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.

Uso de factor VII en el control de sangrados perioperatorios: evidencias actuales / Recombinant activated factor VII for perioperative bleeding

Recombinant activated factor VII (rFVIIa) is a new haemostatic drug, originally used for the treatment of patients with hemophilia A and B. At the present time it is used for other bleeding conditions such as the perioperative period. When used prophylactically there is a reduction in the number of bleeding episodes but no changes in the need for blood transfusion or other blood products. The adverse effects are arterial or venous thromboembolic events that are mostly related to the severity of the underlying disease of the patient and the concurrent administration of other haemostatic agents, rather than the use of rFVIIa. Its use is recommended when there is a persistent bleeding after the reposition of blood products and when surgical causes of bleeding have been discarded. The cost of the medication should also be considered before its use.

Characterization of the mouse matrilin-4 gene: a 5' antiparallel overlap with the gene encoding the transcription factor RBP-l.

We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5' UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5' with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.

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.

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z.

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.

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.

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.

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.

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.

The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix.

A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.

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.

Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.

We developed a method using a single set of degenerate oligonucleotide primers for amplification of the conserved active site of transglutaminases by reverse transcription-polymerase chain reaction (RT-PCR) and identification of the PCR products by cleavage with diagnostic restriction enzymes. We demonstrate amplification of tissue transglutaminase (TGC), keratinocyte transglutaminase (TGK), prostate transglutaminase (TGP), the a-subunit of factor XIII, and band 4.2 protein from different human cells or tissues. Analysis of normal human keratinocytes revealed expression of a transglutaminase different from the expected and characterized transglutaminase gene products. A full-length cDNA for the novel transglutaminase (TGX) was obtained by anchored PCR. The deduced amino acid sequence encoded a protein with 720 amino acids and a molecular mass of approximately 81 kDa. A comparison of TGX to the other members of the gene family revealed that the domain structure and the residues required for enzymatic activity and Ca2+ binding are conserved and showed an overall sequence identity of about 35%. Two transcripts with an apparent size of 2.2 and 2.8 kilobases were detected with a specific probe for TGX on Northern blots of human foreskin keratinocyte mRNA, indicating the presence of alternatively spliced mRNAs. cDNA sequencing revealed a shorter TGX transcript lacking the sequence homologous to that encoded by exon III of other transglutaminase genes. TGX expression increased severalfold when keratinocyte cultures were induced to differentiate by suspension or growth to postconfluency, suggesting that TGX contributes to the formation of the cornified envelope.

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.

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development.

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

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 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.