OBJECTIVE

Coeliac autoimmunity (CA) has a known association with type 1 diabetes mellitus (T1DM) for which screening is routinely recommended but less frequently followed. The impact of CA in T1DM has been variably reported. The aims of this study are as follows: (1) to study the prevalence of CA in patients with T1DM and (2) to study the impact of CA not only on nutritional parameters but also on glycaemic control, endocrine axes and bone health.

METHODS

Eighty-six consecutive patients with T1DM were screened for CA using immunoglobulin A (IgA) tissue transglutaminase as a marker (TTG; IgG anti-gliadin in IgA-deficient case). CA positive (CA+) cases were compared with age-matched and sex-matched CA negative (CA-) T1DM cases for anthropometry, glycaemic control (assessed by glycated haemoglobin (HbA1c) and hypoglycaemic/hyperglycaemic episodes), endocrine (thyroid function, cortisol, growth hormone (GH) axis, gonadal axes), haematological (haemoglobin, iron profile and vitamin B12 status) and calcium metabolism parameters and bone densitometry (by dual-energy X-ray absorptiometry (DXA)). Consenting patients with CA also underwent upper gastrointestinal (GI) endoscopy with duodenal biopsy.

RESULTS

Out of 86 patients, 11 (12.75%) screened positive for CA (seven patients underwent duodenal biopsies which were suggestive of Marsh grade III(2), II(3) and I(2) disease). The CA+ T1DM patients were comparable with CA- T1DM in terms of anthropometry. CA+ patients had higher HbA1c (10.7±1.8 vs. 8.4±1.0 (93±19 vs. 68±11 mmol/mol); p<0.01), more hypoglycaemic episodes (five vs. two; p<0.05), higher prevalence of iron and vitamin B12 deficiency, lower insulin-like growth factor-1 (IGF-1) levels and lower bone mineral density (BMD) z-score at total body (-1.91±1.05 vs. -0.63±0.73; p<0.05) and lumbar spine (-1.69±0.92 vs. -0.36±0.93; p<0.05). The incidence of fractures in the past 3years was also more in CA+ patients than in CA- patients (four vs. one; p<0.05).

CONCLUSIONS

CA has an important autoimmune association with T1DM. The concomitant presence of CA adversely affects stature, bone health, glycaemic control and iron and B12 levels in T1DM. IgA sufficiency should be ensured before using an IgA-based screening test for CA.

BACKGROUND

Palmoplantar pustulosis (PPP) is a chronic inflammatory disease affecting mainly smoking women. Some patients also have psoriasis. A subgroup of patients with psoriasis has been shown to have silent gluten sensitivity with relevance for their psoriasis. Nothing is known about gluten sensitivity in PPP.

OBJECTIVE

To find out whether any patients with PPP are gluten-sensitive and whether this might be relevant for the PPP activity.

METHODS

One hundred and twenty-three patients (113 women) with PPP participated. Screening for IgA antibodies against gliadin and tissue transglutaminase (tTG) was performed, the duodenal mucosa in patients with and without these antibodies was studied and the effect of a gluten-free diet (GFD) was followed up.

RESULTS

Twenty-two patients (18%) had IgA antibodies against gliadin and nine of 94 (10%) against tTG. Twelve patients with antibodies and 11 without underwent gastro-duodenoscopy. Four displayed villous atrophy, whereas all other specimens were judged as essentially normal at routine staining. However, with immunohistochemistry, the numbers of CD3+ and CD8+ lymphocytes in the epithelium were found to be increased in patients with any type of antibody, although they were most numerous in those with both types of antibodies. Seven of 123 patients (6%) had coeliac disease (three previously diagnosed). Patients with antibodies who adhered to the GFD displayed total or nearly total clearance of the skin lesions and normalization of the antibody levels.

CONCLUSIONS

Patients with PPP should be screened for antibodies against gliadin and tTG. Those with antibodies can be much improved on a GFD regardless of the degree of mucosal abnormalities.

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic beta cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.

