Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase: dependence on reaction environment and enzyme fitness

Modification of the enzymatic functions of tissue transglutaminase (TG2) by anti-TG2 autoantibodies may play a role in manifestations of coeliac disease. Our aim was to evaluate the effect of coeliac autoantibodies on reactions catalysed by TG2 by a systematic biochemical approach, and in relation to observed clinical presentation type. Coeliac antibodies did not have significant inhibitory effect on transamidation/deamidation activity of TG2 as measured by amine-incorporation into solid and immobilised casein and by ultraviolet kinetic assay. In contrast, immunoglobulins from patients with severe malabsorption enhanced the reaction velocity to 105.4-242.2%. This activating effect was dose-dependent, most pronounced with immobilised glutamine-acceptor substrates, and correlated inversely with the basal specific activity of the enzyme and with dietary treatment. A similar activation could be demonstrated also with the TG2-specific fraction of autoantibodies and in transamidation activity assays which use fibronectin-bound TG2 and thereby mimic in vivo conditions. These results suggest that coeliac antibodies may stabilise the enzyme in a catalytically advantageous conformation. GTPase activity of TG2 decreased to 67.0-73.4% in the presence of antibodies raising the possibility that inhibition of GTPase activity may affect cellular signalling in case coeliac autoantibodies would reach intracellular compartments.     

Intestinal T Cell Responses to Cereal Proteins in Celiac Disease

Celiac disease is caused by sensitivity to wheat gluten in genetically susceptible individuals. The etiological role of the other wheat-related cereals, barley, rye, and oats, is still debated. In order to investigate this issue further, in this study we examined the immune response of celiac mucosal T cell lines to fractions from all four cereals. Cell stimulation was assessed by measuring proliferation (employing ³H-thymidine incorporation) or cytokine (IL-2, IFN-γ) production. All five T cell lines demonstrated immunoreactivity to protein fractions from the four related cereals. In some cell lines, reactivity to wheat, barley, and rye was only evident when these cereal fractions had been pretreated with tissue transglutaminase. This study confirms the similar T cell antigenic reactivity of these four related cereals and has implications for their exclusion in the gluten-free diet. However, despite oats stimulation of T cell lines, this cereal does not activate a mucosal lesion in most celiac patients.     

Clinical response to gluten withdrawal is not an indicator of coeliac disease

Objective. Although the diagnosis of coeliac disease requires specific histological and serological findings, patients considered to be affected by coeliac disease only on the basis of clinical improvement after gluten withdrawal are commonly referred to our outpatient clinic. The objective of this study was to investigate whether the clinical response of gastrointestinal symptoms to gluten withdrawal and subsequent dietary re-introduction could be an indicator of the presence of coeliac disease. Material and methods. From December 1998 to January 2007, 180 patients on a gluten-free diet because of a diagnosis of coeliac disease not based on proper diagnostic criteria came to our out-patient clinic. In 112 of these patients, gluten was re-introduced into their diet. Subsequent duodenal biopsies and endomysial antibodies confirmed the diagnosis of coeliac disease in 51 of them. The relationship between improvement/worsening of symptoms and withdrawal/re-introduction of dietary gluten was analysed. Results. Gastrointestinal symptoms improved in 64.7% of coeliac patients and 75.0% of non-coeliac patients after gluten withdrawal (χ² test, p=NS). Gluten re-introduction was followed by clinical exacerbation in 71.4% of coeliac patients and 54.2% of non-coeliac patients (χ² test, p=NS). The positive predictive value for clinical improvement after gluten withdrawal was 36%; the positive predictive value for clinical exacerbation after gluten re-introduction was 28%. Conclusions. Clinical response to either withdrawal or re-introduction of dietary gluten has no role in the diagnosis of coeliac disease.     

Potential of transglutaminase 2 as a therapeutic target

Importance of the field: Increased expression and activity of transglutaminase 2 – a calcium-dependent enzyme which catalyzes protein cross-linking, polyamination or deamidation at selective glutamine residues – are involved in the etiopathogenesis of several pathological conditions, such as neurodegenerative disorders, autoimmune diseases and inflammatory diseases. Inhibition of enzyme activity has potential for therapeutic management of these diseases.

