BACKGROUND

Anti-tissue transglutaminase (tTG) antibody is being used increasingly as a diagnostic tool in the serological investigation of coeliac disease. However, positive predictive values of immunoglobulin A (IgA) anti-tTG for coeliac disease in prospective studies have been disappointing and false-positive results are reported.

OBJECTIVE

To assess the clinical utility of cascade testing for anti-tTG and anti-endomysium antibody (AEA).

METHODS

Two unselected retrospective cohorts from routine diagnostic investigation for possible gluten sensitive enteropathy: group 1 comprised 57 cases seropositive for anti-tTG and group 2 comprised 52 cases seronegative for anti-tTG. In both groups, all cases had also undergone small-intestinal biopsy.

METHODS

Patients were assessed for the presence of IgA anti-tTG by enzyme-linked immunosorbent assay and for IgA AEA by immunofluorescence.

RESULTS

The positive predictive value of IgA anti-tTG for biopsy-confirmed coeliac disease was 54%. The positive predictive value of dual positivity for anti-tTG and AEA was 97%. The negative predictive value of IgA anti-tTG was 100%.

CONCLUSIONS

The data presented here support the use of IgA anti-tTG as an initial screen for coeliac disease. Coeliac disease is unlikely when IgA anti-tTG is absent. However, many false-positive results are seen, and clinical utility and diagnostic efficiency are improved markedly if positive results are confirmed with the more accurate, but labour-intensive, AEA assay.

Toxocara canis and T. cati are highly prevalent nematode infections of the intestines of dogs and cats. In paratenic hosts, larvae do not mature in the intestine but instead migrate through the somatic tissues and organs of the body. The presence of these migrating larvae can contribute to pathology. Toxocara larvae can invade the brains of humans, and while case descriptions of cerebral toxocariasis are historically rare, improved diagnosis and greater awareness have contributed to increased detection. Despite this, cerebral or neurological toxocariasis (NT) remains a poorly understood phenomenon. Furthermore, our understanding of cognitive deficits due to toxocariasis in human populations remains particularly deficient. Recent data describe an enhanced expression of biomarkers associated with brain injury, such as GFAP, AβPP, transforming growth factor β1 (TGF-β1), NF-L, S100B, tTG, and p-tau, in mice receiving even low doses of Toxocara ova. Finally, this review outlines a hypothesis to explore the relationship between the presence of T. canis larvae in the brain and the progression of Alzheimer's disease (AD) due to enhanced AD-associated neurodegenerative biomarker expression.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.

OBJECTIVE

The association between diagnosed coeliac disease and malignancy has been established. The present study was conducted to determine whether previously unrecognised and thus untreated adults with screening-identified evidence of coeliac disease carry an increased risk of malignancies.

METHODS

A Finnish population-based adult-representative cohort of 8000 individuals was drawn in 1978-1980. Stored sera of the participants with no history of coeliac disease or any malignancy were tested for immunoglobulin A (IgA) class tissue transglutaminase antibodies (Eu-tTG) in 2001. Positive sera were further analysed by another tissue transglutaminase antibody test (Celikey tTG) and for endomysial antibodies (EMAs). Malignant diseases were extracted from the nationwide database and antibody-positive cases were compared with negative cases during a follow-up of nearly 20 years.

RESULTS

Altogether 565 of all the 6849 analysed serum samples drawn in 1978-80 were Eu-tTG positive. In further analyses, 202 (2.9%) of the participants were Celikey tTG positive and 73 (1.1%) were EMA positive. The overall risk of malignancy was not increased among antibody-positive cases in the follow-up of two decades; the age- and sex-adjusted relative risk was 0.91 (95% CI 0.60 to 1.37) for those who were Celikey tTG positive and 0.67 (95% CI 0.28 to 1.61) for those who were EMA positive.

CONCLUSIONS

The prognosis of adults with unrecognised coeliac disease with positive coeliac disease antibody status is good as regards the overall risk of malignancies. Thus, current diagnostic practice is sufficient and there is no need for earlier diagnosis of coeliac disease by mass screening on the basis of the findings of this study.