OBJECTIVE

The B cell-activating factor of the tumour necrosis factor (TNF) family (BAFF) was recently described as a critical survival factor for B cells, and its expression is increased in several autoimmune diseases. Abnormal production of BAFF disturbs immune tolerance allowing the survival of autoreactive B cells and participates in the progression of B-cell lymphomas. Coeliac disease (CD) is a common autoimmune disorder induced by gluten intake in genetically predisposed individuals, associated with autoantibody production and with an increased risk of lymphoma at follow-up. The purpose of this study was to investigate the possible implications of BAFF in CD.

METHODS

Seventy-three patients with small-bowel biopsies and laboratory-proven diagnosis of CD were included in the study. All serum samples were analysed before the start of a gluten-free diet (GFD). In 12 cases, one or more samples were analysed during follow-up of the GFD. Seventy-seven blood donors were taken as controls. Serum BAFF levels and anti-transglutaminase (a-tTG) antibodies were assessed by ELISA and endomysial antibodies by indirect immunofluorescence.

RESULTS

Serum BAFF levels appeared to be significantly more elevated in CD patients than in controls (p<0.0001) and, compared with other autoimmune diseases where BAFF is increased, a much larger percentage (80.8%) of CD patients presented BAFF levels above the normal range. In addition, serum BAFF levels were found to correlate with a-tTG antibody levels (p =0.0007) and there was a significant reduction of BAFF after introduction of a GFD.

CONCLUSIONS

BAFF may represent a possible pathogenic factor in CD. Its implications for the diagnosis, prognosis and treatment of CD should also be assessed.

BACKGROUND

Autoantibodies to exocrine-pancreatic glycoprotein 2 (anti-GP2) are Crohn's disease (CD) markers. However, CD-specific antibodies have also been found in celiac-disease (CeD) patients, in which type 1 diabetes-specific autoantibodies against endocrine pancreatic targets can be present. We investigated whether anti-GP2 are also present in CeD, a disease like CD which is also characterised by intestinal mucosal inflammation with barrier impairment.

METHODS

Antibodies against GP2, tissue transglutaminase (tTG), deamidated gliadin (dGD), glutamic decarboxylase (GAD), and islet antigen-2 (IA2) were tested in sera from 73 CD patients, 90 blood donors (BD), and 79 (58 de novo) CeD patients (2 consecutive sera were available from 40 patients).

RESULTS

IgA and/or IgG anti-GP2 were found in 15/79 (19.0%) CeD patients on at least one occasion, in 25/73 (34.2%) CD patients, and in 4/90 (4.4%) BD (CeD vs. CD, p=0.042; BD vs. CeD and CD, p<0.001, respectively). Amongst the 58 de novo CeD patients, anti-GP2 IgA and/or IgG were present in 11 (19.0%). Anti-GP2 IgA was significantly less prevalent in CeD compared with CD (p=0.004). Anti-GP2 IgA and IgG in CD patients demonstrated a significantly higher median level compared to patients with CeD (p<0.001, p=0.008, respectively). IgA anti-GP2 levels correlated significantly with IgA anti-tTG and anti-dGD levels in CeD Spearman's coefficient of rank correlation (ρ)=0.42, confidence interval (CI): 0.26-0.56, p<0.001; ρ=0.54, CI 0.39-0.65, p<0.001, respectively.

CONCLUSIONS

The presence of anti-GP2 in CeD patients supports the notion that loss of tolerance to GP2 can probably be a manifestation of an autoinflammatory process in this intestinal disorder.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.

Metal transcription factor-1 (MTF-1) is a ubiquitous transcriptional regulator and chromatin insulator with roles in cellular stress responses and embryonic development. The studies described herein establish for the first time the involvement of MTF-1 in tumor development. Genetically manipulated ras-transformed mouse embryonic fibroblasts (MEFs), wild-type (MTF-1+/+), or nullizygous for MTF-1 (MTF-1-/-) were used to develop fibrosarcoma tumors. Loss of MTF-1 resulted in delayed tumor growth associated with increased matrix collagen deposition and reductions in vasculature density. Molecular consequences of MTF-1 loss include increased expression and activation of the transforming growth factor-beta1 (TGF-beta1) and tissue transglutaminase (tTG), two proteins with documented roles in the production and stabilization of extracellular matrix (ECM). Our findings support the hypothesis that MTF-1 enhances the ability of the developing tumor mass to evade fibrosis and scarring of the tumor, a critical step in tumor cell proliferation.

