more frequently found in diabetic patients with AITD than in controls [OR=4.053, 95% CI=1.607–10.221 (P=0.0017, P_c<0.05)] or than in non-diabetic AITD patients [OR=15.769, 95% CI=1.905–130.530(P=0.002, P_c<0.05)]. In non-diabetic subjects, DQB1*0401 did not provide susceptibility for AITD.

Our results suggest that HLA DQB1*0401 is a predisposing genetic marker for the development of AITD in patients with type 1 DM in Korea.     

Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto''s disease (HD) and Graves'' disease (GD). MicroRNA (miR) bind directly to the 3′ untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-β; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.     

DICER and DROSHA gene expression and polymorphisms in autoimmune thyroid diseases

Dicer and Drosha are RNase III enzymes that are necessary for the biogenesis of most miRNAs. However, there are no reports on the association of Dicer and Drosha with the pathogenesis of autoimmune thyroid disease (AITD). We genotyped DICER rs3742330A/G and rs1057035T/C as well as DROSHA rs644236C/T and rs10719C/T polymorphisms in 255?Hashimoto's disease (HD) patients, in 255 Graves' disease (GD) patients and in 128 healthy controls by the polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) method. We also examined the expression of DICER and DROSHA gene in peripheral blood mononuclear cells (PBMCs) by quantitative RT-PCR (qRT-PCR) methods. The TT genotype of the DICER rs1057035 polymorphism was less frequent in GD patients (p?=?0.0098) than in healthy subjects. The CC genotype of DROSHA rs644236 polymorphism were more frequent in GD patients than in HD patients (p?=?0.0171). The gene expression of DICER was lower in patients with AITD compared with that in control subjects (p?=?0.0064) and was lower in patients with GD in remission than in patients with intractable GD (p?=?0.0213). In addition, the expression of DROSHA was lower in patients with AITD than that in control subjects (p?p?=?0.0440). In conclusion, the DICER rs1057035 TT genotype and DROSHA rs644236?CC genotype were associated with the development of GD and the differentiation between GD and HD, respectively. The expression levels of DICER and DROSHA genes were low in AITD and differed depending on the intractability of GD and the severity of HD, respectively.     

Association of NKG2D gene variants with susceptibility and severity of rheumatoid arthritis

NKG2D (KLRK1) is a C‐type lectin receptor present on natural killer (NK) cells, γδ, CD8⁺ and CD4⁺ T cells. Upon ligand binding, NKG2D mediates activatory and co‐stimulatory signals to NK cells and activated CD4⁺ T cells, respectively. Polymorphisms in NKG2D predispose to infectious diseases, cancer, transplantation and autoimmune disorders. We studied the influence of this NK receptor polymorphism on predisposition to and modification of the disease phenotype in patients with rheumatoid arthritis (RA). Eight different single nucleotide polymorphisms (SNP) in the NKG2 gene were genotyped in 236 patients with RA and 187 controls using Taqman 5' nuclease assays. NKG2D genotype/allele frequency did not differ between patients and controls. Subgroup analysis showed that the frequency of A allele of NKG2D9 and T allele of NKG2D10 was significantly higher in patients with deformities (a marker of severe disease) [11 versus 5%, Pc = 0·03, odds ratio (OR) = 2·44, 95% confidence interval (CI) = 1·09‐5·98 and 10 versus 4%, Pc = 0·04, OR = 2·45, 95% CI = 1·05‐6·39, respectively], while the frequency of alleles G of NKG2D9 and A of NKG2D10 was greater in patients without deformities (Pc = 0·03, OR = 0·41, 95% CI = 0·17‐0·91 and Pc = 0·04, OR = 0·41, 95% CI = 0·16‐0·96). Similar trends of association were observed with deforming phenotype of RA in female patients and deforming young onset RA subgroups. Haplotype analysis revealed that the frequency of haplotype G‐C‐A‐G‐A‐T‐C‐C was higher in patients than in controls (12 versus 8%, P = 0·04, OR = 1·61, 95% CI = 1·01‐2·55), suggesting that it may predispose to RA. Our study suggests that the NKG2D gene polymorphisms may modify the risk of development and severity of RA.     

