Celiac disease IgA modulates vascular permeability in vitro through the activity of transglutaminase 2 and RhoA

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.

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. © 2013 Kalliokoski et al.

Unexpected role of surface transglutaminase type II in celiac disease

Background & Aims: In celiac disease (CD), transglutaminase type II (TG2) has 2 fundamental roles: (1) as the autoantigen recognized by highly specific autoantibodies and (2) the modifier of pathogenic gliadin T-cell epitopes. It follows that inhibition of TG2 might represent an attractive strategy to curb the toxic action of gliadin. Here we studied the validity of this strategy using the organ culture approach. Methods: Duodenal biopsy specimens from 30 treated patients with CD, 33 untreated patients with CD, and 24 controls were cultured with or without gliadin peptides p31-43, pα-9, and deamidated pα-9 for 20 minutes, 3 hours, and 24 hours. In 31 patients with CD and 16 controls, TG2 inhibitor R283 or anti-TG CUB 7402 or anti-surface TG2 (6B9) mAbs were used in cultures. T84 cells were also cultured with or without peptides with or without TG inhibitors. Mucosal modifications after culture were assessed by immunofluorescence, in situ detection of TG activity, confocal microscopy, and fluorescence-activated cell sorter analysis. Results: The enzymatic inhibition of TG2 only controlled gliadin-specific T-cell activation. The binding of surface TG2 contained gliadin-specific T-cell activation and p31-43-induced actin rearrangement, epithelial phosphorylation, and apoptosis, both in organ cultures and T84 cells. Conclusions: These data indicate a novel and unexpected biological role for surface TG2 in the pathogenesis of CD suggesting a third role for TG2 in CD. These results have a specific impact for celiac disease, with wider implications indicating a novel biologic function of TG2 with possible repercussions in other diseases. © 2005 by the American Gastroenterological Association.

Are transglutaminase 2 inhibitors able to reduce gliadin-induced toxicity related to celiac disease? A proof-of-concept study

Purpose Celiac disease is an autoimmune-mediated enteropathy characterized by adaptive and innate immune responses to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), leading to an immune response in the small-intestinal mucosa. TG2 inhibitors have therefore been suggested as putative drugs for celiac disease. In this proof-of-concept study we investigated whether two TG2 inhibitors, cell-impermeable R281 and cell-permeable R283, can prevent the toxic effects of gliadin in vitro and ex vivo. Methods Intestinal epithelial Caco-2 cells were treated with peptic-tryptic-digested gliadin (PT-gliadin) with or without TG2 inhibitors and thereafter direct toxic effects (transepithelial resistance, cytoskeletal rearrangement, junction protein expression and phoshorylation of extracellular-signal-regulated kinase 1/2) were determined. In an organ culture of celiacpatient- derived small-intestinal biopsies we measured secretion of TG2-autoantibodies into the culture medium and the densities of CD25- and interleukin (IL) 15-positive cells, forkhead box P3 (FOXP3)-positive regulatory Tcells (Tregs) and Ki-67- positive proliferating crypt cells. Results Both TG2 inhibitors evinced protective effects against gliadin-induced detrimental effects in Caco-2 cells but the cellimpermeableR281seemedslightlymorepotent. Inaddition,TG2 inhibitor R281 modified the gluten-induced increase in CD25- and IL15-positive cells,Tregs and crypt cell proliferation, but had no effect on antibody secretion in celiac-patient-derived biopsies. Conclusions Our results suggest that TG2 inhibitors are able to reduce certain gliadin-induced effects related to responses in vitro and ex vivo. © Springer Science+Business Media, LLC 2012.

Prevalence of subclinical thyroid dysfunction and its relation to socioeconomic deprivation in the elderly:a community-based cross-sectional survey