The tet(K) gene from Staphylococcus aureus was highly expressed in Escherichia coli by an alteration of its initiation codon from TTG to ATG and its ribosome-binding sequence from GAGG to GGAGG [Noguchi, N. et al. (1994) Biol. Pharm. Bull. 17, 352-355]. The inverted membrane vesicles prepared from the tet(K)-expressing cells showed respiration-dependent [3H]tetracycline transport comparable to the vesicles from the tet(B)-expressing cells. The affinity of Tet(K) vesicles to tetracycline was the same as that of Tet(B) vesicles, whereas the former Vmax value was about 60% of the latter one. Contrary to Tet(B) vesicles, Tet(K) vesicles showed no significant minocycline uptake, which was consistent with the low minocycline resistance of the Tet(K)-producing cells. The tetracycline transport mediated by Tet(K) vesicles was coupled with proton transport and the translocation of 60Co2+ ions as well as in Tet(B) vesicles. This observation indicates that the class K tetracycline resistance determinant from Gram-positive bacteria also encodes a metal-tetracycline/H+ antiporter that is functionally similar to that encoded by tet(B), although there is a considerable difference in the primary sequences and the putative topologies of these Tet proteins.

Whereas specialized frequency-encoding patterns in the human auditory cortex are generally accepted, termed tonotopicity, a similar principle of intensity encoding--amplitopicity--is debated controversially. This functional magnetic resonance imaging study describes the relationship of the activation volume and the spatial distribution of activated clusters under different sound pressure levels (SPL) across the temporal plane including the transverse temporal gyrus (TTG). Nine healthy subjects with no hearing deficiencies were investigated using an echo-planar imaging technique at 1.5 T. A boxcar stimulation paradigm was applied with a 5-Hz pulsed sine tone of 1000 Hz frequency at three SPLs of 70, 82, and 90 dB. Linear cross-correlation analysis (correlation coefficient>> 0.3 corresponding to P < 0.08) of the functional data set revealed bilateral BOLD response within the auditory cortex of the nine subjects with moderate increase of activation volume for higher sound pressure levels. With increasing sound pressure a two-dimensional drift of cortical activation was observed (a) from the ventral to the dorsal edge and (b) from lateral to medial parts of TTG. This latero-medial drift therefore mimics the well-accepted principle of tonotopy for frequency-encoding neurons. This study demonstrates the existence of an amplitopic pattern of intensity-encoding neuronal clusters that in part resembles the tonotopic distribution of frequency-encoding neurons. This finding has to be integrated into the understanding of the auditory organization for the interpretation of higher auditory functions such as sound perception or speech.

BACKGROUND

Celiac disease (CD) occurs in as many as 1 in 80 pregnant women and is associated with poor pregnancy outcome, but it is not known if this is an effect on maternal nutrient absorption or, alternatively, if the placenta is an autoimmune target. The major autoantigen, tissue transglutaminase (tTG), has previously been shown to be present in the maternal-facing syncytiotrophoblast plasma membrane of the placenta.

METHODS

ELISA was used to demonstrate the presence of antibodies to tissue transglutaminase in a panel of CD sera. Immunohistochemistry was used to evaluate the binding of IgA autoantibodies from CD serum to term placenta. In addition, novel direct binding and activity assays were developed to mimic the in vivo exposure of the villous placenta to maternal autoantibody.

CONCLUSIONS

CD IgA autoantibodies located to the syncytial surface of the placenta significantly more than IgA antibodies in control sera (P < 0.0001). The distribution of antigen was similar to that observed using a monoclonal antibody to tissue transglutaminase. Staining was reduced by pre-absorption of CD serum with recombinant human tissue transglutaminase. In direct binding assays, autoimmune immunoglobulin A (IgA) from the maternal compartment became associated with antigen at the syncytial surface of the placenta, as a result of which transglutaminase activity at this site was inhibited.

CONCLUSIONS

These data indicate that direct immune effects in untreated CD women may compromise placental function.

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.

Werner's syndrome is an autosomal recessive disease caused by mutation of the WRN gene, which may lead to DNA repair failure and acceleration of aging. A polymorphism at amino acid 1367 Cys (TTG)/Arg (CTG) reportedly reduces the risk of myocardial infarction in Japanese. We studied the possible involvement of this polymorphism in type 2 diabetes. When polymorphism of the WRN gene was analyzed in 272 randomly recruited type 2 diabetic subjects (age 64.5+/-11.1), we found those with Cys/Arg to be older than those with Cys/Cys (p=0.021) and that the age at diagnosis of diabetes was greater in Cys/Arg than in Cys/Cys subjects (p=0.011). Diabetes-free survival rate over the age, analyzed by Kaplan-Meier method, differed significantly between these two genotype groups (p=0.0125) and the survival curve was shifted to the right in the Cys/Arg group as compared to the Cys/Cys group. No difference in allele frequency was observed between our diabetic (n=272) and non-diabetic subjects (n=171, age 66.0+/-8.0). These results suggest that the 1367 Arg allele of the WRN gene protects against the development of type 2 diabetes mellitus in Japanese.