Association of cytokine Th2 gene polymorphisms with autoimmune thyroid diseases in Tunisian population

Autoimmune thyroid diseases (AITD) including Graves' disease (GD) and Hashimoto's thyroiditis (HT) are complex genetic diseases. Th2 cytokines act on the development of AITD. This study was conducted on Tunisian patients with AITD to investigate the association of Th2 cytokine gene polymorphisms and haplotype combination with GD or HT risk. A total of 156 controls, 160 patients with HT and 88 patients with GD were genotyped for IL‐4 rs2243250, IL‐5 rs2069812, IL‐6 rs1800796 and IL‐13 rs1800925 polymorphisms by PCR‐RFLP. The AITD risk was assessed by a logistic regression analysis using the SNP stats statistical program. False‐positive report probability (FPRP) was estimated to evaluate significant findings. IL‐13 rs1800925 was associated with GD, after adjustment for age and gender, in codominant, dominant and allele genetic models (p = .0072; p = .0018; p = .012, respectively). Significant association of the IL‐6 rs1800796C/G genotype with GD was also detected (p = .025). Furthermore, increased risk of HT was still found for IL‐13 rs1800925T allele (p = .039, OR = 1.39) and for IL‐4 rs2243250T/T genotype both in codominant (p = .033, OR = 2.59) and recessive (p = .011, OR = 2.73) models after adjustment for age and gender. Interestingly, haplotype analysis performed on the IL‐4, IL‐5 and IL‐13 genes revealed a high risk of HT with CTT haplotype (p = .008, OR = 2.12). However, the CCT haplotype is a protective factor (OR = 0.36). Patients carrying the CT haplotype with only one minor allele had a moderate risk of HT (OR = 1.56). The FPRP analysis showed that the association of IL‐13 rs1800925 polymorphism with GD and HT and the association of CTT haplotype with HT were noteworthy. In conclusion, the IL‐4, IL‐5, IL‐6 and IL‐13 polymorphism may play a role in susceptibility to GD and HT in the Tunisian population. Furthermore, gene–gene interaction between the IL‐4, IL‐5 and IL‐13 significantly increases the risk of AITD. Further studies with larger numbers of individuals are needed to confirm the results.     

Detection of enterovirus in the thyroid tissue of patients with graves' disease

The etiology and pathogenesis of Graves' disease (GD) are still unknown, although it is thought that both genetic and environmental factors are important. Some indirect evidence implies that a viral infection may be a possible etiologic factor in autoimmunity. The main objective of this study was to examine direct evidence of the presence of enteroviruses (EVs) in the thyroid tissue of patients with GD. Thyroid tissue from 22 patients with newly diagnosed GD was obtained by core needle biopsy, while tissue from 24 patients with chronic GD and 24 control subjects without any autoimmune thyroid diseases was collected during neck surgery. Formalin‐fixed, paraffin‐embedded thyroid tissue samples were examined for the presence of enterovirus capsid protein using immunohistochemistry and for enterovirus RNA using in situ hybridization. Enterovirus capsid protein was detected in 17 (37%) patients and in 4 (17%) control subjects (P = 0.103). Enterovirus RNA was identified in thyroid tissue from nine (20%) patients, but in none of the control subjects (P = 0.016). Eight (90%) of the nine virus RNA positive patients were also positive for enterovirus protein. This is the first study to analyze thyroid tissue for EVs, including patients with untreated, newly diagnosed GD. The results suggest that EVs are more frequently present in thyroid tissue of patients than controls. Further studies are indicated to explore this association to find out if a low‐grade chronic enteroviral infection might be involved in the pathogenesis of GD and if this could offer new therapeutic and preventive opportunities. J. Med. Virol. 85:512–518, 2013. © 2012 Wiley Periodicals, Inc.     

Urinary haptoglobin,alpha‐1 anti‐chymotrypsin and retinol binding protein identified by proteomics as potential biomarkers for lupus nephritis

The study was aimed at identification by proteomics and validation by enzyme‐linked immunosorbent assay (ELISA) of potential urinary biomarkers for lupus nephritis. Study subjects comprised 88 systemic lupus erythematosus (SLE) patients and 60 controls (rheumatoid arthritis, diabetes mellitus and healthy individuals). Based on the SLE disease activity index (SLEDAI), patients were classified as active renal (AR), active non‐renal (ANR) or inactive disease (ID). Urinary proteins from a group of patients with AR or ID were resolved by two‐dimensional gel electrophoresis and identified by matrix‐assisted laser desorption ionization–time of flight–mass spectrometry (MALDI‐TOF‐MS/MS). The selected biomarkers were validated by ELISA using samples from all patients and controls. AR patients were followed‐up for 12 months after start of therapy. Three urinary proteins, alpha‐1 anti‐chymotrypsin (ACT), haptoglobin (HAP) and retinol binding protein (RBP), were detected in patients with AR and not ID. Upon validation, ACT levels were higher in AR patients than the other groups (P < 0·001) and showed good correlation with renal SLEDAI (r = 0·577, P < 0·001) as well as SLEDAI (r = 0·461, P < 0·001). Similarly, HAP levels were > 10‐fold higher in AR than other groups (P < 0·001) and correlated well with renal SLEDAI (r = 0·594, P < 0·001) and SLEDAI (r = 0·371, P < 0·01). RBP levels were also higher in AR patients than in other groups (P < 0·05), except diabetes, and showed moderate correlation with renal SLEDAI (r = 0·284, P < 0·008) and SLEDAI (r = 0·316, P < 0·003). Upon follow‐up with treatment, levels of all three proteins declined at 6 and 12 months (P < 0·01). Multiple logistic regression identified ACT as the best marker to differentiate AR from ANR. Urinary HAP, ACT and RBP are potential biomarkers for lupus nephritis activity.     