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.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.

Covalent attachment of synthetic and biological molecules to tissue surfaces can be used to enhance local drug delivery, reduce adhesions after surgery, and attach reconstructive biomaterials and tissue-engineered matrices to tissues. We present here a mild approach to coupling polymers to tissue surfaces through an enzyme catalyzed reaction between peptide modified polymer and native protein components of the tissue extracellular matrix (ECM). Tissue transglutaminase (tTG), a Ca2+-dependent enzyme that catalyzes the reaction between lysine and glutamine residues to form a epsilon(gamma-glutaminyl) lysine isopeptide bond, was incubated with cartilage in the presence of lysine (FKG-NH2) and glutamine (GQQQLG-NH2) peptides as well as peptide functionalized poly(ethylene glycol) (PEG). Immunohistochemistry was used to detect the presence of covalently bound PEG polymer at the tissue surface as well as to a depth of as much as 10 microm below the surface. Collagen II, fibronectin, osteopontin and osteonectin were found to react with the peptides and peptide modified PEG in the presence of tTG in solution, suggesting these cartilage ECM components as being substrates in the tissue reaction. The results illustrate the use of tTG as a simple, effective and biologically compatible method of coupling synthetic and biological molecules to cartilage and other tissues containing ECM proteins that are substrates of tTG.

OBJECTIVE

We present a systematic review on the performance of currently available methods for serological diagnosis of celiac disease (CD) and the role of human leukocyte antigen (HLA) typing.

OBJECTIVE

A literature survey was conducted using PubMed, MeSH database, Web of Science as well as manual searches.

RESULTS

Tissue transglutaminase antibodies (tTG) (IgA) (tested in nine studies) show sensitivities and specificities in the range of 0.76-0.968 and 0.909-0.98, and deamidated gliadin peptide (DGP) (IgA and IgG) (tested in eight studies) show sensitivities and specificities in the range of 0.69-0.984 and 0.903-1. Endomysial antibodies (EMA) (tested in five studies) show sensitivities and specificities in the range of 0.61-0.937 and 0.98-1, respectively. Combination assays (tested in three studies) using DGP + tTG and DGP (IgA + IgG) show sensitivities and specificities in the range of 0.87-1 and 0.8-1, respectively. HLA DQ2/DQ8 may be necessary for the development of CD-HLA DQ2 in particular. A possible close correlation may also exist between CD and HLA-G.

CONCLUSIONS

DGP and tTG for serological testing for CD show equivalent diagnostic performance. More studies with, in particular, DGP alone and in combination with tTG are necessary before a firm recommendation can be made. HLA typing to exclude CD may still be controversial. It still seems premature to diagnose celiac disease in adults based on serology alone.

Selection of patients for diagnostic biopsy concerning celiac disease (CD) is mainly guided by the results with serological screening tests like anti-tissue-transglutaminase (tTG), anti-endomysium (EmA) and anti-gliadin (AGA) IgA. New tests using deamidated gliadin-derived peptides (DGP) including both IgA and IgG antibodies have been developed, to cover the IgA-deficient sera. In addition, a combined IgA and IgG DGP test, with or without human erythrocyte-derived tTG, offers possible advantages. In order to explore the screening accuracy of the new combination tests sera from 167 children below 3 years of age were assayed. Biopsy had been taken in connection with serology in 32 of these children, 24 with histopathological CD. The results with the DGP and the combined test were congruent with the IgA antibody tests for tTG, EmA and AGA, all identifying 21 of 24 of the CD cases. Two of the CD patients were AGA-IgA positive only (2/24), while 2 of 24 sera were AGA-IgA negative but positive in all the other tests. These results raises the question whether the modifications of the gliadin antigen not only decrease false positivity but also give more false-negative results, a major drawback for a screening test for an important disease. Further studies have to be undertaken to explore this. Our results also stress that serologic screening of CD in children cannot be based on one test only.

