Tuula Halttunen

Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease

BACKGROUND & AIMS Tissue transglutaminase has been reported to be the target for endomysial antibodies in celiac disease. We sought to establish whether immunoglobulin (Ig) A class tissue transglutaminase autoantibodies can be considered specific for celiac disease. METHODS Serum samples from 136 patients with untreated celiac disease (diagnosed according to the criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition) and 207 disease controls were studied. Enzyme-linked immunosorbent assay (ELISA) and Western blots were performed using calcium-treated and untreated tissue transglutaminase as antigen. Reticulin, endomysial, and mouse monoclonal tissue transglutaminase antibodies were studied by an indirect immunofluorescence method and gliadin antibodies with ELISA. RESULTS The calcium-activated tissue transglutaminase autoantibody ELISA was highly sensitive (129 of 136) and specific (194 of 207) in detecting celiac disease. The new autoantibody ELISA test correlated well with the endomysial antibody test. Tissue transglutaminase autoantibody ELISA showed a clearly better predictive potential than the IgA class gliadin antibody ELISA. Immunoblots and ELISA blocking studies showed that calcium is needed for the specific antigen-antibody reaction to occur. Double immunofluorescence staining in human umbilical cord with sera from patients with celiac disease and with monoclonal tissue transglutaminase antibodies showed complete overlap. CONCLUSIONS Calcium-activated tissue transglutaminase autoantibody ELISA is highly accurate in detecting untreated celiac disease. Tissue transglutaminase seems to be the target self-antigen for endomysial antibodies.

In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies.

Background: IgA class serum autoantibodies against type 2 (tissue) transglutaminase (TG2) bind to both intestinal and extraintestinal normal tissue sections in vitro, eliciting endomysial, reticulin, and jejunal antibody reactions. It is not known whether similar binding also occurs in coeliac patients in vivo, and may thereby contribute to disease manifestations. Aims: To investigate intestinal and extraintestinal coeliac tissues for the presence of in vivo bound TG2 specific IgA and its relation to small intestinal mucosal atrophy. Patients: We investigated jejunal samples with normal villous morphology from 10 patients with developing coeliac disease who subsequently progressed to a flat lesion, from 11 patients with dermatitis herpetiformis, and from 12 non-coeliac controls. Six extrajejunal biopsy samples (liver, lymph node, muscle, appendix), obtained based on independent clinical indications from patients with active coeliac disease, were also studied. Methods: Double colour immunofluorescent studies for in situ IgA, TG2, and laminin were performed. IgA was eluted from tissue sections and tested for TG2 specificity by enzyme linked immunosorbent assay and indirect immunofluorescence. Results: IgA (in one IgA deficient case IgG) deposition on extracellularly located TG2 was detected in jejunal and extrajejunal specimens of all coeliac patients, and also in seven of 11 dermatitis herpetiformis patients, of whom two had no circulating endomysial antibodies. IgA eluted from extraintestinal coeliac tissues was targeted against TG2. Conclusions: Coeliac IgA targets jejunal TG2 early in disease development even when endomysial antibodies are not present in the circulation. Extraintestinal target sites of coeliac IgA further indicate that humoral immunity may have a pathogenetic role.

Serum immunoglobulin a from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation

BACKGROUND & AIMS Celiac disease is characterized by disturbed jejunal crypt-villus axis biology with immunoglobulin (Ig) A deposits underlining the epithelium. The aim of this study was to test whether celiac disease serum IgA (reticulin/endomysial autoantibodies) interferes with the mesenchymal-epithelial cell cross-talk. METHODS Differentiation of T84 epithelial cells was induced with IMR-90 fibroblasts or transforming growth factor beta in three-dimensional collagen gel cultures. The effects of purified celiac IgA and monoclonal tissue transglutaminase antibodies (CUB7402) were studied by adding the antibodies to the cocultures. RESULTS Active celiac disease IgA, reactive for tissue transglutaminase, significantly inhibited T84 epithelial cell differentiation (P < 0.001) and increased epithelial cell proliferation (P = 0.024). Similar effects were obtained with antibodies against tissue transglutaminase. CONCLUSIONS Celiac disease-associated IgA class antibodies disturb transforming growth factor beta-mediated fibroblast-epithelial cell cross-talk in this in vitro crypt-villus axis model. This primary finding indicates that celiac disease-specific autoantibodies may also contribute to the formation of the gluten-triggered jejunal mucosal lesion in celiac disease.

Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study.