OBJECTIVE

Subclinical gut inflammation has been described in patients with ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Joint involvement has also been reported related to celiac disease. We investigated IgA antibodies to bovine tissue tranglutaminase (tTg) and IgA and IgG antibodies to human tTg and to Saccharomyces cerevisiae (ASCA) in patients with AS and PsA.

METHODS

We evaluated the frequency of IgA antibodies to bovine tTg, and of IgA and IgG antibodies to human tTg and to ASCA in 43 patients with AS and 75 with PsA. As control groups we considered 79 patients with rheumatoid arthritis (RA) and 78 healthy blood donors.

RESULTS

We detected antibodies as follows: IgA antibodies to bovine tTg in 1/43 patients with AS, 3/75 with PsA, 1/79 with RA, and in 9/78 healthy controls; IgA antibodies to human tTg in 1/43 patients with AS, 1/75 with PsA, 1/79 with RA, and in 3/78 healthy controls; IgG antibodies to human tTg in 1/43 patients with AS, 4/75 with PsA, 5/79 with RA, and in 7/78 healthy controls. IgA ASCA were confirmed in 10/43 patients with AS, 7/75 with PsA, 14/79 with RA, and in 7/78 healthy controls; IgG ASCA were present in 5/43 patients with AS, 4/75 with PsA, 8/79 with RA, and in 8/78 healthy controls. No statistically significant difference was observed in the prevalence of IgA or IgG antibodies to bovine and human tTg and in the frequency and in mean level of IgA or IgG ASCA between the studied groups or between each group and healthy controls.

CONCLUSIONS

Our data fail to show an increased prevalence of autoantibodies associated with celiac and Crohn's disease in patients with AS and PsA.

Tissue transglutaminase (tTG) post-translationally modifies proteins in a calcium-dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild-type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid-treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid-induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.

OBJECTIVE

To investigate the effectiveness of treatment with transdermal testosterone gel (TTG) before controlled ovarian stimulation (COS) using GnRH antagonist multiple-dose protocol (MDP) in low responders undergoing IVF/intracytoplasmic sperm injection (ICSI).

METHODS

Prospective randomized controlled trial.

METHODS

University-affiliated infertility clinic.

METHODS

A total of 110 low responders, who were defined as patients who failed to produce ≤ 3 follicles with a mean diameter of ≥ 16 mm with the result that ≤ 3 oocytes were retrieved despite the use of a high gonadotropin dose (>2,500 IU) in a previous failed IVF/ICSI cycle.

METHODS

Patients were randomized into TTG pretreatment group or control group. For TTG pretreatment group, 12.5 mg TTG were applied daily for 21 days in the cycle preceding COS for IVF.

METHODS

COS results and IVF outcome.

RESULTS

There were no differences in patients' characteristics between the two groups. Total dose and days of rhFSH used were significantly fewer in the TTG pretreatment group than in the control group. The numbers of oocytes retrieved, mature oocytes, fertilized oocytes, and good-quality embryos were significantly higher in the TTG pretreatment group. Embryo implantation rate and clinical pregnancy rate per cycle initiated also were significantly higher in the women pretreated with TTG. No patient reported adverse effects attributed to TTG use.

CONCLUSIONS

TTG pretreatment might be beneficial in improving both response to COS and IVF outcome in low responders undergoing IVF/ICSI.

The nucleotide sequences of the tRNASer (trnS), pseudo-tRNA, NADH dehydrogenase subunit 3 (nad3), and ribosomal protein S12 (rps12) genes from rice mitochondrial DNA (mtDNA) were determined. Both trnS and nad3 were confirmed to be single copy genes by Southern blot analysis. The nad3 and rps12 genes were arranged in tandem, and the two were co-transcribed. The order of the above four genes in rice mtDNA differed from the linear order observed for the wheat and maize genes. In rice mitochondria, the trnS and pseudo-tRNA genes were found upstream of the cytochrome c oxidase subunit I gene, instead of the nad3 and rps12 genes as observed in maize and wheat. Additionally, while the rice nad3 and rps12 genes remain paired, they too are in a different sequence environment from the wheat and maize genes. The apparent split of the two pairs of genes indicates the occurrence of a mitochondrial intramolecular recombinational event. Another peculiarity is that the sequence upstream of the translational initiation codon of the rice nad3 gene is different from that of the wheat and maize versions. The ATG initiation codon of wheat and maize nad3 is replaced by TTG in the rice nad3. A subsequent deduction of the amino acid sequence, accompanied by a primer extension analysis, indicates that the predicted rice NAD3 protein has an additional 37 amino acid residues at its N-terminus compared to the wheat and maize NAD3 proteins. cDNA sequence analysis showed no introns or the occurrence of RNA editing at the newly replaced TTG codon.

