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

Autoantibodies against MHC class I polypeptide-related sequence A are associated with increased risk of concomitant autoimmune diseases in celiac patients

Background: Overexpression of autologous proteins can lead to the formation of autoantibodies and autoimmune diseases. MHC class I polypeptide-related sequence A (MICA) is highly expressed in the enterocytes of patients with celiac disease, which arises in response to gluten. The aim of this study was to investigate anti-MICA antibody formation in patients with celiac disease and its association with other autoimmune processes. Methods: We tested serum samples from 383 patients with celiac disease, obtained before they took up a gluten-free diet, 428 patients with diverse autoimmune diseases, and 200 controls for anti-MICA antibodies. All samples were also tested for anti-endomysium and anti-transglutaminase antibodies. Results: Antibodies against MICA were detected in samples from 41.7% of patients with celiac disease but in only 3.5% of those from controls (P <0.0001) and 8.2% from patients with autoimmune disease (P <0.0001). These antibodies disappeared after the instauration of a gluten-free diet. Anti-MICA antibodies were significantly prevalent in younger patients (P <0.01). Fifty-eight patients with celiac disease (15.1%) presented a concomitant autoimmune disease. Anti-MICA-positive patients had a higher risk of autoimmune disease than MICA antibody-negative patients (P <0.0001; odds ratio = 6.11). The risk was even higher when we also controlled for age (odds ratio = 11.69). Finally, we found that the associated risk of developing additional autoimmune diseases was 16 and 10 times as high in pediatric patients and adults with anti-MICA, respectively, as in those without. Conclusions: The development of anti-MICA antibodies could be related to a gluten-containing diet, and seems to be involved in the development of autoimmune diseases in patients with celiac disease, especially younger ones

The role of tissue transglutaminase (TG2) in regulating the tumour progression of the mouse colon carcinoma CT26

The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-?B. TG2-transfected cells showed increased expression of integrin ß3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with ß3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with ß3 integrins. All cells expressed the same level of TGFß receptors I and II, but only cells transfected with active TG2 had increased levels of TGFß1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFß1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature

Coeliac disease-specific autoantibodies targeted against transglutaminase 2 disturb angiogenesis

Coeliac disease is characterized by immunoglobulin-A (IgA)-class autoantibodies targeted against transglutaminase 2 (TG2), a multi-functional protein also with a role in angiogenesis. These antibodies are present in patient serum but are also found bound to TG2 below the epithelial basement membrane and around capillaries in the small intestinal mucosa. Based on these facts and the information that the mucosal vasculature of coeliac patients on a gluten-containing diet is disorganized, we studied whether the coeliac disease-specific autoantibodies targeted against TG2 would disturb angiogenesis. The effects of coeliac disease-specific autoantibodies on in vitro angiogenesis were studied in angiogenic cell cultures. The binding of the antibodies to cells, endothelial sprouting, migration of both endothelial and vascular mesenchymal cells, the integrity of the actin cytoskeleton in both cell types and the differentiation of vascular mesenchymal cells were recorded. In vitro, IgA derived from coeliac disease patients on a gluten-containing diet binds to surface TG2 on endothelial and vascular mesenchymal cells and this binding can be inhibited by the removal of TG2. In addition, coeliac disease-specific autoantibodies targeting TG2 disturb several steps of angiogenesis: endothelial sprouting and the migration of both endothelial and vascular mesenchymal cells. Furthermore, the autoantibodies cause disorganization of the actin cytoskeleton in both capillary cell types that account most probably for the defective cellular migration. We conclude that coeliac disease-specific autoantibodies recognizing TG2 inhibit angiogenesis in vitro. This disturbance of the angiogenic process could lead in vivo to the disruption of the mucosal vasculature seen in coeliac disease patients on a gluten-containing diet

Decreased soil microbial nitrogen under vegetation 'shrubification' in the subarctic forest–tundra ecotone : the potential role of increasing nutrient competition between plants and soil microorganisms

The consequences of warming-induced ‘shrubification’ on Arctic soil carbon storage are receiving increased attention, as the majority of ecosystem carbon in these systems is stored in soils. Soil carbon cycles in these ecosystems are usually tightly coupled with nitrogen availability. Soil microbial responses to ‘shrubification’ may depend on the traits of the shrub species that increase in response to warming. Increase in deciduous shrubs such as Betula nana likely promotes a loss of soil carbon, whereas the opposite may be true if evergreen shrubs such as Empetrum hermaphroditum increase. We analyzed soil organic matter stocks and 13C NMR fractions, microbial CO2 respiration, biomass, extracellular enzyme activities (EEAs), and their association with shrub density in northern Sweden after 20 years of experimental warming using open top chambers (OTCs). Our study sites were located in a tundra heath that stores high soil carbon quantities and where the OTCs had increased deciduous shrubs, and in a mountain birch forest that stores lower soil carbon quantities and where the OTCs had increased evergreen shrubs. We predicted that organic matter stocks should be lower and respiration and EEAs higher inside the OTCs than untreated plots in the tundra, whereas no effect should be detected in the forest. Soil organic matter stocks and 13C NMR fractions remained unaffected at both sites. When expressed as per gram microbial biomass, respiration and EEAs for carbohydrate and chitin degradation were higher inside the OTCs, and contrasting our prediction, this effect was stronger in the forest. Unexpectedly, the OTCs also led to a substantially lower microbial biomass carbon and nitrogen irrespective of habitat. The decline in the microbial biomass counteracted increased activities resulting in no effect of the OTCs on respiration and a lower phenol oxidase activity per gram soil. Microbial biomass nitrogen correlated negatively with evergreen shrub density at both sites, indicating that ‘shrubification’ may have intensified nutrient competition between plants and soil microorganisms. Nutrient limitation could also underlie increased respiration per gram microbial biomass through limiting C assimilation into biomass. We hypothesize that increasing nutrient immobilization into long-lived evergreen shrubs could over time induce microbial nutrient limitation that contributes to a stability of accumulated soil organic matter stocks under climate warming

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

OBJECTIVE: Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. MATERIAL AND METHODS: The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. RESULTS: Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. CONCLUSIONS: Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonalTG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliacautoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activityand whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliacpatient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a threedimensionalangiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cellenzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced byrecombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When thisenzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitroangiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelialcell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targetedautoantibodies are exerted by enhanced enzymatic activity of TG2

RhoB is associated with the anti-angiogenic effects of celiac patient transglutaminase 2-targeted autoantibodies.

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies

Localization services for online common operational picture and situation awareness

Many operations, be they military, police, rescue, or other field operations, require localization services and online situation awareness to make them effective. Questions such as how many people are inside a building and their locations are essential. In this paper, an online localization and situation awareness system is presented, called Mobile Urban Situation Awareness System (MUSAS), for gathering and maintaining localization information, to form a common operational picture. The MUSAS provides multiple localization services, as well as visualization of other sensor data, in a common frame of reference. The information and common operational picture of the system is conveyed to all parties involved in the operation, the field team, and people in the command post. In this paper, a general system architecture for enabling localization based situation awareness is designed and the MUSAS system solution is presented. The developed subsystem components and forming of the common operational picture are summarized, and the future potential of the system for various scenarios is discussed. In the demonstration, the MUSAS is deployed to an unknown building, in an ad hoc fashion, to provide situation awareness in an urban indoor military operation.©2013 by the authors. Published by IEEE. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license, http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.fi=vertaisarvioitu|en=peerReviewed