Objective In coeliac disease, autoantibodies directed against transglutaminase 2 are produced in small-bowel mucosa, and they have been found to be deposited extracellularly. The aim of this study was to investigate whether such mucosal IgA deposits are important in the diagnostic work-up of early-stage coeliac disease without small-bowel mucosal villous atrophy. Material and methods Forty-one adults suspected of coeliac disease owing to increased density of mucosal γδ+ intraepithelial lymphocytes but normal villous morphology were randomized to gluten challenge or a gluten-free diet for 6 months. Clinically and histologically verified gluten dependency was compared with existence of small-bowel mucosal transglutaminase 2-specific extracellular IgA deposits and (coeliac disease-type) HLA DQ2 and DQ8; 34 non-coeliac subjects and 18 patients with classical coeliac disease served as controls. Results Of the 41 patients, 5 in the challenge group and 6 in the gluten-free diet group were clinically gluten sensitive; all 11 had HLA DQ2 or DQ8. Ten of these 11 patients showed transglutaminase 2-targeted mucosal IgA deposits, which were dependent on gluten consumption. Minimal IgA deposits were seen in only 3 out of 30 patients with suspected coeliac disease without any clinically detected gluten dependency. The deposits were found in all classical coeliac patients and in none of the non-coeliac control subjects. Conclusions Clinically pertinent coeliac disease exists despite normal small-bowel mucosal villous architecture. Mucosal transglutaminase 2-specific IgA deposits can be utilized in detecting such patients with genetic gluten intolerance.

Design, Synthesis, and Evaluation of Gluten Peptide Analogs as Selective Inhibitors of Human Tissue Transglutaminase

Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.

Missing endomysial and reticulin binding of coeliac antibodies in transglutaminase 2 knockout tissues

Background: Autoantibodies against transglutaminase 2 (TG2) are thought to be responsible for the endomysial (EMA), reticulin (ARA), and jejunal antibody (JEA) tissue binding of serum samples from coeliac patients but the exclusive role of TG2 in these staining patterns has not yet been established. Aims: To evaluate whether antigens other than TG2 contribute to EMA/ARA/JEA reactions. Patients: Serum samples from 61 EMA/ARA/JEA positive untreated patients with coeliac disease, 40 dermatitis herpetiformis patients, and 34 EMA/ARA/JEA negative non-coeliac controls were tested. Methods: TG2 knockout (TG2−/−) and wild-type mouse oesophagus, jejunum, liver, and kidney sections, and TG2−/− sections coated with human recombinant TG2 were used as substrates in single and double immunofluorescent studies for patient IgA binding and tissue localisation of TG2, fibronectin, actin, and calreticulin. Results: None of the patient serum samples elicited EMA, ARA, or JEA binding in TG2−/− morphologically normal tissues. In contrast, 96 of 101 gluten sensitive patient samples (95%) reacted with wild-type mouse tissues and all 101 reacted in EMA/ARA/JEA patterns with TG2−/− mouse tissues coated with human TG2. Serum IgA binding to TG2−/− smooth muscle cells was observed in low titres in 31.1%, 27.5%, and 20.5%, and to TG2−/− epithelium in 26.3%, 5.0%, and 8.8% of coeliac, dermatitis herpetiformis, and control samples, respectively. These positivities partly colocalised with actin and calreticulin but not with TG2 or fibronectin. Conclusions: EMA/ARA/JEA antibody binding patterns are exclusively TG2 dependent both in coeliac and dermatitis herpetiformis patients. Actin antibodies are responsible for some positivities which are not part of the EMA/ARA/JEA reactions.

Measurement of total and local bone morphogenetic protein concentration in bone tumours.

Summary. Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.Résumé. Il a été démontré que la protéine morphogénétique osseuse (BMP) est un facteur important dans le pronostic des affections osseuses. Dans un développement osseux normal, cette protéine induit la formation de nouveau tissu osseux et contribue, par exemple, dans les fractures, à la consolidation. Dans les affections malignes, la fonction précise de la BMP reste inconnue. Dans la présente étude, la concentration de protéine morphogénétique osseuse a été mesurée dans des tumeurs osseuses primaires à l’aide de deux méthodes. L’intensité de coloration locale de la BMP a été détectée immunohistochimiquement par la méthode avidine-biotine-péroxydase, déterminant le titre le plus élevé de dilution d’antisérum par rapport à la BMP bovine (bBMP); le résultat de la coloration a été positif. Le total de protéine morphogénétique osseuse présent dans un échantillon de tumeur a été mesuréà l’aide d’un dosage enzymatique immuno-absorbant (ELISA) aprés digestion du tissu avec de la collagénase pour retirer les protéines du tissu conjonctif. La coloration immunohistochimique a montré que la BMP se localisait dans les cytoplasmes et dans le tissu osseux réactif formé par les cellules malignes. La plus forte concentration locale de BMP a été trouvée dans le tissu des cellules géantes de la tumeur dans chondrosarcome, l’ostéosarcome et les tumeurs osseuses bénignes. Le total de BMP présent dans les tumeurs osseuses malignes était 2.4 fois supérier à celui décelé dans les tumeurs osseuses bénignes.

Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate

BACKGROUND Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type matrix metalloproteinase [MT1-MMP] or MMP-14). MATERIAL AND METHODS We evaluated the effects of a bisphosphonate (clodronate) on MT1-MMP mRNA expression and protein production, catalytic activity and proteolytic activation of proMMP-2 by cultured human MG-63 osteosarcoma cells. RESULTS Clodronate, at therapeutically attainable noncytotoxic concentrations, dose-dependently inhibited phorbol myristic acetate (PMA)-induced proteolytic activation of proMMP-2 by human MG-63 osteosarcoma cells. Clodronate also downregulated the PMA-induced expression of MT1-MMP mRNA and protein production in human MG-63 osteosarcoma cells, as evidenced by Northern analysis and fluorescent immunohistochemistry. Furthermore, clodronate inhibited directly and dose-dependently MT1-MMP activity, and the MT1-MMP inhibition by clodronate was reduced in the presence of an increased (5 mM) Ca(2+) concentrations when compared to physiological (1 mM) Ca(2+) concentrations. CONCLUSION We conclude that (1) the extracellular/cell-associated mechanism of bisphosphonate involves inhibition of MT1-MMP catalytic activity eventually by chelation, and that (2) intracellular mechanism involves downregulation of induced MT1-MMP mRNA and protein expression. The inhibition and downregulation of MT1-MMP by clodronate can be related to their ability to reduce MG-63 osteosarcoma cell invasion and spread. These findings may, at least in part, explain at molecular level the antitumor and antibone resorption activities of clodronate observed in clinical studies.

The role of transforming growth factor-β on retarded osteoblastic differentiation in vitro

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.

Blisters in the small intestinal mucosa of coeliac patients contain T cells positive for cyclooxygenase 2

Background and aims: Coeliac disease is characterised by atrophy of the villi and hyperplasia of the crypts in the mucosa of the small intestine. It is caused by an environmental trigger, cereal gluten, which induces infiltration of the mucosa by inflammatory cells. We hypothesised that these inflammatory cells express cyclooxygenase 2 (COX-2), an enzyme that contributes to the synthesis of pro and anti-inflammatory prostaglandins and is known to be expressed at sites of inflammation in the stomach and colon. We have investigated expression of COX-2 in the coeliac disease affected small intestinal mucosa where it may be an indicator of either disease induction or mucosal restoration processes. Patients and methods: Small intestinal biopsy samples from 15 coeliac patients and 15 non-coeliac individuals were stained immunohistochemically for COX-2. Samples from 10 of the patients were also stained after these patients had been on a gluten free diet for 6–24 months. Various cell type marker antigens were used for immunohistochemical identification of the type of cell that expressed COX-2. To further verify colocalisation of the cell type marker and COX-2, double immunoperoxidase and immunofluorescence methods were employed. Immunoelectron microscopy was used to investigate the subcellular location of COX-2. Results: In all samples taken from coeliac patients, clusters of cells with strong immunoreactivity for COX-2 were found in those areas of the lamina propria where the epithelium seemed to blister or was totally detached from the basement membrane. These clusters were reduced in number or totally absent in samples taken after a gluten free diet. No such clusters were seen in any control samples. The density of COX-2 positive cells lining the differentiated epithelium decreased significantly from 13.5 (5.1) cells/105 μm2 (mean (SD)) in the untreated patient samples to 6.5 (2.0) cells/105 μm2 after a gluten free diet (p<0.001), and was 3.3 (1.9) cells/105 μm2 in control samples (p<0.001 compared with untreated or diet treated coeliac samples). Staining for COX-2 was localised to CD3+ T cells and CD68+ macrophages in the mucosal lesions but not all of these cells were positive for COX-2. Immunoelectron microscopy revealed that the ultrastructure of the COX-2 positive cells resembled that of lymphocytes, and the immunoreaction was localised to the rough endoplasmic reticulum and the nuclear envelope. Conclusions: Our results show that in coeliac disease, blistering of small intestinal epithelial cells is associated with accumulation of COX-2 positive T cells, and the number of these cells decreases after a gluten free diet. These observations suggest that COX-2 mediated prostanoid synthesis contributes to healing of the coeliac mucosa and may be involved in maintenance of intestinal integrity.