OBJECTIVE

Assays for antibodies against native gliadin (anti-nGli) are still often assumed to perform better in the diagnosis of coeliac disease in young children than tests for antibodies to deamidated gliadin (anti-dGli), tissue transglutaminase (anti-tTG), and endomysium (EmA). We compared the performance of assays for anti-nGli, anti-dGli, anti-tTG, and EmA in this age group.

METHODS

We investigated retrospectively 184 children (42 with coeliac disease under normal diet and 142 controls) up to 2 years of age. Immunoglobulin (Ig) A- and IgG-anti-dGli, IgA- and IgG-anti-nGli, IgA- and IgG-anti-tTG, and IgA-EmA were measured in serum. Areas under receiver operating characteristics curves, sensitivities, specificities, positive and negative predictive values, positive and negative likelihood ratios, as well as diagnostic odds ratios were calculated.

RESULTS

From all of the tests investigated, only assays for IgG-anti-dGli, IgA-anti-tTG, and IgA-EmA had high specificity (≥ 0.96) connected with high sensitivity (≥ 0.86), with high positive predictive values (≥ 0.52 and ≥ 0.69 at pretest probabilities of 0.05 and 0.1, respectively) and negative predictive values (≥ 0.99 and ≥ 0.98 at pretest probabilities of 0.05 and 0.1, respectively). These assays also showed high positive likelihood ratio (≥ 24) at low negative likelihood ratio (≤ 0.15) and high diagnostic odds ratios (≥ 136).

CONCLUSIONS

Our results do not support the use of assays of anti-nGli to diagnose coeliac disease in young children. IgA-anti-tTG, IgA-EmA, and IgG-anti-dGli perform better than anti-nGli.

OBJECTIVE

Some recent studies showed that alteration of upper-gut motility in coeliac disease may be related to dysfunction of autonomic nervous system. The aim of our study was to investigate whether autonomic nervous system was altered in untreated and unselected coeliac disease patients.

METHODS

We studied 8 untreated and consecutive coeliac disease patients (2 males and 6 females, age range 37+/-14.5 years). Histological evaluation of duodenal mucosa, anti-gliadin antibodies (AGA), antiendomysial antibodies (EMA) and anti-tTG antibodies and sorbitol H2 breath test were performed in all patients. Extrinsic autonomic neuropathy was assessed by the standardized measurement of cardiovascular reflexes (lying-to-standing, Valsalva manoeuvre, deep breathing, sustained handgrip). The results obtained were compared with a healthy, asymptomatic control group (6 males and 7 females, age range 42.3+/-13.5 years).

RESULTS

Coeliac patients exhibited a lower increase of PAS as a response to isometric effort, a reduction of spectral power LF as a response to clinostatic position, but without statistical significance. Also they showed a lower tolerance to orthostatic position, associated with a latent disequilibrium of sympathetic-vagal balance, a relative prevalence of parasympathetic component of the autonomic function. However, these results were not statistically significant when compared with control group (P = n.s.). And they were unchanged after 6 and 12 mo of gluten-free diet.

CONCLUSIONS

This study failed to confirm a significant correlation between autonomic dysfunction and coeliac disease, yet we could not exclude a role of autonomic dysfunction in the genesis of systemic symptoms in some coeliacs.

OBJECTIVE

In celiac disease (CD), there is increased mRNA coding for tissue transglutaminase (tTG) and interferon gamma (IFNα). In seronegative celiac patients, the mucosal immune complexes anti-tTG IgA/tTG are found. We assayed tTG- and IFNα-mRNA in the mucosa of patients with a clinical suspicion of seronegative CD and correlated the values with intraepithelial CD3 lymphocytes (IELs).

