Transglutaminase Type Research Articles

Type 2 Transglutaminase, mitochondria and Huntington's disease: Menage a trois

Mitochondria produce the bulk of cellular energy and work as decisional “hubs” for cellular responses by integrating different input signals. The determinant in the physiopathology of mammals, they attract major attention, nowadays, for their contribution to brain degeneration. How they can withstand or succumb to insults leading to neuronal death is an object of great attention increasing the need for a better understanding of the interplay between inner and outer mitochondrial pathways residing in the cytosol. Of the latter, those dictating protein metabolism and therefore influencing the quality function and control of the organelle are of our most immediate interest and here we describe the Transglutaminase type 2 (TG2) contribution to mitochondrial function, dysfunction and neurodegeneration. Besides reviewing the latest evidences we share also the novel ones on the IF1 pathway depicting a molecular conduit governing mitochondrial turnover and homeostasis relevant to envisaging preventive and therapeutic strategies to respectively predict and counteract deficiencies associated with deregulated mitochondrial function in neuropathology.

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states.

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293TG) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.

A novel approach for improving the yield of Bacillus subtilis transglutaminase in heterologous strains.

The transglutaminase (BTG) from Bacillus subtilis is considered to be a new type of transglutaminase for the food industry. Given that the BTG gene only encodes a mature peptide, the expression of BTG in heterologous microbial hosts could affect their normal growth due to BTG's typical transglutaminase activity which can catalyze cross-linking of proteins in the cells. Therefore, we developed a novel approach to suppress BTG activity and reduce the toxicity on microbial hosts, thus improving BTG yield. Genes encoding the respective regions of transglutaminase propeptide from seven species of Streptomyces were fused to the N-terminal of the BTG gene to produce fusion proteins. We found that all the fused propeptides could suppress BTG activity. Importantly, BTG activity could be completely restored after the removal of the propeptides by proteolytic cleavage. Of the seven propeptides tested, the propeptide proD from Streptomyces caniferus had the strongest suppressive effect on BTG activity (70 % of the activity suppressed). Moreover, fusion protein proD-BTG (containing proD) also exhibited the highest yield which was more than twofold of the expression level of BTG in an active form in Escherichia coli. Secretion expression of BTG and proD-BTG in Corynebacterium glutamicum further showed that our novel approach was suitable for the efficient BTG expression, thus providing a valuable platform for further optimization of large-scale BTG production.

The second type of transglutaminase regulates immune and stress responses in white shrimp, Litopenaeus vannamei

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopenaeus vannamei, (7.5 ± 0.5 g) were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or different dsRNA at 3 days of injection. In addition, haemolymph glucose and lactate, and haemocytes crustacean hyperglycemic hormone (CHH), transglutaminase I (TGI), transglutaminase II (TGII) and clottable protein (CP) mRNA expression were determined for the shrimp that received DEPC-H2O and different dsRNA after 3 days, and then transferred to 22 and 28 °C from 28 °C. Results showed that respiratory burst, phagocytic activity and clearance efficiency significantly decreased, but hyaline cells significantly increased in the shrimp received LvTGII dsRNA after 3 days. In hypothermal stress studies, LvTGI and CHH were significantly up-regulated in LvTGII-depleted shrimp following exposure to 28 and 22 °C, and haemolymph glucose and lactate were significantly enhanced in LvTGII-depleted shrimp. The injection of LvTGII dsRNA also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. These results suggest that LvTGII is an important component on the immune resistance of shrimp, and is involved in the regulation of some immune parameters and carbohydrate metabolites, as well as has a complementary effect with LvTGI in immunological and physiological response of shrimp.

Germline sequence variants in TGM3 and RGS22 confer risk of basal cell carcinoma

To search for new sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conducted a genome-wide association study of 38.5 million single nucleotide polymorphisms (SNPs) and small indels identified through whole-genome sequencing of 2230 Icelanders. We imputed genotypes for 4208 BCC patients and 109 408 controls using Illumina SNP chip typing data, carried out association tests and replicated the findings in independent population samples. We found new BCC susceptibility loci at TGM3 (rs214782[G], P = 5.5 × 10−17, OR = 1.29) and RGS22 (rs7006527[C], P = 8.7 × 10−13, OR = 0.77). TGM3 encodes transglutaminase type 3, which plays a key role in production of the cornified envelope during epidermal differentiation.

Syndecan-4 knockout leads to reduced extracellular transglutaminase-2 and protects against tubulointerstitial fibrosis.

Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. The interaction of TG2 with the heparan sulfate proteoglycan syndecan-4 (Sdc4) regulates the cell surface trafficking, localization, and activity of TG2 in vitro but remains unstudied in vivo. We tested the hypothesis that Sdc4 is required for cell surface targeting of TG2 and the development of kidney fibrosis in CKD. Wild-type and Sdc4-null mice were subjected to unilateral ureteric obstruction and aristolochic acid nephropathy (AAN) as experimental models of kidney fibrosis. Analysis of renal scarring by Masson trichrome staining, kidney hydroxyproline levels, and collagen immunofluorescence demonstrated progressive fibrosis associated with increases in extracellular TG2 and TG activity in the tubulointerstitium in both models. Knockout of Sdc-4 reduced these effects and prevented AAN-induced increases in total and active TGF-β1. In wild-type mice subjected to AAN, extracellular TG2 colocalized with Sdc4 in the tubular interstitium and basement membrane, where TG2 also colocalized with heparan sulfate chains. Heparitinase I, which selectively cleaves heparan sulfate, completely abolished extracellular TG2 in normal and diseased kidney sections. In conclusion, the lack of Sdc4 heparan sulfate chains in the kidneys of Sdc4-null mice abrogates injury-induced externalization of TG2, thereby preventing profibrotic crosslinking of extracellular matrix and recruitment of large latent TGF-β1. This finding suggests that targeting the TG2-Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD.

Abstract C58: The transglutaminase 2 (TG2) as a new target of p53

Abstract The transglutaminase type 2 (TG2) is a multifunctional protein that has been found previously to be upregulated in lung and breast cancer cells in response to DNA damage. Since the tumor suppressor p53 stabilizes and accumulates in the nucleus upon DNA damage, we investigated the relationship of TG2 and p53 in this study. We demonstrated that TG2 is a downstream target of p53. Knockdown of p53 down-regulated the expression of TG2 mRNA and protein. Similarly, over-expression of p53 upregulated the level of TG2 under normal cell culture condition and also with doxorubicin treatment. More importantly, we found that loss of TG2 enables the transformation of primary mammary epithelial cells in a sensitized colony forming assay. In this system, we used primary human mammary epithelial cells (HMECs) that over-express hTERT, SV40 Small T and RasV12; these cells depend only on the loss of p53 activity for full transformation. Interestingly, we observed a synergy in colony formation when both p21 and TG2 were knock-down, and this effect is even more pronounced when there is a deprivation of growth factors. We discovered that insulin and EGF can overcome the effects of p53 in colony formation and the combined loss of TG2 and p21 can replace the absence of insulin in the transformation of HMECs, similar to that of p53 knock-down. This suggests that loss of TG2 potentiates insulin signaling that could be important in cell transformation. Indeed, we showed that loss of TG2 increase the phosphorylation of 4EBP1 at Thr37/46. Our findings indicate that TG2 functions like a tumor suppressor and is an important target of p53. The loss of TG2 results in cell transformation by mimicking insulin signaling. Citation Format: Shi Yun Yeo. The transglutaminase 2 (TG2) as a new target of p53. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C58.

The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis

Astakine is an important cytokine that is involved in crustacean hematopoiesis. Interestingly, the protein levels of astakine increased dramatically in plasma of LPS-injected shrimp while mRNA levels remained unchanged. Here, we investigated the involvement of astakine 3′-untranslated region (UTR) in its protein expression. The 3′-UTR of astakine down-regulated the expression of reporter protein but the mRNA stability of reporter gene was unaffected. We identified the functional regulatory elements of astakine 3′-UTR, where 3′-UTR242–483 acted as repressor. The electrophoresis mobility shift assay (EMSA), RNA pull-down assay and LC/MS/MS were performed to identify the protein association. We noted that crustin Pm4 and shrimp transglutaminase I (STG I) were associated to astakine 3′-UTR242–483, while two other proteins have yet to be revealed. Depletion of hemocytic crustin Pm4 and STG I significantly increased the protein level of astakine while astakine mRNA level remained unaffected. Lipopolysaccharide (LPS) stimulated the secretion of crustin Pm4 and STG I from hemocytes to plasma and increased the astakine level to stimulate the hemocytes proliferation. Altogether, we identified the shrimp crustin Pm4 and STG I as novel RNA binding proteins that play an important role in down-regulating astakine expression at post-transcriptional level and are crucial for the maintenance of hematopoiesis.

