GAD antibody-associated neurological illness and its relationship to gluten sensitivity.

BACKGROUND: The high prevalence of gluten sensitivity in patients with stiff-person syndrome (SPS) lead us to investigate the relationship between gluten sensitivity and GAD-antibody-associated diseases. METHODS: We used ELISA assays for anti-GAD and for serological markers of gluten sensitivity. Patients were recruited from clinics based at the Royal Hallamshire hospital, Sheffield, UK. Patients with gluten sensitivity were followed up after the introduction of a gluten-free diet and serological testing was repeated. RESULTS: Six of seven (86%) patients with SPS were positive for anti-GAD, mean titre 109 U/ml; This compared with 9/90 (11%) patients with idiopathic sporadic ataxia, mean titre 32 U/ml, 16/40 (40%) patients with gluten ataxia, mean titre 25 U/ml, and 6/10 patients with type 1 diabetes only, mean titre 8 U/ml. None of 32 patients with celiac disease only, and of 40 patients with genetic ataxia were positive for anti-GAD. The titre of anti-GAD reduced following the introduction of a gluten-free diet in patients with SPS who had serological evidence of gluten sensitivity. The same was observed in patients with gluten ataxia and anti-GAD antibodies. This was also associated with clinical improvement. CONCLUSION: These findings suggest a link between gluten sensitivity and GAD antibody-associated diseases.

Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes.

Calcifying cartilages show a restricted expression of tissue transglutaminase. Immunostaining of newborn rat paw bones reveals expression only in the epiphyseal growth plate. Tissue transglutaminase appears first intracellularly in the proliferation/maturation zone and remains until calcification of the tissue in the lower hypertrophic zone. Externalization occurs before mineralization. Subsequently, the enzyme is present in the interterritorial matrix during provisional calcification and in the calcified cartilage cores of bone trabeculae. In trachea, mineralization occurring with maturation in the center of the cartilage is accompanied by expression of tissue transglutaminase at the border of the hydroxyapatite deposits. Transglutaminase activity also shows a restricted distribution in cartilage, similar to the one observed for tissue transglutaminase protein. Analysis of tissue homogenates showed that the enzyme is present in growth plate cartilage, but not in articular cartilage, and recognizes a limited set of substrate proteins. Osteonectin is coexpressed with tissue transglutaminase both in the growth plate and in calcifying tracheal cartilage and is a specific substrate for tissue transglutaminase in vitro. Tissue transglutaminase expression in skeletal tissues is strictly regulated, correlates with chondrocyte differentiation, precedes cartilage calcification, and could lead to cross-linking of the mineralizing matrix.

Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.

Cross-linking of the dermo-epidermal junction of skin regenerating from keratinocyte autografts. Anchoring fibrils are a target for tissue transglutaminase.

Since transglutaminases create covalent gamma-glutamyl-epsilon-lysine cross-links between extracellular matrix proteins they are prime candidates for stabilizing tissue during wound healing. Therefore, we studied the temporo-spatial expression of transglutaminase activity in skin regenerating from cultured epithelial autografts in severely burned children by the specific incorporation of monodansylcadaverine into cryostat sections from skin biopsies obtained between 5 d to 17 mo after grafting. The dansyl label was subsequently immunolocalized in the epidermis, dermal connective tissue, and along the basement membrane. Incubation of cryosections of normal and regenerating skin with purified tissue transglutaminase confirmed the dermo-epidermal junction and the papillary dermis as targets for this enzyme and revealed that in regenerating skin transamidation of the basement membrane zone was completed only 4-5 mo after grafting. Immunoelectron microscopy revealed that three distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo formation of anchoring fibrils in regenerating skin. Biochemically, we identified collagen VII as potential substrate for tissue transglutaminase. Thus, tissue transglutaminase appears to play an important role not only in cross-linking of the papillary dermis but also of the dermo-epidermal junction in particular.

Expression and activation of tissue transglutaminase in apoptotic cells of involuting rodent mammary tissue.

Apoptosis is a form of cell death in which cellular integrity is maintained and neither cytoplasmic nor nuclear content is released. The Ca(2+)-dependent tissue transglutaminase (tTG) is an enzyme that forms protein cross-links between specific glutamyl and lysyl side-chains of intra- and extracellular proteins, therefore it may be responsible for the structural stabilization observed during the death process. In this study, the expression of tTG was investigated following the physiological process of forced weaning which results in an almost complete elimination of secretory epithelium by apoptosis and remodelling of the tissue structure. A dramatic induction of tTG was detected by immunoblotting of total mammary gland homogenates prepared from the involuting glands. The concentration of epsilon(gamma-glutamyl)-lysine crosslinks was also elevated in these samples, showing that the enzyme is activated during apoptosis. To determine the distribution of tTG and its relationship to apoptotic cells, paraffin-embedded specimens were studied by RNA in situ hybridization and immunohistochemical methods as well as using in situ labeling for nuclear fragmentation. All of these approaches indicated that mammary secretory epithelium expressed tissue transglutaminase coincident with the onset of apoptosis. The apoptotic and tTG-expressing cells were found to be identical as demonstrated by a histological double-labeling technique.

