Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study

Objective To evaluate the feasibility and diagnostic accuracy of screening for coeliac disease by rapid detection of IgA antibodies to tissue transglutaminase performed in primary care.Design District nurses screened 6 year old children using rapid antibody testing of finger prick blood. They also collected capillary blood samples for laboratory determination of IgA and IgG antibodies to endomysium and IgA antibodies to tissue transglutaminase. Children with positive rapid test results were directly sent for biopsy of the small intestine.Setting Primary care in Jász-Nagykun-Szolnok county, Hungary.Participants 2690 children (77% of 6 year olds living in the county) and 120 nurses.Main outcome measures Positivity for antibodies to endomysium or transglutaminase in the laboratory and coeliac disease confirmed at biopsy.Results 37 children (1.4%, 95% confidence interval 0.9% to 1.8%) had biopsy confirmed coeliac disease. Only five of these children had been diagnosed clinically before screening. Rapid testing had a 78.1% sensitivity (70.0% to 89.3%) and 100% specificity (88.4% to 100%) for a final diagnosis of coeliac disease by biopsy. Sensitivity was 65.1% (50.2% to 77.6%) and specificity was 100% (99.8% to 100%) compared with combined results of IgA and IgG laboratory tests. Trained laboratory workers detected 30 of the 31 newly diagnosed IgA competent patients with the rapid test kit used blindly. Median time to biopsy after a positive rapid test result was significantly shorter (20 days, range 4-148) than after a positive laboratory result (142 days, 70-256; P<0.001). Children with coeliac disease detected at screening were smaller and had worse health status than their peers but they improved on a gluten-free diet.Conclusions A simple rapid antibody test enabled primary care nurses to detect patients with coeliac disease in the community who were not picked up in clinical care. Extra training is needed to improve sensitivity.     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

A role for anti-transglutaminase 2 autoantibodies in the pathogenesis of coeliac disease?

Coeliac disease is an autoimmune-mediated disorder with both innate and adaptive immune components. The disease is triggered by dietary gluten, which provokes the development of a massive immune reaction leading to the destruction of the small-intestinal mucosal morphology and intestinal dysfunction. Besides the typical small-bowel symptoms extraintestinal manifestations may also arise in a subset of coeliac disease patients. In addition, gluten evokes the production of antibodies mainly targeting deamidated gluten peptides or transglutaminase 2. Although coeliac disease has traditionally been regarded as a T cell-mediated disorder, this review discusses the role of the gluten-induced disease-specific anti-transglutaminase 2-autoantibodies in the pathogenesis of the disease.     

Extracellular transglutaminase 2 has a role in cell adhesion, whereas intracellular transglutaminase 2 is involved in regulation of endothelial cell proliferation and apoptosis

Objective: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. Materials and methods: The effect of small interfering RNA (siRNA)‐TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting. Results: We found that siRNA‐TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G₁ phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA‐TG2‐induced apoptosis. Conclusion: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.     

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.     

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.     

Effects of a prostacyclin analog iloprost on kidney function, renin-angiotensin and kallikrein-kinin systems, prostanoids and catecholamines in man

Iloprost (ZK 36 374), a stable analog of carbaprostacyclin, was infused for 72 h to nine patients with advanced obliterative arterial disease. Iloprost caused a marked vasodilation and a compensatory increase in cardiac output. The glomerular filtration rate increased by 45% and tubular reabsorption of sodium and water were reduced by 80% and 107%, respectively. The urine excretion rate increased by 122%. Tubular handling of potassium and calcium were not influenced by iloprost but magnesium reabsorption was stimulated. The renin-angiotensin system was not activated while serum angiotensin converting enzyme activity was decreased. Kallikrein excretion in urine was increased 4.4-fold but plasma kininogen, a substrate for kallikrein in producing vasoactive kinins, was unaffected by the drug. Plasma levels of 6-keto-PGF1 alpha and TxB2 were decreased and their excretion in urine increased. Plasma catecholamines were not changed by iloprost. Several of the changes persisted for at least the first postinfusion day. The results indicate that iloprost increases urine excretion rate by increasing glomerular blood flow and by inhibiting sodium and water reabsorptions. The kinin-forming system, but not the renin-angiotensin system or plasma catecholamines, may be activated. The decrease in plasma level of prostanoids can be, at least partly, due to their increased excretions in urine.     

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lotta Koskinen and Jihane Romanos are authors with equal contribution.     

Thioredoxin Is Involved in Endothelial Cell Extracellular Transglutaminase 2 Activation Mediated by Celiac Disease Patient IgA

Purpose

To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation.

Methods

TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study.

Results

We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity.

Conclusions

Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.