Context: Population-based screening has been advocated for subclinical thyroid dysfunction in the elderly because the disorder is perceived to be common, and health benefits may be accrued by detection and treatment. Objective: The objective of the study was to determine the prevalence of subclinical thyroid dysfunction and unidentified overt thyroid dysfunction in an elderly population. Design, Setting, and Participants: A cross-sectional survey of a community sample of participants aged 65 yr and older registered with 20 family practices in the United Kingdom. Exclusions: Exclusions included current therapy for thyroid disease, thyroid surgery, or treatment within 12 months. Outcome Measure: Tests of thyroid function (TSH concentration and free T 4 concentration in all, with measurement of free T3 in those with low TSH) were conducted. Explanatory Variables: These included all current medical diagnoses and drug therapies, age, gender, and socioeconomic deprivation (Index of Multiple Deprivation, 2004) Analysis: Standardized prevalence rates were analyzed. Logistic regression modeling was used to determine factors associated with the presence of subclinical thyroid dysfunction Results: A total of 5960 attended for screening. Using biochemical definitions, 94.2% [95% confidence interval (CI) 93.8-94.6%] were euthyroid. Unidentified overt hyper- and hypothyroidism were uncommon (0.3, 0.4%, respectively). Subclinical hyperthyroidism and hypothyroidism were identified with similar frequency (2.1%, 95% CI 1.8-2.3%; 2.9%, 95% CI 2.6-3.1%, respectively). Subclinical thyroid dysfunction was more common in females (P < 0.001) and with increasing age (P < 0.001). After allowing for comorbidities, concurrent drug therapies, age, and gender, an association between subclinical hyperthyroidism and a composite measure of socioeconomic deprivation remained. Conclusions: Undiagnosed overt thyroid dysfunction is uncommon. The prevalence of subclinical thyroid dysfunction is 5%. We have, for the first time, identified an independent association between the prevalence of subclinical thyroid dysfunction and deprivation that cannot be explained solely by the greater burden of chronic disease and/or consequent drug therapies in the deprived population. Copyright © 2006 by The Endocrine Society.

Prevalence of the use of cancer related self-tests by members of the public:A community survey

Background: Self-tests are those where an individual can obtain a result without recourse to a health professional, by getting a result immediately or by sending a sample to a laboratory that returns the result directly. Self-tests can be diagnostic, for disease monitoring, or both. There are currently tests for more than 20 different conditions available to the UK public, and self-testing is marketed as a way of alerting people to serious health problems so they can seek medical help. Almost nothing is known about the extent to which people self-test for cancer or why they do this. Self-tests for cancer could alter perceptions of risk and health behaviour, cause psychological morbidity and have a significant impact on the demand for healthcare. This study aims to gain an understanding of the frequency of self-testing for cancer and characteristics of users. Methods: Cross-sectional survey. Adults registered in participating general practices in the West Midlands Region, will be asked to complete a questionnaire that will collect socio-demographic information and basic data regarding previous and potential future use of self-test kits. The only exclusions will be people who the GP feels it would be inappropriate to send a questionnaire, for example because they are unable to give informed consent. Freepost envelopes will be included and non-responders will receive one reminder. Standardised prevalence rates will be estimated. Discussion: Cancer related self-tests, currently available from pharmacies or over the Internet, include faecal occult blood tests (related to bowel cancer), prostate specific antigen tests (related to prostate cancer), breast cancer kits (self examination guide) and haematuria tests (related to urinary tract cancers). The effect of an increase in self-testing for cancer is unknown but may be considerable: it may affect the delivery of population based screening programmes; empower patients or cause unnecessary anxiety; reduce costs on existing healthcare services or increase demand to investigate patients with positive test results. It is important that more is known about the characteristics of those who are using self-tests if we are to determine the potential impact on health services and the public. © 2006 Wilson et al; licensee BioMed Central Ltd.

Higher prevalence of retinopathy in diabetic patients of South Asian ethnicity compared with white Europeans in the community:a cross-sectional study