In vitro and rapid in situ transglutaminase assays for congenital ichthyoses--a comparative study.

Autosomal recessive congenital ichthyoses are a heterogeneous group of disfiguring skin diseases. They are generally characterized by variable scaling and erythroderma, and patients are frequently collodion babies at birth. Autosomal recessive congenital ichthyoses are represented in 25 of our 50 families by a defective keratinocyte transglutaminase (TGK). Pathogenic classification is difficult to assess on clinical grounds for autosomal recessive congenital ichthyoses and impossible for collodion babies. Thus, we have established a rapid TGK assay in situ on frozen skin sections using incorporation of dansyl-cadaverin to assess transglutaminase (TG) activity in combination with immunohistochemistry for TGK protein. Results were compared with TG activity levels measured in cultured differentiating keratinocytes. Sixteen of 26 patients, including a collodion baby, had strongly diminished TG activity in the cell periphery of differentiating keratinocytes and membrane-bound TG activities in vitro, ranging from 2.2 to 281.3 pmol per h mg. Nine of 26 patients, including a collodion baby, showed strong TG activity in the cell periphery of differentiating keratinocytes in situ and membrane-bound TG activities in vitro ranged from 1519 to 10917 pmol per h mg. In one case, TG assay in situ was ambiguous; however, membranous TG activity in vitro was very low at 76.9 pmol/h x mg. Our results demonstrate an excellent correlation of TG assays in vitro and in situ. In addition, we present a novel test with prognostic value for the collodion baby phenotype. This assay allows rapid pathogenic classification of autosomal recessive congenital ichthyoses with only one caveat that in rare ambiguous cases it might be necessary for proper classification to assess membrane-bound TG activity in vitro.

Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

Expression and initial characterization of recombinant mouse thrombospondin 1 and thrombospondin 3.

To analyze the function of TSP family members, we have expressed and purified mouse TSP1 and TSP3 encoded by recombinant baculoviruses in Spodoptera frugiperda cells and compared these TSPs to mouse TSP2 prepared in a similar way. Yields of purified TSP1 and TSP3 were 5-15 and 2-4 micrograms, respectively, per ml of conditioned medium. Mature, secreted mouse T41 and TSP3 had the previously predicted NH2-terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS-PAGE and rotary shadowing electron microscopy indicated that TSP1 and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide-bonded pentamer. Binding assays with 45Ca2+ as ligand and immobilized TSP1, TSP2 and TSP3 demonstrated that all three TSPs are calcium-binding proteins. These results are consistent with previous studies of TSP structure and demonstrate that Spodoptera cells process and secrete TSPs having the same subunit organizations and structure as the native vertebrate molecules.

Transglutaminase activity in the eye: cross-linking in epithelia and connective tissue structures.

PURPOSE: To assess the distribution of transglutaminase (TGase) activity in ocular tissues and the target structures for cross-linking. METHODS: Cryosections from human and cynomolgus monkey eyes were incubated with the biotinylated amine donor substrate cadaverine (biotC), which was subsequently visualized with streptavidin-peroxidase. Confocal laser scanning was used to colocalize biotC and fibrillin, a major component of elastic microfibrils and the zonular fibers in particular. Cryosections and isolated bovine zonules were treated with purified TGase 2 and biotC. The distribution of different TGases (1, 2, 3, and factor XIII) was confirmed immunohistochemically. RESULTS: Virtually all ocular tissues showed TGase activity with a remarkable preponderance for the ciliary body, zonular fibers, and blood vessel walls. Confocal laser scanning revealed fibrillin-containing microfibrils as a major target for TGase activity, in particular the ciliary zonules. Corneal epithelium and basement membrane showed a TGase cross-linking pattern similar to skin. Treatment of cryosections and isolated bovine zonular fibers with purified TGase 2 led to additional incorporation of biotC into extracellular matrix, particularly zonular fibers. The immunohistochemically predominant TGase 2 was associated with epithelia and particularly with connective tissue fibers. TGase 1 was restricted to the corneal epithelium, whereas factor XIII was found to be associated only with blood vessels. TGase 3 was absent. CONCLUSIONS: TGase 2 appears to be an important cross-linker and thus stabilizer of ocular connective tissue. In particular, the zonular fibers are a major target for TGase 2. This is of relevance in hereditary microfibrillopathies such as Marfan syndrome, which exhibits distinct ocular manifestations such as elongated bulbus, retinal detachment, and subluxation of the lens. Purified or recombinant TGase might be of therapeutic use in the future.

