Enzyme Tissue Transglutaminase Research Articles

БОЛЕЗНИ ТОНКОЙ КИШКИ. ЧАСТЬ 4. ЦЕЛИАКИЯ И ДРУГИЕ РАССТРОЙСТВА, СВЯЗАННЫЕ С ГЛЮТЕНОМ

In the fourth part of our literary review devoted to diseases of the small intestine, information is provided about celiac disease, i.e. about "suffering from intestinal disorder" (translated from Greek). According to the classification, autoimmune and allergic conditions associated with gluten, as well as non-celiac gluten sensitivity are considered. The modern possibilities of diagnosis and differential diagnosis of these disorders are presented. Gluten itself, an important fraction of wheat, rye and barley, is characterized. Accurate diagnosis of celiac disease at the present stage is the possibility of conducting second–order tests to detect antibodies to deaminated gliadin peptides, antibodies to endomysium and its main antigen – the enzyme tissue transglutaminase. Histological changes by stages, the role of HLA-DQ2/DQ8 in the pathogenesis of celiac disease are presented. The basis of treatment is compliance with an elimination diet. Further described are herpetiform dermatitis, gluten ataxia, variants of wheat allergy, which includes more than three hundred proteins, thirty of them are recognized as allergenic. Modern tests for determining specific IgE, available for prescribing and detecting allergens. Non–celiac sensitivity (NCCHG) to gluten is the diagnosis of exclusion. NCCHG triggers. Solerno criteria. Risk groups for celiac disease.

Absence of tissue transglutaminase reduces amyloid-beta pathology in APP23 mice.

AimsAlzheimer's disease (AD) is characterised by amyloid‐beta (Aβ) aggregates in the brain. Targeting Aβ aggregates is a major approach for AD therapies, although attempts have had little to no success so far. A novel treatment option is to focus on blocking the actual formation of Aβ multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post‐translational modifications in Aβ resulting in covalently cross‐linked, stable and neurotoxic Aβ oligomers.In vivo absence of TG2 in the APP23 mouse model may provide evidence that TG2 plays a key role in development and/or progression of Aβ‐related pathology.MethodsHere, we compared the effects on Aβ pathology in the presence or absence of TG2 using 12‐month‐old wild type, APP23 and a crossbreed of the TG2−/− mouse model and APP23 mice (APP23/TG2−/−).ResultsUsing immunohistochemistry, we found that the number of Aβ deposits was significantly reduced in the absence of TG2 compared with age‐matched APP23 mice. To pinpoint possible TG2‐associated mechanisms involved in this observation, we analysed soluble brain Aβ1–40, Aβ1–42 and/or Aβ40/42 ratio, and mRNA levels of human APP and TG2 family members present in brain of the various mouse models. In addition, using immunohistochemistry, both beta‐pleated sheet formation in Aβ deposits and the presence of reactive astrocytes associated with Aβ deposits were analysed.ConclusionsWe found that absence of TG2 reduces the formation of Aβ pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aβ deposition in AD.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1–10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

2029-P: Role of Adipose Tissue Macrophage–Derived F13A1 in Regulating Preadipocyte Hyperplasia during Obesity

The epidemic of DM has increased in the last decades and is the leading cause of decreased life expectancy. AT adapts to nutrient excess by balancing hypertrophy and hyperplasia that contribute to AT expansion. Unhealthy AT expansion seen in a person with diabetes is characterized by decreased preadipocyte (PA) content, and increased adipocyte size. AT macrophages (ATM) contribute to unhealthy AT expansion by blocking adipogenesis, promoting adipocyte hypertrophy, fibrosis and insulin resistance (IR). Many of the cellular and molecular mechanisms by which adipocyte numbers and size is regulated by ATMs in vivo are still unknown, especially in human obesity. This study focused on the hypotheses that ATM expression of F13A1, a tissue transglutaminase enzyme shown to regulate fibrin networks during tissue growth: 1) modulates abundance of preadipocyte (PA) populations during obesity and DM states, and 2) contributes to adipocyte hypertrophic states and potential metabolic dysfunction. The premise for these studies was based on evidence that F13A1 is increased in Visceral AT (VAT) of DM individuals compared to non-DM and that it is produced by a tissue remodeling ATM subset increased in DM. We utilized nondiabetic (NDM) and DM human secondary PA treated with F13A1 and assessed the following: 1) F13A1 expression requirement for the inhibition of healthy PA expansion in obesity, and 2) the mechanisms by which its function leads to arrested proliferation. Results showed that F13A1 impairs PA proliferation through its exerted effects on PA ECM fibronectin crosslink leading to: 1) downregulation of downstream gene proliferative signaling pathways and 2) defaulted adipocyte differentiation with hypertrophic profiles as an antagonistic response to decreased proliferation. These studies have advanced our understanding of ATM-derived F13A1 as a potential immunotherapeutic target to improve AT function. Disclosure D.M. Elizondo: None. L.M. Geletka: None. C.G. Flesher: None. C.N. Lumeng: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (T32DK101357)

Tissue Transglutaminase Promotes Early Differentiation of Oligodendrocyte Progenitor Cells.