Association of the T-cell regulatory gene CTLA4 with Graves’ disease and autoimmune thyroid disease in the Japanese

Autoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigen. The cytotoxic T-lymphocyte antigen-4 (CTLA4) gene, encoding a negative regulator of the T-lymphocyte immune response, had been reported to be associated and/or linked to AITD. Recently, AITD susceptibility in the Caucasians was mapped to the 6.1-kb 3UTR of the CTLA4 gene, in which the three single-nucleotide polymorphisms (SNPs) CT60, JO31, and JO30 were strongly associated with AITD. In order to determine the association of the CTLA4 gene with AITD in the Japanese, case-control association analysis for the four SNPs of the CTLA4 gene using 380 AITD patients and 266 healthy controls was done. Among the SNPs examined, the SNP JO31 was most significantly associated with AITD in the Japanese, whereas the association of the JO30 with AITD was not observed. The frequency of the disease-susceptible G allele of the JO31 of the Japanese control was higher than that of the Caucasians (67.1% vs 50.2%); however, the G allele of the JO31 was associated with Graves disease (GD) (67.1% vs 76.3%, P=0.0013) and AITD in the Japanese (67.1% vs 74.2%, P=0.0055). Furthermore, the G allele of the JO31 was associated with the increased risk for GD [P=0.0051, odds ratio (OR)=1.7] and AITD (P=0.016, OR=1.5) in a dominant model. These results suggested that the CTLA4 gene is involved in the susceptibility for GD and AITD in the Japanese.     

Increased prevalence of autoimmunity in Turner syndrome – influence of age

Individuals with Turner syndrome (TS) are prone to develop autoimmune conditions such as coeliac disease (CD), thyroiditis and type 1 diabetes (T1DM). The objective of the present study was to examine TS of various karyotypes for autoantibodies and corresponding diseases. This was investigated in a prospective cross‐sectional study of Danish TS patients (n = 107, median age 36·7 years, range: 6–60 years). A medical history was recorded and a blood sample was analysed for autoantibodies against gliadin, transglutaminase, adrenal cortex, intrinsic factor, anti‐thyroid peroxidase (anti‐TPO) and glutamic‐acid‐decarboxylase 65 (GAD‐65). Autoantibodies were present in 58% (n = 61) of all patients, whereof 18% (11) had autoantibodies targeting more than one organ. Patients with autoantibodies were significantly older than those without (P = 0·001). Anti‐TPO was present in 45% (48) of patients, of whom 33% (16) were hypothyroid. Overall, 18% (19) presented with CD autoantibodies, of whom 26% (five) had CD. Anti‐TPO and CD autoantibodies co‐existed in 9% (10). Immunoglobulin A deficiency was found in 3% (three) of patients, who all had CD autoantibodies without disease. Among four patients with anti‐GAD‐65 none had T1DM, but two were classified as having T2DM. One patient had adrenocortical autoantibodies but not adrenal failure. Autoantibodies against intrinsic factor were absent. Anti‐GAD‐65 was increased in isochromosomal karyotypes (3/23 versus 1/84, P = 0·008) with no other association found between autoantibodies and karyotype. In conclusion, TS girls and women face a high prevalence of autoimmunity and associated disease with a preponderance towards hypothyroidism and CD. Thus, health care providers dealing with this patient group should be observant and test liberally for these conditions even before clinical symptoms emerge.     