Analysis of changes in mRNA levels of myoblast- and fibroblast-derived gene products in healing skeletal muscle using quantitative reverse transcription-polymerase chain reaction.

Changes in expression of type III alpha1-collagen and myosin II heavy chains were characterized in rabbit skeletal muscle following single stretch injury using quantitative reverse transcription-polymerase chain reaction. Collagen III expression was highly elevated in the injured leg compared with the control limb both at the myotendinous junction and in the distal muscle belly. While upregulation of collagen III expression at the myotendinous junction was maximal on day 1, collagen III expression in the distal muscle belly was unchanged on day 1 but highly elevated by day 3. Over the initial 7-day period, there was on average a 94% increase in collagen III expression at the myotendinous junction and a 42% increase in the distal muscle belly. On the other hand, there was little difference, in fact, slightly less expression of myosin II isoforms, in the injured leg compared with the control side. Immunohistochemical analysis of injured muscle showed significant collagen III deposition at the myotendinous junction beginning at day 3 post-injury and still evident by day 14. Focal deposits of type I and III collagen were first apparent in the distal muscle belly by day 3 and striking by day 7. Taken together, the data suggest the formation of connective tissue scar at the injury site and the absence of significant muscle regeneration following muscle stretch. Furthermore, microinjuries distant to the primary site of injury may result in more general muscle fibrosis and scarring.

Expression patterns of extracellular matrix components in native and cultured normal human thyroid tissue and in human toxic adenoma tissue.

The extracellular matrix (ECM) and basement membranes (BM, a specialized form of ECM) greatly influence proliferation, differentiation, and function of cells and the structure of tissues. While a considerable amount of information is available on thyroid cellular proliferation, differentiation and function, much less is known about thyroid ECM and BM. In this study the presence of the ECM/BM components fibronectin, collagen IV, alpha1, beta1, gamma1 laminin, several laminin variants, osteonectin, and perlecan was demonstrated in cryosections of nonadenomatous and toxic adenoma human thyroid tissue. Also, positive immunohistochemical staining for collagen IV, laminin, perlecan, and fibronectin was obtained in sections of human thyroid tissue cultured in a three-dimensional (alginate) culture system. The present study provides methods and data that will facilitate the investigation of the interaction between cells and ECM in thyroid tissue.

A new biological glue for cartilage-cartilage interfaces: tissue transglutaminase.

In this study, we used an in vitro model to test the capacity of tissue transglutaminase to increase the adhesive strength at a cartilage-cartilage interface. Full-thickness cartilage-bone cylinders were prepared from fresh adult bovine shoulder joints, and the superficial half of the hyaline cartilage was then removed to provide a plane surface. Tissue transglutaminase was applied to the freshly cut surface of one cylinder, and a calcium-chloride solution (to act as an activating agent) was applied to that of the other. The cartilage surfaces were immediately apposed, one on top of the other, and an eighty-gram weight was applied to the upper cylinder for ten minutes at 37 degrees Celsius under defined humidity conditions. A measured force was then applied transversely to the upper cylinder until it was displaced from the lower one (which was clamped in a holding device), and the force recorded at this point was taken as a measure of the adhesive strength achieved at the cartilage-cartilage interface. The adhesive strength increased linearly with an increasing concentration of tissue transglutaminase (0.25 to 2.75 milligrams per milliliter) and was enhanced by increasing the duration of incubation, but it was not influenced by the level of humidity. The adhesive strength was improved by as much as 40 per cent when the cartilage surfaces had been pretreated with chondroitinase AC or hyaluronidase to remove glycosaminoglycan chains of proteoglycans, which are largely responsible for the intrinsic anti-adhesive properties of cartilage.

Cross-linking of laminin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization.

The laminin-nidogen complex, a major component of basement membranes, incorporates [3H]putrescine and monodansylcadaverine in the presence of guinea pig liver transglutaminase. Label was detected in nidogen in the isolated, as well as in the complexed form, but not in laminin. The incorporation proceeds in a time-dependent manner at a rate similar to that achieved with N,N-dimethylcasein, a well characterized transglutaminase substrate. Saturation of incorporation site(s), as well as comparison with the incorporation level in reference proteins, indicated the presence of one high affinity amine acceptor site in nidogen. Electron microscopy of the reaction products showed that the laminin-nidogen complexes become stabilized in a head-to-head arrangement, characteristic of Ca(2+)-induced self-aggregation. Indirect immunofluorescence and detection of transglutaminase activity on unfixed cryosections revealed an extracellular distribution of tissue transglutaminase. Intensive staining was observed in collagen-rich connective tissue. Codistribution with nidogen was not a ubiquitous feature, but was observed in many locations.