Areas covered in this review: The major results achieved in the last twelve years of research in the field of inhibition of tranglutaminase activity using cell cultures as well as in vivo models of high-social-impact or widespread diseases, such as CNS neurodegenerative disorders, celiac sprue, cancer and fibrotic diseases.

What the reader will gain: Beneficial effects of enzyme activity inhibition have been observed in neurodegeneration and fibrosis in vivo models by delivery of the competitive inhibitor cystamine and more recently designed inhibitors, such as thiomidaziolium or norleucine derivatives, which irreversibly bind the active site cysteine residue. Transglutaminase 2 targeting with specific antibodies has also been shown to be a promising tool for celiac disease treatment.

Take home message: New insights from transglutaminase inhibition studies dealing with side effects of in vivo administration of pan-transglutaminase inhibitors will help in design of novel therapeutic approaches to various diseases.     

A novel hemostatic sealant composed of gelatin,transglutaminase and thrombin effectively controls liver trauma-induced bleeding in dogs

Aim:

A novel hemostatic sealant based on the in situ gel formation of gelatin catalyzed by thrombin and crosslinked by transglutaminase was designed. The aim of this study was to investigate the efficacy of the hemostatic sealant in control of bleeding caused by liver trauma in dogs.

Methods:

Hepatic trauma that mimicked the grade III–IV rupture of liver was made in 20 dogs. The traumatic lesion was topically administered the hemostatic sealant (treatment group, n=10), or a thrombin solution (control group, n=10). The time to achieve hemostasis and the blood loss were measured. Contrast-enhanced ultrasound (CEUS) examination was performed in each animal on d 3, d 7, and d 10 d postoperatively to study the healing of the lesions.

Results:

The mean time to achieve hemostasis in the treatment group was significantly shorter than that in the control group (1.20±0.33 vs 6.70±0.64 min, P<0.05). The mean blood loss in the treatment group was significantly less than that in the control group (47.22±8.61 vs 79.29±11.97 mL, P<0.05). In CEUS examination, the traumatic lesions in the treatment group became significantly smaller on d 3, and disappeared on d 7, whereas the lesions in the control group still existed on d 10. Ascites were never found during 10 d follow-up in the treatment group but were observed on d 3 and d 7 in the control group.

Conclusion:

Compared with thrombin, the novel hemostatic sealant shows much better efficacy in hemostasis and may promote wound healing in dog liver trauma.     

New developments in the area of factor XIII

To cite this article: Schroeder V, Kohler HP. New developments in the area of factor XIII. J Thromb Haemost 2013; 11 : 234–44. Summary.  Coagulation factor (F)XIII is best known for its role in fibrin stabilization and cross‐linking of antifibrinolytic proteins to the fibrin clot. From patients with congenital FXIII deficiency, it is known that FXIII also has important functions in wound healing and maintaining pregnancy. Over the last decade more and more research groups with different backgrounds have studied FXIII and have unveiled putative novel functions for FXIII. FXIII, with its unique role as a transglutaminase among the other serine protease coagulation factors, is now recognized as a multifunctional protein involved in regulatory mechanisms and construction and repair processes beyond hemostasis with possible implications in many areas of medicine. The aim of this review was to give an overview of exciting novel findings and to highlight the remarkable diversity of functions attributed to FXIII. Of course, more research into the underlying mechanisms and (patho‐)physiological relevance of the many described functions of FXIII is needed. It will be exciting to observe future developments in this area and to see if and how these interesting findings may be translated into clinical practice in the future.     