OBJECTIVE—The purpose of this study was to compare prevalence and risk factors for diabetic retinopathy among U.K. residents of South Asian or white European ethnicity. RESEARCH DESIGN AND METHODS—This was a community-based cross-sectional study involving 10 general practices; 1,035 patients with type 2 diabetes were studied: 421 of South Asian and 614 of white European ethnicity. Diabetic retinopathy, sight-threatening retinopathy, maculopathy, and previous laser photocoagulation therapy were assessed after grading of retinal photographs. Data were collected on risk factors including age, duration, and treatment of diabetes, blood pressures, serum total cholesterol, and A1C. RESULTS—Patients of South Asian ethnicity had significantly higher systolic (144 vs. 137 mmHg, P < 0.0001) and diastolic (84 vs. 74 mmHg, P < 0.0001) blood pressure, A1C (7.9 vs. 7.5%, P < 0.0001), and total cholesterol (4.5 vs. 4.2 mmol/l, P < 0.0001). Diabetic retinopathy was detected in 414 (40%) patients (189 South Asian [45%] versus 225 white European [37%]; P = 0.0078). Sight-threatening retinopathy was detected in 142 (14%) patients (68 South Asian [16%] versus 74 white European [12%]; P = 0.0597). After adjustment for confounders, there were significantly elevated risks of any retinopathy and maculopathy for South Asian versus white European patients. CONCLUSIONS—Patients of South Asian ethnicity had a significantly higher prevalence of diabetic retinopathy and maculopathy, with significantly elevated systolic and diastolic blood pressure, A1C, and total cholesterol; lower attained age; and younger age at diagnosis. Earlier onset of disease and higher levels of modifiable risk factors make early detection of diabetes, annual referral for retinal screening, and intensive risk factor control key elements in addressing this health inequality.

What is the prevalence of diabetic macular oedema involving the centre of the macula in patients undergoing digital diabetic retinopathy screening

Free Paper Sessions Design. Retrospective analysis. Purpose. To assess the prevalence of center-involving diabetic macular oedema (CIDMO) and risk factors. Methods. Retrospective review of patients who were screen positive for maculopathy (M1) during 2010 in East and North Birmingham. The CIDMO was diagnosed by qualitative identification of definite foveal oedema on optical coherence tomography (OCT). Results. Out of a total of 15,234 patients screened, 1194 (7.8%) were screen positive for M1 (64% bilateral). A total of 137 (11.5% of M1s) were diagnosed with macular oedema after clinical assessment. The OCT results were available for 123/137; 69 (56.1%) of these had CI-DMO (30 bilateral) which is 0.5% of total screens and 5.8% of those screen positive for M1. In those with CIDMO 60.9% were male and 63.8% Caucasian; 90% had type 2 diabetes and mean diabetes duration was 20 years (SD 9.7, range 2-48). Mean HbA1c was 8.34%±1.69, with 25% having an HbA1c =9%. Furthermore, 62% were on insulin, 67% were on antihypertensive therapy, and 64% were on a cholesterol-lowering drug. A total of 37.7% had an eGFR between 30% and 60% and 5.8% had eGFR <30. The only significant difference between the CIDMO and non-CIDMO group was mean age (67.83±12.26 vs 59.69±15.82; p=0.002). A total of 65.2% of those with CIDMO also had proliferative or preproliferative retinopathy in the worst eye and 68.1% had subsequently been treated with macular laser at the time of data review. Conclusions. The results show that the prevalence of CIDMO in our diabetic population was 0.5%. A significant proportion of macula oedema patients were found to have type 2 diabetes with long disease duration, suboptimal glycemic and hypertensive control, and low eGFR. The data support that medical and diabetic review of CIDMO patients is warranted particularly in the substantial number with poor glycemic control and if intravitreal therapies are indicated.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

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. © 2013 Nadalutti et al.

The prevalence of tympanic membrane and related middle ear pathology in children:a large longitudinal cohort study followed from birth to age ten

OBJECTIVE: To record with video-otoscopy the appearance of the tympanic membranes of a cross section of children aged 9 to 10 years. STUDY DESIGN: Cross-sectional study nested within an established longitudinal study of childhood development, the Avon Longitudinal Study of Parents and Children. SETTING: South West England, U.K. PARTICIPANTS: Approximately 6908 of 7261 children with ages ranging from 105 to 140 months born between April 1, 1991, and December 31, 1992, were examined by trained technicians with video-otoscopy. MAIN OUTCOME MEASURES: Two photographs were taken of each child's tympanic membranes to show the features of the pars tensa and the pars flaccida. RESULTS: In just less than three quarters of the children, both ears were normal. Retraction of the pars flaccida was present in 9.6% of children, and that of the pars tensa was present in 7.9%. Most of these changes were mild with few severe retractions. There were 15 cases of overt or suspected cholesteatoma. CONCLUSION: The tympanic membrane changes reflect most of the middle ear disease seen in 9- to 10-year-old children. The prevalence is low, and few children have serious disease at this stage.