Glucose Homeostasis in Mice Is Transglutaminase 2 Independent

Transglutaminase type 2 (TG2) has been reported to be a candidate gene for maturity onset diabetes of the young (MODY) because three different mutations that impair TG2 transamidase activity have been found in 3 families with MODY. TG2 null (TG2−/−) mice have been reported to be glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Here we rigorously evaluated the role of TG2 in glucose metabolism using independently generated murine models of genetic TG2 disruption, which show no compensatory enhanced expression of other TGs in pancreatic islets or other tissues. First, we subjected chow- or fat-fed congenic SV129 or C57BL/6 wild type (WT) and TG2−/− littermates, to oral glucose gavage. Blood glucose and serum insulin levels were similar for both genotypes. Pancreatic islets isolated from these animals and analysed in vitro for GSIS and cholinergic potentiation of GSIS, showed no significant difference between genotypes. Results from intraperitoneal glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were similar for both genotypes. Second, we directly investigated the role of TG2 transamidase activity in insulin secretion using a coisogenic model that expresses a mutant form of TG2 (TG2R579A), which is constitutively active for transamidase activity. Intraperitoneal GTTs and ITTs revealed no significant differences between WT and TG2R579A/R579A mice. Given that neither deletion nor constitutive activation of TG2 transamidase activity altered basal responses, or responses to a glucose or insulin challenge, our data indicate that glucose homeostasis in mice is TG2 independent, and question a link between TG2 and diabetes.

Extracellular matrix proteins and displacement of cultured fibroblasts from duodenal biopsies in celiac patients and controls

BackgroundCeliac disease (CD) is mainly characterised by villous atrophy and mucosal architectural rearrangement. The fibroblasts (FBs) are the most abundant mesenchymal cell type in the intestinal mucosa and are responsible for both the architectural arrangement of the villi and the formation of the extracellular matrix (ECM). This study aimed at the evaluation of both the intracellular distribution of different proteins involved in ECM and FBs characterisation, and the cellular displacement of primary FBs obtained from duodenal endoscopic biopsies of healthy subjects and celiac patients.MethodsPrimary healthy and celiac duodenal FBs were evaluated by means of immuno-fluorescence assay for collagen type I and IV, fibronectin, actin, alpha-Smooth Muscle Actin (alpha-SMA), Fibroblast Surface Protein (FSP) and transglutaminase type 2 (TG2). The geometric indexes of the fluorescence signals were investigated by image analysis software (Image J, NIH). Both morphology and kinetic were evaluated during a 72 hours time course movie. TG2 medium activity was evaluated by means of ELISA.ResultsAll the cells examined were immunopositive for FSP, alpha-SMA, actin, collagen I, collagen IV and TG2. CD cells showed a signet collagen-I and collagen-IV pattern, as compared to the controls being characterised by a spindle geometry. Moreover, the collagen signals in CD FBs showed a significantly higher circularity index (major orthogonal diameter ratio) than the controls (p < 0.0001), whereas the perimeter and area ratio were significantly lower (p < 0.0001). The TG2 signal had a decreased area (p < 0.05), but a two-fold increased medium activity. The time course highlighted a reduction of the displacement of CD FBs.ConclusionsThe isolated primary CD FBs showed a different collagen and TG2 pattern of distribution associated with a different cellular displacement. The reasons for such CD cell peculiar characteristics are yet unknown but they might represent a factor in the progression of the intestinal damage.

Transglutaminase type 2 in human abdominal aortic aneurysm is a potential factor in the stabilization of extracellular matrix

The aim of this study was to evaluate transglutaminase type 2 (TG2) expression in human abdominal aortic aneurysm (AAA) tissue and to elucidate a potential role of TG2 in AAA formation. TG2, which is a Ca(2+)-dependent cross-linking enzyme, has been proven important for stabilizing the extracellular matrix. However, there is no evidence of the effect of TG2 on AAA formation in a human model. Aortic wall tissues were obtained during surgery in AAA patients (n = 38) and in patients with aortoiliac occlusive disease (Control; n = 4) in the Asan Medical Center from March 2011 to February 2012. In each AAA patient, the aortic neck (Neck) and maximally dilated portion (Max) of the aneurysm were sampled for analysis. TG2 expression was evaluated using immunohistochemistry and Western blotting. In addition, ex vivo experiments of isolated AAA tissue culture with the TG2 inhibitor cystamine and recombinant human TG2 were performed. Among 38 AAA patients, 11 had ruptured (contained or free) AAAs. The mean maximal diameter of AAAs was 6.09 ± 1.46 cm. TG2 expressions of Max were significantly increased compared with those of Control (1.7-fold increase of Control; P = .00). Compared with Control, the intensities of tissue necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitors of metalloproteinase-2 were significantly upregulated in Max (1.7-fold, 1.5-fold, 1.3-fold, and 1.6-fold increases of Control; P = .00, P = .004, P = .046, and P = .007, respectively). Furthermore, double immunofluorescent staining showed that colocalization of TG2/transforming growth factor-β or TG2/fibronectin was prominent in Max compared with those of Neck or Control. In addition, MMP-2 intensity was upregulated in ruptured AAAs compared with unruptured AAAs, with marginal significance (P = .078). Ex vivo experiments showed that protein expressions of tissue necrosis factor-α, MMP-2, and MMP-9 in cultured AAA tissue were decreased by recombinant human TG2 but were increased by exogenous cystamine. The TG2 expression in the maximally dilated portion of AAAs was enhanced compared with that of nondilated aorta. It is suggested that TG2 has a potential effect in stabilization of extracellular matrix by inhibition of proinflammatory cytokines and MMPs or by interaction with fibronectin and transforming growth factor-β.