Protein crosslinking in assembly and remodelling of extracellular matrices: the role of transglutaminases.

Transglutaminases form a family of proteins that have evolved for specialized functions such as protein crosslinking in haemostasis, semen coagulation, or keratinocyte cornified envelope formation. In contrast to the other members of this protein family, tissue transglutaminase is a multifunctional enzyme apparently involved in very disparate biological processes. By virtue of its reciprocal Ca2+-dependent crosslinking activity or GTP-dependent signal transducing activity, tissue transglutaminase exhibits true multifunctionality at the molecular level. The crosslinking activity can subserve disparate biological phenomena depending on the location of the target proteins. Intracellular activation of tissue transglutaminase can give rise to crosslinked protein envelopes in apoptotic cells, whereas extracellular activation contributes to stabilization of the extracellular matrix and promotes cell-substrate interaction. While tissue transglutaminase synthesis and activation is normally part of a protective cellular response contributing to tissue homeostasis, the enzyme has also been implicated in a number of pathological conditions including fibrosis, atherosclerosis, neurodegenerative diseases, celiac disease, and cancer metastasis. This review discusses the role of transglutaminases in extracellular matrix crosslinking with a focus on the multifunctional enzyme tissue transglutaminase.

New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels.

Biodegradable materials for spatially and temporally controlled delivery of bioactive agents such as drugs, growth factors, or cytokines are key to facilitating tissue repair. We have developed a versatile method for chemical crosslinking high-molecular-weight hyaluronic acid under physiological conditions yielding biocompatible and biodegradable hydrogels. The method is based on the introduction of functional groups onto hyaluronic acid by formation of an active ester at the carboxylate of the glucuronic acid moiety and subsequent substitution with a side chain containing a nucleophilic group on one end and a (protected) functional group on the other. We have formed hyaluronic acid with amino or aldehyde functionality, and subsequently hydrogels with these hyaluronic acid derivatives and bifunctional crosslinkers or mixtures of the hyaluronic acid derivatives carrying different functionalities using active ester- or aldehyde-mediated reactions. Size analysis of the hyaluronic acid derivatives showed that the chemical modification did not lead to fragmentation of the polysaccharide. Hydrogels formed with hyaluronic acid derivatized to a varying degree and crosslinked with low- or high-molecular-weight crosslinkers were evaluated for biodegradability by digestion with hyaluronidase and for biocompatibility and ectopic bone formation by subcutaneous implantation in rats. Several hydrogel formulations showed excellent cell infiltration and chondro-osseous differentiation when loaded with bone morphogenetic protein-2 (BMP-2). Synergistic action of insulin-like growth factor-1 with BMP-2 promoted cartilage formation in this model, while addition of transforming growth factor-beta and BMP-2 led to rapid replacement of the matrix by bone.

Identification of Gln726 in nidogen as the amine acceptor in transglutaminase-catalyzed cross-linking of laminin-nidogen complexes.

The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.

Tissue transglutaminase and factor XIII in cartilage and bone remodeling.

While it is well established that factor XIII functions in crosslinking of the fibrin clot during blood coagulation and in wound healing, the physiological role of tissue transglutaminase is still unclear. Recent studies suggest that the expression of tissue transglutaminase correlates with (terminal) differentiation of cells and that the enzyme may play a role in extracellular matrix remodeling. In cartilage, tissue transglutaminase expression is restricted to hypertrophic chondrocytes and the enzyme is externalized at a distinct step in the chondrocyte maturation program. Upon activation by Ca2+, the transglutaminase modifies matrix constituents in a way that might predispose the matrix for the subsequent mineralization. Crosslinks of the structure gamma-glutamyl-epsilon-lysine are also abundant in bone matrix, but the transglutaminase expressed by osteoblasts and presumably involved in crosslinking of newly formed osteoid is likely to be distinct from both tissue transglutaminase and factor XIII. Matrix proteins thought to be crosslinked by transglutaminases in cartilage and bone matrix include glycoproteins such as osteonectin, osteopontin, fibronectin, fibrillin, and collagens II, III, V, and XI. Expression of the A subunit of factor XIII is restricted to megakaryocytes in the bone marrow cavity, and factor XIIIa is abundant in platelets that probably provide the major source for factor XIII in plasma.