METHODS

Distal duodenum specimens from 67 patients were retrieved and re-evaluated for immunohistochemically proven CD3 IELs. Five 10 μm sections were used for the extraction and assay of tTG and IFNα coding mRNA levels using reverse transcriptase real-time polymerase chain reaction (RT-PCR). Samples from 15 seropositive CD patients and 15 healthy subjects were used as positive and negative controls. Results were expressed as fold-change.

RESULTS

Our series was divided into three groups based on IEL count: >25 (14 patients: group A), 15-25 (26 patients: group B), and 0-15 (27 patients: Group C). tTG-mRNA levels were (mean ± SD): CD = 9.8 ± 2.6; group A = 10.04 ± 4.7; group B = 4.99 ± 2.3; group C = 2.26 ± 0.8, controls = 1.04 ± 0.2 (CD = group A>> group B>> group C = controls). IFNα-mRNA levels were: CD = 13.4 ± 3.6; group A = 7.28 ± 3.6; group B = 4.45 ± 2.9; group C = 2.06 ± 1.21, controls = 1.04 ± 0.4.

CONCLUSIONS

Our results suggest that tTG- and IFNγmRNA levels are increased in both seropositive and potential seronegative patients with CD, showing a strong correlation with the CD3 IEL count at stage Marsh 1. An increase in both molecules is found even when IELs are in the range 15-25 (Marsh 0), suggesting the possibility of a "gray zone" inhabited by patients which should be closely followed up in gluten-related disorders.

BACKGROUND

Serum radioimmunoassay (RIA) tissue transglutaminase autoantibodies (tTG-Abs) proved to be a sensitive test also during coeliac disease (CD) follow-up. We demonstrated that RIA tTG-Abs could be detected in human saliva.

OBJECTIVE

To evaluate salivary RIA tTG-Abs in coeliac children on gluten-free diet (GFD).

METHODS

Saliva and serum samples from 109 coeliac children were evaluated at diagnosis (group 1: 71 females, median age 9.4 years) and 58 of them on GFD: 36 after 3-6 months (group 2a), 34 at 9 months or more (group 2b). Two gender- and age-matched control groups: 89 gastroenterological patients (group 3) and 49 healthy subjects (group 4) participated in the study. Saliva and serum tTG-Abs were detected by RIA and compared with serum tTG-Abs ELISA and IgA anti-endomysium antibodies (EMA).

RESULTS

Salivary RIA tTG-Abs were found in 94.5%, 66.7% and 50.0% of groups 1, 2a and 2b CD patients and in 98.2%, 72.2% and 50.0% of corresponding serum samples, respectively. tTG-Abs decreased with GFD progression and a correlation was found between saliva and serum titres (r = 0.75, P = 0.0001). During the CD follow-up, salivary and serum RIA sensitivities were comparable, and higher with respect to EMA and ELISA.

CONCLUSIONS

This study demonstrates that it is possible to detect salivary tTG-Abs with high sensitivity not only at CD diagnosis, but also during GFD.

Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca(2+) mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca(2+) release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca(2+), respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca(2+) level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca(2+) and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.