Immunological effects of transglutaminase-treated gluten in coeliac disease

Coeliac disease pathogenesis is characterized by an immune response triggered, in genetically predisposed subjects, by ingested gluten and its withdrawal from the diet is the only available therapy. However, enzymatic modification of gluten through the insertion of lysine to avoid antigen presentation could represent a new therapeutical approach for patients. Sixty-six duodenal biopsies from 17 coeliac patients were cultured for 48h with gluten or enzymatically-modified gluten (treated with human recombinant transglutaminase type 2 or bacterial transglutaminase, with or without lysine). Interferonγ, anti endomisium and anti transglutaminase IgA antibodies, lactate dehydrogenase and transglutaminase activity were measured in the culture medium. Transglutaminase type 2 expression was evaluated on biopsies by immunohistochemistry. Gluten and transglutaminase-treated gluten increased by 13-15 fold interferon γ release, as well as antibodies, transglutaminase activity, and the immunohistochemical expression of transglutaminase type 2. Addition of lysine to the enzymatic modification of gluten normalized interferon γ, antibodies, transglutaminase activity and immunohistochemical expression of transglutaminase type 2. Lactate dehydrogenase did not differ among the studied groups. Enzymatic modification of gluten by transglutaminase plus lysine prevents the immunologic effects on cultured duodenal biopsies from coeliac patients and could be tested as an alternative therapy in coeliac disease.     

Serum autoantibodies directed against transglutaminase‐2 have a low avidity compared with alloantibodies against gliadin in coeliac disease

Coeliac disease is characterized by intolerance to gliadin and related gluten components present in wheat, barley and rye. Coeliac disease patients harbour antibodies directed against alloantigens such as gliadin, but also against the autoantigen transglutaminase‐2 (TG2). The type and quality of antibody responses provides insight into the underlying immune activation processes. Therefore, in this study we have analysed the avidity of the antibody response directed against the autoantigen TG2 and compared this with antibody responses against the alloantigens gliadin and Escherichia coli. We observed that the immunoglobulin (Ig)A autoantibody response directed against TG2 is of low avidity compared with the IgA response against the alloantigens gliadin and E. coli in the same patients; the same was true for IgG, both in IgA‐deficient and in ‐sufficient coeliac patients. The observed avidities appear not to be related to disease stage, antibody levels, age or duration of exposure to gluten. In conclusion, in coeliac disease there is a clear difference in avidity of the antibody responses directed against the auto‐ and alloantigens, indicating different regulation or site of initiation of these responses.     

The Absence of a Mucosal Lesion on Standard Histological Examination Does Not Exclude Diagnosis of Celiac Disease

Some patients with undiagnosed celiac disease have minor mucosal lesions that may not be apparent during routine histological analysis. Twenty-five such patients of our institution were discharged to their primary-care physicians despite having positive endomysial antibody serology. To re-evaluate diagnosis for these patients, immunohistological staining with antibodies to CD2, CD3, CD7, CD8, CD69, and Ki67 was conducted on original biopsies from twenty patients. Clinical, serological, and histological investigations were offered to all fourteen patients who attended for review. We observed a significantly greater (P < 0.0001) numbers of intraepithelial lymphocytes and Ki67-positive enterocytes in sections from these twenty patients than for normal controls. Of the fourteen patients who attended for further review, firm diagnosis of celiac disease was made for seven patients and diagnosis was likely for another two. Our study clearly revealed that over-reliance on standard histological findings results in failure to diagnose celiac disease.     

Tissue transglutaminase: a novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases

Alzheimer's disease, Parkinson's disease and Huntington's disease are neurodegenerative diseases, characterized by the accumulation and deposition of neurotoxic protein aggregates. The capacity of specific proteins to self-interact and form neurotoxic aggregates seems to be a common underlying mechanism leading to pathology in these neurodegenerative diseases. This process might be initiated and/or accelerated by proteins that interact with these aggregating proteins. The transglutaminase (TG) family of proteins are calcium-dependent enzymes that catalyze the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds, which can result in both intra- and intermolecular cross-links. Intramolecular cross-links might modify self-interacting proteins, and make them more prone to aggregate. In addition, intermolecular cross-links could link self-aggregating proteins and thereby initiate and/or stimulate the aggregation process. So far, increased levels and activity of tissue transglutaminase (tTG), the best characterized member of the TG family, have been observed in many neurodegenerative diseases, and the self-interacting proteins, characteristic of Alzheimer's disease, Parkinson's disease and Huntington's disease, are known substrates of tTG. Here, we focus on the role of tTG in the initiation of the aggregation process of self-interacting proteins in these diseases, and promote the notion that tTG might be an attractive novel target for treatment of neurodegenerative diseases.