Role of proneurotensin as marker of paediatric coeliac disease

Neurotensin (NT) is a gut hormone functioning proinflammatory through nuclear factor kappa B (NF‐κB) and interleukin (IL)?8 secretion or anti‐inflammatory through epidermal growth factor receptors. NT mRNA is down‐regulated in duodenal biopsies of children with untreated coeliac disease. The aim of this study was to investigate if plasma pro‐NT levels correlated with the degree of intestinal mucosal damage and tissue transglutaminase autoantibody (tTGA) levels in children with coeliac disease. Fasting plasma samples from 96 children with coeliac disease and 89 non‐coeliac disease controls were analysed for NT precursor fragment pro‐NT 1–117 by a chemiluminometric immunoassay. Pro‐NT levels were compared with NT mRNA from duodenal biopsies, assessed previously with quantitative polymerase chain reaction (PCR). Illumina core exome arrays were used for human leucocyte antigen (HLA) typing and the Marsh criteria applied to score mucosal damage. Tissue TGA was measured by radio binding assay. A general linear model compared pro‐NT levels with diagnosis of coeliac disease, Marsh score and HLA DQ haplotype. Spearman's rank test was used to compare pro‐NT levels with tTGA, age and duodenal NT mRNA levels, respectively. Plasma pro‐NT levels were elevated in children with coeliac disease (median 23 pmol/l higher, P = 0·003) and in those with severe intestinal mucosal damage (median 24 pmol/l higher for ≥ Marsh 3b versus not, P = 0·0004). Pro‐NT levels correlated further with tTGA (r² = 0·22, P = 0·002), but not with duodenal NTS mRNA levels (r² = ?0·12, P = 0·14). Pro‐NT was not associated with any of the HLA risk‐haplotypes. Elevated peripheral pro‐NT levels reflect more severe forms of active coeliac disease, indicating a potential role of NT in intestinal inflammation.     

Functional TSH receptor antibodies in children with autoimmune thyroid diseases

Introduction: The diagnostic value of the level of TSH receptor antibodies (TSHR-Ab) in the population of children with autoimmune thyroid diseases (AITDs) is still unknown. The aim of this cross-sectional study was to investigate the prevalence of TSHR-Ab in a paediatric cohort with AITD and healthy controls.

Materials and methods: A total of 240 serum samples were obtained from 205 patients with AITD, type 1 diabetes (T1D), juvenile arthritis (JA), and healthy controls (C). TSHR stimulating (TSI) and -blocking (TBI) immunoglobulins were measured in cell-based bioassays using CHO cells expressing a chimeric TSHR and a c-AMP response-element-dependent luciferase. TSI was reported as percentage of specimen-to-reference ratio (cutoff 140SRR%). Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (40% inhibition).

Results: C as well as children with JA and T1D were both TSI and TBI negative. In contrast, children with Graves’ disease (GD) were positive for TSI in 47/53 samples (88.7%) while those with thyroidal and orbital GD showed TSI positivity in 95.8% (23/24 samples). Serum TSI levels were SRR% 320?±?157 and 417?±?135 in GD and GD?+?orbitopathy, respectively (p?=?.02). Children with Hashimoto’s thyroiditis (HT) were TSI positive in 4/83 (4.8%) samples, including two with orbital involvement. TSI levels were increased in HT children with vs. those without eye disease (SRR% 177 vs. 51, p?Conclusions: In conclusion, TSI is prevalent in children with GD while the highest serum TSI levels were noted in children with AITD and orbitopathy.     

The level of urinary secretory immunoglobulin A (sIgA) of patients with IgA nephropathy is elevated and associated with pathological phenotypes

Recent studies have demonstrated deposition of secretory immunoglobulin A (sIgA) in glomeruli of some patients with IgA nephropathy (IgAN). The aim of this study is to investigate the levels of urinary sIgA in IgAN patients with different pathological phenotypes and whether it could be used as a non‐invasive biomarker for assessment of kidney injury in IgAN. Urine samples from 202 patients with IgAN were collected on the day of renal biopsy. Forty‐eight fulfilled the histopathological criteria of Haas‐I or II (group 1), 60 fulfilled Haas‐III (group 2) and 94 patients fulfilled Haas‐IV or V (group 3). Urine samples from 60 healthy sex‐ and age‐matched volunteers with negative urinalysis were collected as normal controls. Urinary sIgA was detected by sandwich enzyme‐linked immunosorbent assay and was corrected by urinary creatinine. In comparison with normal controls, the levels of urinary sIgA were significantly higher in IgAN [2·22 (0–43·82) μg/mg Cr versus 1·08 (0–16·49) μg/mg Cr, P < 0·001]. The levels of urinary sIgA were significantly higher in group 3 than that in group 2 and group 1 [3·54 (0–43·82) μg/mg Cr versus 1·63 (0–15·88) μg/mg Cr versus 0·91 (0–11·79), P < 0·001], and group 2 than group 1 (P = 0·014). The levels of urinary sIgA were associated positively with proteinuria (r = 0·443, P < 0·001), serum creatinine (r = 0·376, P < 0·001) and histopathological parameters, such as ratio of global sclerosis (r = 0·356, P < 0·001), ratio of total crescents (r = 0·339, P < 0·001) and ratios of cellular crescents (r = 0·231, P < 0·001). The levels of urinary sIgA were associated closely with histopathological phenotypes of IgAN and might be used as a non‐invasive biomarker to evaluate kidney injury in IgAN.