The first association of the synapse as a potential site of neurodegenerative disease burden was suggested for Alzheimer's disease (AD) almost 30 years ago. Since then protease:protease inhibitor (P:PI) systems were first linked to functional regulation of synaptogenesis and synapse withdrawal at the neuromuscular junction (NMJ) more than 20 years ago. Confirmatory evidence for the involvement of the synapse, the rate-limiting or key unit in neural function, in AD did not become clear until the beginning of the 1990s. However, over the past 15 years evidence for participation of thrombin, related serine proteases and neural PIs, homologous and even identical to those of the plasma clot cascade, has been mounting. Throughout development a balance between stabilization forces, on the one hand, and breakdown influences, on the other, becomes established at synaptic junctions, just as it does in plasma clot proteins. The formation of protease-resistant cross-links by the transglutaminase (TGase) family of enzymes may add to the stability for this balance. The TGase family includes coagulation factor XIIIA and 8 other different genes, some of which may also influence the persistence of neural connections. Synaptic location of protease-activated, G-protein-coupled receptors (PARs) for thrombin and related proteases, their serpin and Kunitz-type PIs such as protease nexin I (PNI), alpha1-antichymotrypsin (alpha-ACT), and the Kunitz protease inhibitor (KPI)-containing secreted forms of beta-amyloid protein precursor (beta-APP), along with the TGases and their putative substrates, have all been amply documented. These findings strongly add to the conclusion that these molecules participate in the eventual structural stability of synaptic connections, as they do in coagulation cascades, and focus trophic activity on surviving terminals during periods of selective contact elimination. In disease states, this imbalance is likely to be shifted in favor of destabilizing forces: increased and/or altered protease activity, enhanced PAR influence, decreased and/or altered protease inhibitor function, reduction and/or alteration in tTG expression and activity, and alteration in its substrate profile. This imbalance further initiates a cascade of events leading to inappropriate programmed cell death and may well be considered evidence of synaptic apoptosis.

An open reading frame (orfx) in mitochondria of the higher plants Oenothera berteriana and Arabidopsis thaliana is homologous to orf244 in the mitochondrial genome of Marchantia polymorpha. Homologous sequences are also present in carrot, potato and sugar beet. Profile analysis revealed similarity to two overlapping reading frames in the Escherichia coli genome. Potential translation initiation at conserved ATA (isoleucine) and TTG (leucine) codons is discussed. Transcripts of the open reading frame are altered by RNA editing in Arabidopsis and Oenothera downstream of these codons, suggesting this to be the functionally important region.

A previously characterised promoter region upstream from the Bacillus subtilis argC gene was sequenced. The in vivo position of transcription start point (+1), was determined by mung-bean-nuclease mapping. The nucleotide (nt) sequences in the '-10' (TATAAT) and '-35' (TTGAAT) regions closely resemble consensus promoter sequences recognised by B. subtilis sigma 43 and Escherichia coli sigma 70 RNA polymerases. Between +9 and -64 are three imperfect inverted repeats with high homology to the E. coli arginine biosynthetic gene putative operator sequences (ARG boxes) [Cunin et al., Nucl. Acids Res. II (1985) 5007-5019] and which contain variable intra-repeat distances. Upstream from the '-35' region, extending as far as -71, is a 97% AT-rich sequence. The argC mRNA has a short leader region containing a B. subtilis ribosome-binding site 8 nt upstream from a TTG start codon for an open reading frame (ORF). The deduced amino acid sequence for this ORF contains regions of homology to that for the E. coli argC N-terminal region.

BACKGROUND

In premenopausal women, iron-deficiency anaemia is common and menstrual flow is often held responsible, but it is not clear whether these women should be submitted to gastrointestinal (GI) evaluation. We aim to prospectively investigate whether premenopausal women with iron-deficiency anaemia benefit from GI evaluation regardless of menstrual flow.

METHODS

The study population comprised 59 consecutive premenopausal women with iron-deficiency anaemia. Excluded were women with obvious or suspected causes of anaemia and those < or = 21 years. Heavy menstrual loss was not considered an exclusion criterion. All subjects had: complete blood count, ferritin, non-invasive testing by faecal occult blood (FOB), 13C-urea breath test (13C-UBT), anti-tissue transglutaminase antibodies (tTG) and gastrin levels. Gastroscopy with antral (n = 3), corporal (n = 3) and duodenal (n = 2) biopsies was performed in women with positive 13C-UBT or tTG titre or hypergastrinaemia.

RESULTS

Heavy menstrual loss was present in 50.8%. Non-invasive tests were positive in 40/59 (67.8%): 30 had positive 13C-UBT, 12 had hypergastrinaemia, 7 had positive tTG and 3 had positive FOB. Women tested positive were similar to those tested negative as far as concerned age, haemoglobin and ferritin levels and heavy menstrual flow (55% versus 42.1%). All 40 women tested positive underwent gastroscopy with biopsies. Four (10%) had bleeding-associated lesions and 34 (85%) had non-bleeding-associated lesions. As regards upper GI findings, no differences were observed between women with normal and those with heavy menstrual flow. No lower GI tract lesions were detected in the three women with positive FOB.