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular epsilon(gamma-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.

OBJECTIVE

No comprehensive information exists nationwide about the familial prevalence and age of rearranged during transfection (RET) germline mutations. The current investigation was undertaken to provide such natural history data which are urgently needed to enable factual decision-making about DNA-based screening programmes for RET germline mutations.

METHODS

Descriptive study drawn on countrywide referrals to a specialist surgical centre.

METHODS

Included were 452 patients from 141 German RET families: 277 carriers referred for pre-emptive or therapeutic surgery, and 175 additional carriers or relatives with endocrine tumours associated with multiple endocrine neoplasia type 2 (MEN2).

METHODS

Key variables included familial prevalence, phenotype and latest year of birth of RET germline mutations. RESULTS A total of 26 different RET germline mutations were identified among the 141 RET families: C634R (21%); M918T (15%); C634Y (9%); L790F/TTG->>TTT (8%), Y791F (7%), V804M (6%); C620R and C634F (5% each); L790F/TTG->>TTC and C634S/TGC->>TCC (4% each); C618S/TGC->>AGC, C634G and S891A (2% each); C618F and E768D (1% each); and in < 1% each: C609G, C611F, C611Y, C618G, C618Y, C620S/TGC->>AGC, C620S/TGC->>TCC, C620Y, C630R, D631Y and V804L. Most of these differences in prevalence rates, seemingly, were caused by recent spontaneous mutations in the germline. With rare exceptions, longstanding transmission was noted in at least one RET family per affected codon. Many germline mutations were traceable back to the early 20th, and a few even to the 19th century.

CONCLUSIONS

These data reveal the potential of DNA-based screening of all relevant RET exons, especially for index patients with solitary, seemingly sporadic disease.

The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.

OBJECTIVE

So far the reliability of the anti-tissue transglutaminase (anti-tTG) test for the diagnosis of coeliac disease has mostly been evaluated using slightly different enzyme-linked immunosorbent assays (ELISAs) in selected and usually small groups of patients. The aims of this study were: (1) to evaluate the reliability of the IgA anti-tTG antibodies for the diagnosis of coeliac disease; and (2) to define the sensitivity and specificity of a commercially available kit for the anti-tTG antibodies' quantitative determination.

METHODS

Each centre in this international multi-centre study collected sera from three groups of subjects: coeliac disease patients at the onset of (1) or on a gluten-free diet for at least 12 months (2); disease and healthy controls (3).

METHODS

The anti-tTG antibodies were determined in duplicate using an ELISA-based commercially available kit (Eu-tTG Eurospital, Trieste, Italy).

RESULTS

The following overall cases and controls have been enrolled: (1) 399 subjects with active coeliac disease; (2) 351 treated coeliac disease cases; (3) 432 controls. The centralized re-testing was performed on: (1) group a: 176 patients with active coeliac disease (mean anti-tTG, 21 arbitrary units [AU]); (2) group b: 172 treated coeliac disease cases (mean anti-tTG, 5 AU); (3) group c: 206 controls (mean anti-tTG, 3 AU). In active coeliacs, the anti-tTG antibodies showed a significant progressive decrease with age, while in controls an opposite trend was found. In active coeliac disease patients, the anti-tTG antibodies were significantly higher in coeliacs with a grade III enteropathy than in those showing a grade II lesion. In treated coeliacs, the mean anti-tTG values were significantly lower in patients strictly adhering to a gluten-free diet than in those reporting dietary transgressions. The sensitivity and the specificity of the Eu-tTG assay were 90% and 96%, respectively.

CONCLUSIONS

The results of this study show that the commercially available test for the anti-tTG antibodies' determination is a reproducible and valuable tool for the diagnosis and follow up of coeliac disease.

BACKGROUND

It has been suggested that serological screening for coeliac disease (CD) should be performed in patients with chronic unexplained hypertransaminasaemia.

OBJECTIVE

To evaluate the specificity for CD diagnosis of serum IgA antitissue transglutaminase (tTG) determination in consecutive patients with chronic hypertransaminasaemia using the most widely utilised ELISA based on tTG from guinea pig as the antigen.

METHODS

We studied 98 patients with chronic hypertransaminasaemia, evaluated for the first time in a hepatology clinic. Serum anti-tTG and antiendomysial (EmA) assays were performed. Patients positive for EmA and/or anti-tTG were proposed for intestinal biopsy. Finally, all sera were reassayed for anti-tTG using an ELISA based on human recombinant tTG as the antigen.