Does Treatment with Corticosteroids and Cyclosporine Reduce Transglutaminase Type 2 Expression in the Renal Tissue of Patients with Membranous Nephropathy?

Background/Aims: Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults. Transglutaminase type 2 (TG2) contributes to renal scarring through altering extracellular matrix homeostasis. In this study we hypothesized that immunosuppressive treatment would downregulate TG2 expression leading to reduced fibrosis, and subsequently TG2 would have value as a biomarker of progression of MN. Methods: TG2 expression was studied by immunofluorescence in kidney biopsy sections from 32 patients with MN and was compared to control biopsies. All patients were subsequently treated by a combination of cyclosporine and prednisolone for at least 24 months with a repeat biopsy taken in 14 patients. Results: Twenty-two out of 32 patients showed stable renal function, whereas 10 showed doubling of baseline serum creatinine and 5 of them reached end-stage renal disease during the 5-year follow-up. At the end of the follow-up, 22 out of 32 patients were in remission of nephrotic syndrome. TG2 immunostaining was increased in sections from patients with MN compared to healthy controls (p = 0.0002). TG2 at diagnosis was more intense in patients with severer interstitial fibrosis and advanced glomerular sclerosis. TG2 significantly increased in most patients in the repeat biopsies after treatment (p < 0.0001), whereas patients who showed a marked increase in interstitial fibrosis in the repeat biopsy had significantly more TG2 expression in the first biopsy (p = 0.02). Conclusion: TG2 expression is increased in MN patients and continues to increase despite immunosuppressive treatment. However, early detection of TG2 might be of value in MN since increased TG2 production seems to precede extensive interstitial fibrosis.

Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots.

Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis.

Using FLIM-FRET to Measure Conformational Changes of Transglutaminase Type 2 in Live Cells

Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.

A Randomized, Double-Blind Study of Larazotide Acetate to Prevent the Activation of Celiac Disease During Gluten Challenge

OBJECTIVES:In patients with celiac disease, enteropathy is caused by the entry of gluten peptides into the lamina propria of the intestine, in which their immunogenicity is potentiated by tissue transglutaminase (tTG) and T-helper type 1–mediated immune responses are triggered. Tight junction disassembly and paracellular permeability are believed to have an important role in the transport of gluten peptides to the lamina propria. Larazotide acetate is a tight-junction regulator peptide that, in vitro, prevents the opening of intestinal epithelial tight junctions. The aim of this study was to evaluate the efficacy and tolerability of larazotide acetate in protecting against gluten-induced intestinal permeability and gastrointestinal symptom severity in patients with celiac disease.METHODS:In this dose-ranging, placebo-controlled study, 86 patients with celiac disease controlled through diet were randomly assigned to larazotide acetate (0.25, 1, 4, or 8 mg) or placebo three times per day with or without gluten challenge (2.4 g/day) for 14 days. The primary efficacy outcome was the urinary lactulose/mannitol (LAMA) fractional excretion ratio. Secondary endpoints included gastrointestinal symptom severity, quality-of-life measures, and antibodies to tTG.RESULTS:LAMA measurements were highly variable in the outpatient setting. The increase in LAMA ratio associated with the gluten challenge was not statistically significantly greater than the increase in the gluten-free control. Among patients receiving the gluten challenge, the difference in the LAMA ratios for the larazotide acetate and placebo groups was not statistically significant. However, larazotide acetate appeared to limit gluten-induced worsening of gastrointestinal symptom severity as measured by the Gastrointestinal Symptom Rating Scale at some lower doses but not at the higher dose. Symptoms worsened significantly in the gluten challenge–placebo arm compared with the placebo–placebo arm, suggesting that 2.4 g of gluten per day is sufficient to induce reproducible gluten toxicity. Larazotide acetate was generally well tolerated. No serious adverse events were observed. The most common adverse events were headache and urinary tract infection.CONCLUSIONS:LAMA variability in the outpatient setting precluded accurate assessment of the effect of larazotide acetate on intestinal permeability. However, some lower doses of larazotide acetate appeared to prevent the increase in gastrointestinal symptom severity induced by gluten challenge.