CONCLUSIONS

Our data suggest that premenopausal women with iron-deficiency anaemia benefit from endoscopic evaluation of the upper GI tract irrespective of menstrual flow.

In patients with coeliac disease, a regression of intestinal damage without a gluten-free diet is a very rare event. We describe a young child with diarrhoea, intestinal mucosa atrophy and positive serum anti-endomysial and anti-tissue transglutaminase (anti-tTG) antibodies during intestinal giardiasis infection. He showed normal intestinal mucosa architecture and negative anti-endomysial and anti-tTG antibodies after his giardiasis was cured, although he continued to assume a normal diet. Re-evaluations on a 6-monthly basis showed that he was symptom free, and all haemato-chemical parameters were within normal limits. Three years after the initial diagnosis, a third intestinal biopsy showed: normal mucosa architecture; an increase in the intra-epithelial CD3+ and gamma/delta+ lymphocyte counts; and immunoglobulin-A anti-endomysial antibody detection in the supernatant of the intestinal mucosa culture incubated with gliadin. An active coeliac disease status, with intestinal mucosa atrophy, may regress to a latent coeliac disease status with normal intestinal mucosa histology after removal of the environmental factors that have presumably precipitated mucosa damage. Serum anti-endomysial and anti-tTG antibody behaviour is not a permanent, life-long feature and this must recommend the repetition of anti-endomysial or anti-tTG antibody assays in the same patient whenever coeliac disease diagnosis is again suspected, irrespective of previous negativity.

Tissue transglutaminase (tTg) has been identified as the major autoantigen in coeliac disease (CD). ELISA methods have been developed for measuring the autoantibody. There are divergent reports on the effects of calcium on the antibody binding to tTg. Furthermore, zinc is a potent inhibitor of tTg. To better understand the role of transglutaminase in CD, we have studied the stability of commercial tTG, the effect of CD serum on tTg-activity and the effects of calcium and zinc on the antibody binding. The inclusion of calcium during the coating of the ELISA plates significantly increases the binding of the antibody, while zinc at physiological concentrations inhibits the binding. Moreover, our results show that commercial guinea pig liver Tg treated with calcium contains at least four major antigenic molecules and is a labile enzyme, which is degraded rapidly by contaminating proteases. Human serum contains anti-proteases that protect the enzyme. Probably, the labile character of commercial tTG explains the divergent reports on the effects of calcium on antibody binding. Finally, antibodies in serum from a CD patient do not seem to inhibit tTg activity. Hypothetically, low, intestinal Zn2+ -levels facilitate Ca2+-activation of tTg, which deamidates gliadin. A complex between tTg and modified gliadin forms the antigen and triggers the immune reaction leading to manifest CD. Hypozincaemia secondary to villous atrophy aggravates the induced disease.

OBJECTIVE

To evaluate the presence of celiac disease in patients with systemic sclerosis (SSc). The association of autoimmune diseases with celiac disease has been reported, but few publications deal with the combination of SSc and celiac disease.

METHODS

We investigated the presence of anti-tissue transglutaminase (anti-tTG) antibodies and serum antiendomysial antibodies (anti-EMA) in 50 patients with SSc. All subjects were on a gluten-containing diet. Duodenal mucosa histology and biopsy culture were performed in anti-tTG-positive patients; anti-EMA and IgA, IgG1 anti-tTG were detected in culture supernatants.

RESULTS

The incidence of celiac disease in patients with SSc was found to be 8%. Serum anti-tTG antibody-positive results were detectable in 5 out of 50 patients with SSc, but only in 4 of them was the diagnosis confirmed by histological results (Marsh classification).

CONCLUSIONS

Our data show an increased prevalence of celiac disease in patients with SSc.