Demyelinated lesions of the central nervous system are characteristic for multiple sclerosis (MS). Remyelination is not very effective, particular at later stages of the disease, which results in a chronic neurodegenerative character with worsening of symptoms. Previously, we have shown that the enzyme Tissue Transglutaminase (TG2) is downregulated upon differentiation of oligodendrocyte progenitor cells (OPCs) into myelin-forming oligodendrocytes and that TG2 knock-out mice lag behind in remyelination after cuprizone-induced demyelination. Here, we examined whether astrocytic or oligodendroglial TG2 affects OPCs in a cell-specific manner to modulate their differentiation, and therefore myelination. Our findings indicate that human TG2-expressing astrocytes did not modulate OPC differentiation and myelination. In contrast, persistent TG2 expression upon OPC maturation or exogenously added recombinant TG2 accelerated OPC differentiation and myelin membrane formation. Continuous exposure of recombinant TG2 to OPCs at different consecutive developmental stages, however, decreased OPC differentiation and myelin membrane formation, while it enhanced myelination in dorsal root ganglion neuron-OPC co-cultures. In MS lesions, TG2 is absent in OPCs, while human OPCs show TG2 immunoreactivity during brain development. Exposure to the MS-relevant pro-inflammatory cytokine IFN-γ increased TG2 expression in OPCs and prolonged expression of endogenous TG2 upon differentiation. However, despite the increased TG2 levels, OPC maturation was not accelerated, indicating that TG2-mediated OPC differentiation may be counteracted by other pathways. Together, our data show that TG2, either endogenously expressed, or exogenously supplied to OPCs, accelerates early OPC differentiation. A better understanding of the role of TG2 in the OPC differentiation process during MS is of therapeutic interest to overcome remyelination failure.

Prevalence of anti-tissue Transglutaminase antibodies among patients with alopecia areata in Nassiriah city.

Background: alopecia areata is an autoimmune disease in which hair is lost from some or all areas of the body, usually from the scalp due to the body's failure to recognize its own body cells and subsequent destruction of its own tissue as if it were an aggressor. The Anti- tissue transglutaminase antibodies, antibodies against the enzyme tissue transglutaminase (tTG), are found in the blood of some patients with alopecia areata. Serological blood tests such as anti- tissue transglutaminase antibodies are the first-line non-invasive investigation required to make a diagnosis of celiac disease. Aims: the aim of the study is to establish the prevalence of Anti-tissue transglutaminase antibodies in patients with alopecia areata. Methods: we evaluated 96 serum samples. The samples were collected from two groups; group I of sixty five patients with alopecia areata and group II of thirty one control patients who were referred to dermatology department in al-hussaien teaching hospital for different dermatological diseases other than alopecia areata. All participants were evaluated for anti-tissue transglutaminase IgA and anti-tissue transglutaminase IgG antibodies. Results: out of the 65 patients with alopecia areata included in the study, tTGA was positive in 13 patients (20%) and tTGG was positive in 8 patient (12.4%). The prevalence of positivity in patients with alopecia areata was higher compared to the control group in which tTGA was positive in 3 patients (9.7%) and the tTGG was positive in 2 patients (6%). Conclusion: we conclude that patients with alopecia areata have higher prevalence of serum anti-tissue transglutaminase antibodies compared with control which support the autoimmune basis for alopecia areata, however, we were unable to demonstrate an increased risk of celiac disease in those patients.

Tissue Transglutaminase Appears in Monocytes and Macrophages but Not in Lymphocytes in White Matter Multiple Sclerosis Lesions.