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 48 h 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.

Ethyl acetate fraction of Radix rubiae inhibits cell growth and promotes terminal differentiation in cultured human keratinocytes

Ethnopharmacological relevanceIn Chinese medicine practice, Radix rubiae, the dry root of Rubia cordifolia L. is commonly used for the treatment of psoriasis. Aim of the studyPsoriasis is a chronic inflammatory skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes. Our previous studies identified Radix rubiae to have potent antiproliferative action on cultured HaCaT keratinocytes and to induce keratinocyte differentiation in mouse tail model. The present study aimed to investigate whether Radix rubiae could also induce terminal differentiation in cultured human keratinocytes. Methods and resultsThe cornified envelope (CE) formation assay showed that ethyl acetate (EA) fraction of Radix rubiae significantly accentuated the CE formation, a well-recognized marker of terminal differentiation, in cultured HEK and HaCaT cells in a dose and time dependent manner. Western blot analyses demonstrated that EA fraction of Radix rubiae at a concentration of 3.2μg/ml significantly increased transglutaminase type I and involucrin expression in both HEK and HaCaT keratinocytes after 96h treatment, a response similar to that of Ca2+ positive control. Moreover, the expression level of cytokeratin 5/14, which is specifically related to cell proliferation, was significantly downregulated while terminal differentiation markers cytokeratin 1/10 were markedly increased by Radix rubiae treatment in both HEK and HaCaT cells. ConclusionThe present experimental findings unequivocally confirmed the keratinocyte terminal differentiation promoting capacity of Radix rubiae, and strongly suggest that Radix rubiae is a promising antipsoriatic agent warranting further clinical development for psoriasis treatment.

Usefulness of tissue transglutaminase type 2 antibodies in early pregnancy

Celiac disease (CD), an autoimmune disease triggered by dietary gluten, is a multi-systemic disorder that primarily results in mucosal damage of the small intestine. Reproductive disorders and pregnancy complications have been associated with CD. Conflicting results have been published concerning CD and the risk of impaired fetal growth with reduced birthweight. The aim of our multicentric, perspective, case–control study was to determine the prevalence of undiagnosed CD in mothers of small for gestational age (SGA) newborns in two regions of Italy. The study included 480 mothers: group A consisted of 284 SGA newborns’ mothers and group B consisted of 196 appropriate for gestational age (AGA) newborns’ mothers. Tissue transglutaminase type 2 antibodies (TG2) IgA and IgG were measured in blood samples. We diagnosed two new cases of CD in asymptomatic mothers. It may be appropriate to include the TG2 to the panel of prenatal blood test.

European Society for Pediatric Gastroenterology, Hepatology, and Nutrition Guidelines for the Diagnosis of Coeliac Disease

Diagnostic criteria for coeliac disease (CD) from the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) were published in 1990. Since then, the autoantigen in CD, tissue transglutaminase, has been identified; the perception of CD has changed from that of a rather uncommon enteropathy to a common multiorgan disease strongly dependent on the haplotypes human leukocyte antigen (HLA)-DQ2 and HLA-DQ8; and CD-specific antibody tests have improved. A panel of 17 experts defined CD and developed new diagnostic criteria based on the Delphi process. Two groups of patients were defined with different diagnostic approaches to diagnose CD: children with symptoms suggestive of CD (group 1) and asymptomatic children at increased risk for CD (group 2). The 2004 National Institutes of Health/Agency for Healthcare Research and Quality report and a systematic literature search on antibody tests for CD in paediatric patients covering the years 2004 to 2009 was the basis for the evidence-based recommendations on CD-specific antibody testing. In group 1, the diagnosis of CD is based on symptoms, positive serology, and histology that is consistent with CD. If immunoglobulin A anti-tissue transglutaminase type 2 antibody titers are high (>10 times the upper limit of normal), then the option is to diagnose CD without duodenal biopsies by applying a strict protocol with further laboratory tests. In group 2, the diagnosis of CD is based on positive serology and histology. HLA-DQ2 and HLA-DQ8 testing is valuable because CD is unlikely if both haplotypes are negative. The aim of the new guidelines was to achieve a high diagnostic accuracy and to reduce the burden for patients and their families. The performance of these guidelines in clinical practice should be evaluated prospectively.

Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.