Leukocyte infiltration is an important pathological hallmark of multiple sclerosis (MS) and is therefore targeted by current MS therapies. The enzyme tissue transglutaminase (TG2) contributes to monocyte/macrophage migration and is present in MS lesions and could be a potential therapeutic target. We examined the cellular identity of TG2-expressing cells by immunohistochemistry in white matter lesions of 13 MS patients; 9 active and chronic active lesions from 4 patients were analyzed in detail. In these active MS lesions, TG2 is predominantly expressed in leukocytes (CD45+) but not in cells of the lymphocyte lineage, that is, T cells (CD3+) and B cells (CD20+). In general, cells of the monocyte/macrophage lineage (CD11b+ or CD68+) are TG2+ but no further distinction could be made regarding pro- or anti-inflammatory macrophage subtypes. In conclusion, TG2 is abundantly present in cells of the monocyte/macrophage lineage in active white matter MS lesions. We consider that TG2 can play a role in MS as it is associated with macrophage infiltration into the CNS. As such, TG2 potentially presents a novel target for therapeutic intervention that can support available MS therapies targeting lymphocyte infiltration.

A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells.

The protein crosslinking enzyme tissue transglutaminase (tTG) is an acyltransferase which catalyzes transamidation reactions between two proteins, or between a protein and a polyamine. It is frequently overexpressed in several different types of human cancer cells, where it has been shown to contribute to their growth, survival, and invasiveness. tTG is capable of adopting two distinct conformational states: a protein crosslinking active (“open”) state, and a GTP-bound, crosslinking inactive (“closed”) state. We have previously shown that the ectopic expression of mutant forms of tTG, which constitutively adopt the open conformation, are toxic to cells. This raises the possibility that strategies directed toward causing tTG to maintain an open state could potentially provide a therapeutic benefit for cancers in which tTG is highly expressed. Here, we report the identification of a small molecule, TTGM 5826, which stabilizes the open conformation of tTG. Treatment of breast and brain cancer cell lines, as well as glioma stem cells, with this molecule broadly inhibits their transformed phenotypes. Thus, TTGM 5826 represents the lead compound for a new class of small molecules that promote the toxicity of cancer cells by stabilizing the open state of tTG.

An integrated IoT-Wi-Fi board for remote data acquisition and sharing from innovative immunosensors. Case of study: Diagnosis of celiac disease

A new compact diagnostic device exploiting the integration of screen printed electrode-based immunosensors and remote-controlled IoT-WiFi acquisition board has been realized and validated for diagnosis of Celiac Disease as case of study. The immunodevice is based on chemisorption of open tissue transglutaminase enzyme on the surface of gold nanoparticles-functionalized carbon screen printed electrodes. IgA and IgG anti-tissue transglutaminase target antibodies are recognized by the immobilized bioreceptor as highly specific biomarkers related to Celiac Disease. The signal from the amperometric sensor is acquired and processed through on-purpose developed IoT-WiFi integrated board, allowing for real-time data sharing on cloud services to directly notify all users (physicians, caregivers, etc.) on device outcome. The proposed solution does not require customized hardware or software.The analytical performances of the immunosensors were optimized by experimental design, obtaining diagnostically useful limit of detection (LOD) and limit of quantitation (LOQ) values (LODIgA = 3.2 AU mL−1; LODIgG = 1.4 AU mL−1; LOQIgA = 4.6 AU mL−1; LOQIgG = 2.3 AU mL−1) as well as good intermediate precision (RSD < 5%).The high discrimination capability of the IoT-Wi-Fi device between positive and negative serum control resulted to be suitable for diagnostic purposes, with outstanding statistical significance (p < 0,001)

In vivo evaluation of two tissue transglutaminase PET tracers in an orthotopic tumour xenograft model

BackgroundThe protein cross-linking enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including cancer. Recently, the synthesis and initial evaluation of two high-potential radiolabelled irreversible TG2 inhibitors were reported by us. In the present study, these two compounds were evaluated further in a breast cancer (MDA-MB-231) tumour xenograft model for imaging active tissue transglutaminase in vivo.ResultsThe metabolic stability of [11C]1 and [18F]2 in SCID mice was comparable to the previously reported stability in Wistar rats. Quantitative real-time polymerase chain reaction analysis on MDA-MB-231 cells and isolated tumours showed a high level of TG2 expression with very low expression of other transglutaminases. PET imaging showed low tumour uptake of [11C]1 (approx. 0.5 percentage of the injected dose per gram (%ID/g) at 40–60 min p.i.) and with relatively fast washout. Tumour uptake for [18F]2 was steadily increasing over time (approx. 1.7 %ID/g at 40–60 min p.i.). Pretreatment of the animals with the TG2 inhibitor ERW1041E resulted in lower tumour activity concentrations, and this inhibitory effect was enhanced using unlabelled 2.ConclusionsWhereas the TG2 targeting potential of [11C]1 in this model seems inadequate, targeting of TG2 using [18F]2 was achieved. As such, [18F]2 could be used in future studies to clarify the role of active tissue transglutaminase in disease.

Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

The neurodegenerative disease multiple sclerosis (MS) is pathologically characterized by the massive influx of immune cells into the central nervous system. This contributes to demyelination and axonal damage which causes symptoms such as motor and cognitive dysfunctions. The migration of leukocytes from the blood vessel is orchestrated by a multitude of factors whose determination is essential in reducing cellular influx in MS patients and the experimental autoimmune encephalomyelitis (EAE) animal model. The here studied enzyme tissue Transglutaminase (TG2) is present intracellularly, on the cell surface and extracellularly. There it contributes to cellular adhesion and migration via its transamidation activity and possibly by facilitating cellular interaction with the extracellular matrix. Previous data from our group showed reduced motor symptoms and cellular infiltration after using a pharmacological TG2 transamidation activity inhibitor in a rat EAE model. However, it remained elusive if the cross-linking activity of the enzyme resulted in the observed effects. To follow-up, we now characterized two new small molecule TG2 activity inhibitors, BJJF078 and ERW1041E. Both compounds are potent inhibitor of recombinant human and mouse Transglutaminase enzyme activity, mainly TG2 and the close related enzyme TG1. In addition they did not affect the binding of TG2 to the extracellular matrix substrate fibronectin, a process via which TG2 promotes cellular adhesion and migration. We found, that ERW1041E but not BJJF078 resulted in reduced EAE disease motor-symptoms while neither caused apparent changes in pathology (cellular influx), Transglutaminase activity or expression of inflammation related markers in the spinal cord, compared to vehicle treated controls. Although we cannot exclude issues on bioavailability and in vivo efficacy of the used compounds, we hypothesize that extracellular TG1/TG2 activity is of greater importance than (intra-)cellular activity in mouse EAE pathology.

Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates.

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive "binders" for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein - osteopontin (OPN) - appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates.

Alterations in the Calcitonin and Calcitonin modulated proteins, E-cadherin and the enzyme tissueTransglutaminase II during the window of implantation in a baboon model of endometriosis

Endometriosis is a gynecological condition associated with infertility. We have previously demonstrated dysregulation of several molecular markers of uterine receptivity in a baboon model of induced endometriosis. Specifically, reduced levels of endometrial progesterone receptor (PR) and progesterone (P)-regulated genes, HOXA10 and FKBP52 were observed during the window of uterine receptivity in baboons with endometriosis, suggesting that this disease results in the development of an endometrial P resistance. In this study calcitonin (CALC) and CALC-modulated proteins, E-cadherin (E-Cad) and tissue Transglutaminase (tTgase-2) were evaluated in the eutopic endometrium of endometriotic animals throughout disease progression. Endometriosis was induced in normal cycling baboons by intraperitoneal inoculation of menstrual endometrium. Eutopic and ectopic endometrium was harvested consecutively from each animal at 1, 6 and 15 months of disease, during the window of receptivity. Control eutopic endometrium was s...

Monocyte-derived tissue transglutaminase in multiple sclerosis patients: reflecting an anti-inflammatory status and function of the cells?

BackgroundLeukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes.MethodsPrimary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes.ResultsTG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes.ConclusionsTG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.

The approach to Celiac Disease in children

Celiac Disease (CD) is a permanent, irreversible but treatable multifactorial disease triggered by the ingestion of gluten (a plant storage protein contained in wheat, barley and rye) in genetically predisposed individuals and resulting in an autoimmune small intestinal inflammation with systemic implications. While the gastrointestinal manifestations secondary to an inflammatory enteropathy with variable degrees of severity are what defined it for many decades, CD is in fact characterized also by a wide range of extra-intestinal complaints and elevated titers of celiac-specific autoantibodies. 1.1. Epidemiology and pathogenesis The prevalence of CD is increasing at a remarkable pace during the past few decades [1], [2], [3]. Once thought to be a rare condition, affecting no more than 1/10,000 people, thanks to the availability and widespread use of specific and sensitive serological markers, CD is now recognized worldwide as a common disorder, with a prevalence varying between 0.3 and 3 per 100. Only a limited portion of the expected celiac patients are however actually identified, with proportions varying between different countries: in the USA, even though overall CD prevalence is estimated to be around 1%, only about 15% of this population (including children and adults) has been diagnosed and can therefore be treated [4]. This phenomenon of under-diagnosis is likely due to a combination of inadequate awareness and a high prevalence of asymptomatic or oligo-symptomatic patients. Like most multifactorial disorders, CD is the result of a complex interaction between genes, immune status of the host, and environmental triggers. Gluten is a heterogeneous molecule. The gluten fractions that are toxic to celiac patients are a mixture of alcohol-soluble proteins called gliadins. Gliadins are rich in glutamine and proline residues, which even the healthy human intestine cannot fully digest. As a result, intact gliadin peptides are left in the lumen, and some cross the intestinal barrier. These fragments come into contact with the intracellular enzyme tissue transglutaminase (tTG), which deamidates them, leading to a change in shape and increased negative charge. This creates peptides that can easily be captured by the HLA-DQ2 and/or DQ8 molecules expressed on the surface of the lamina propria-associated antigen-presenting cells (APCs) and are presented to CD4+ T cells triggering an inflammatory reaction [5]. The end result of this autoimmune-triggered inflammatory reaction is a varied degree of small intestinal mucosal damage, typically more severe proximally than distally. The role of additional environmental factors is still the object of an intense research. Recently, our group showed evidence for a plausible role of an otherwise innocuous viral infection (Reovirus) in creating a pro-inflammatory milieu conducive to the development of overt CD [6].

A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies.

Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology.

Development of fluorine-18 labeled peptidic PET tracers for imaging active tissue transglutaminase

IntroductionThe protein–protein crosslinking activity of the enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including celiac disease, lung-, liver- and kidney fibrosis, cancer and neurodegenerative diseases. This study aims at developing a TG2 PET tracer based on the peptidic irreversible TG2 inhibitor Z006. MethodsInitially, the carbon-11 labeling of Z006 at the diazoketone position was explored. Subsequently, a set of analogues that allow for fluorine-18 labeling was synthesized. Two potent analogues, 6f and 6g, were radiolabeled with fluorine-18 and biodistribution and metabolite analysis in Wistar rats was performed. The identity of the main metabolite of [18F]6g was elucidated using LC–MS/MS. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 breast cancer tissue using [18F]6g was performed. Results[18F]6f and [18F]6g were obtained in 20 and 9% yields, respectively. Following administration to healthy Wistar rats, rapid metabolism of both tracers was observed. Remarkably, full conversion to just one single metabolite was observed for one of the tracers, [18F]6g. By LC–MS/MS analysis this metabolite was identified as C-terminally saponified [18F]6g. This metabolite was also found to be a potent TG2 inhibitor in vitro. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 tumor sections using [18F]6g demonstrated high specific and selective binding of [18F]6g to active TG2. ConclusionsWhereas based on the intensive metabolism [18F]6f seems unsuitable as a TG2 PET tracer, the results warrant further evaluation of [18F]6gin vivo.

Celiac disease : Pathogenesis, clinics, epidemiology, diagnostics, therapy

Celiac disease is induced by the consumption of gluten containing cereals (wheat, spelt, barley, rye). With aprevalence of ~1 %, it is the most common non-infectious chronic inflammatory intestinal disease worldwide. It manifests in all age groups, either classically with abdominal pain, diarrhoea and growth failure or weight loss, more commonly with indirect consequences of malabsorption, such as anaemia and osteoporosis, or with associated autoimmune diseases like type1 diabetes, autoimmune thyroiditis or dermatitis herpetiformis. The pathogenesis of celiac disease is well explored. Gluten, the cereal storage protein, is not completely digested and reaches the intestinal mucosa where it activates inflammatory Tcells, which cause atrophy of the resorptive villi. This T‑cell activation requires agenetic predisposition (the molecules HLA-DQ2 or -DQ8 on antigen-presenting immune cells). Moreover, the enzyme tissue transglutaminase (TG2) which is released in the mucosa increases the immunogenicity of the gluten peptides by adeamidation reaction. The test for serum antibodies to the autoantigen TG2 is one of the best diagnostic markers in medicine, which in combination with endoscopically obtained biopsies, secures the diagnosis of celiac disease. Despite these tools celiac disease is severely underdiagnosed, with 80-90 % of those affected being undetected. The untreated condition can lead to grave complications. These include the consequences of malabsorption, cancers (especially intestinal T‑cell lymphoma), and likely also the promotion of autoimmune diseases. The therapy of celiac disease, astrict gluten-free diet, is difficult to maintain and not always effective. Alternative, supporting pharmacological therapies are urgently needed and are currently in development.

Inherent ER stress in pancreatic islet β cells causes self-recognition by autoreactive T cells in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic β cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in β cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4+ diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This β cell ER stress increased activity of the calcium (Ca2+)-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, β cell ER stress induced by chemical and physiological triggers leads to β cell immunogenicity through Ca2+-dependent PTM. These findings elucidate a mechanism of how β cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing β cell recognition by autoreactive T cells.