RESULTS

A total of 94/98 hypertransaminasaemic patients were positive for hepatitis virus markers, with 82/98 (83%) positive for anti-hepatitis C virus. Liver histology showed that most patients had mild or moderate chronic hepatitis while severe fibrosis or overt liver cirrhosis was found in 20/98. CD screening showed that 15/98 (16%) hypertransaminasaemic subjects had anti-tTG values in the same range as CD patients; however, IgA EmA were positive in only 2/98 (2%). Distal duodenal biopsy, performed in nine patients, showed subtotal villous atrophy in the two EmA+/anti-tTG+ patients but was normal in 7/7 EmA-/anti-tTG+ subjects. The presence of anti-tTG+ values in EmA- patients was unrelated to particular gastrointestinal symptoms, other associated diseases, severity of liver histology, or distribution of viral hepatitis markers. There was a significantly higher frequency of positive serum autoantibodies (antinuclear, antimitochondrial, antismooth muscle, and anti-liver-kidney microsomal antibodies) in anti-tTG+/EmA- patients than in the other subjects (9/13 v 10/83; p<0.003). Also, a correlation was found between serum gamma globulin and anti-tTG values (p<0.01). When sera were tested with the ELISA based on human tTG as the antigen, no false positive results were observed: only the two EmA+ patients with atrophy of the intestinal mucosa were positive for anti-tTG while all others were negative, including those false positive in the ELISA based on guinea pig tTG as the antigen.

CONCLUSIONS

In patients with elevated transaminases and chronic liver disease there was a high frequency of false positive anti-tTG results using the ELISA based on tTG from guinea pig as the antigen. Indeed, the presence of anti-tTG did not correlate with the presence of EmA or CD. These false positives depend on the presence of hepatic proteins in the commercial tTG obtained from guinea pig liver and disappear when human tTG is used as the antigen in the ELISA system. We suggest that the commonly used tTG ELISA based on guinea pig antigen should not be used as a screening tool for CD in patients with chronic liver disease.

OBJECTIVE

Coeliac disease (CD) is characterised by the presence of autoantibodies against tissue transglutaminase (tTG), the endomysial autoantigen. This study was performed to determine the effect of purified autoantibodies on the enzymatic activity of tTG.

METHODS

Total IgA and IgG class antibodies and purified anti-tTG autoantibodies were isolated from sera of untreated patients with CD and controls. The inhibitory capacity of the antibodies on tTG activity was checked by a fluorometric assay based on the incorporation of monodansyl cadaverine into casein and by tTG-catalysed cross linking of biotinylated cadaverine to gliadin.

RESULTS

The enriched IgA and IgG fractions of five patients with CD and three controls resulted in no significantly different inhibition of enzymatic activity. In contrast, the use of affinity purified anti-tTG autoantibodies of 12 patients with CD led to a dose dependent reduction of tTG activity, compared to control immunoglobulins (n=6). However, the remaining activity was sufficient for cross linking of cadaverine into gliadin, and enzymatic tTG activity was only blocked completely by high concentrations of a monoclonal antibody, which is directed to the active centre of tTG.

CONCLUSIONS

Despite a partial inhibitory effect of isolated anti-tTG autoantibodies from patients with CD, residual enzymatic activity remains sufficiently high to cast doubt on their in vivo relevance.

OBJECTIVE

Tissue transglutaminase (tTG) was recently identified as the major autoantigen in coeliac disease. The aim of this multicentre study was to evaluate the impact of a new immunoenzymatic assay for the detection of IgA anti-tGT antibodies.

METHODS

Seventy four Italian and French clinical laboratories participated in this study; anti-tTG IgA with an enzyme linked immunosorbent assay (ELISA) method using guinea pig liver extract as the coating antigen, anti-endomysium IgA autoantibodies (EMA), and total serum IgA were determined in 7948 patients, 1162 of whom had coeliac disease (737 untreated cases and 425 on a gluten free diet). A proportion of the sera were then sent to a reference laboratory for anti-tTG retesting with an ELISA method using recombinant human tTG antigen.

RESULTS

Seven thousand four hundred and fifty eight (93.8%) sera were EMA/antiguinea pig tTG concordant (positive or negative); 490 (6.2%) were non-concordant. The sensitivity of EMA and antiguinea pig tTG in the 737 untreated patients with coeliac disease was 92.1% and 94.8%, respectively, and the specificity was 99.8% and 99.2%, respectively. Retesting of the discordant sera showed that of the 162 sera classified as EMA negative/antiguinea pig tTG positive, only 49 were positive for human recombinant anti-tTG, and that 39 of these were also EMA positive. Furthermore, of the 36 sera classified as EMA positive/antiguinea pig tTG negative, only two were confirmed as EMA positive.

CONCLUSIONS

The antiguinea pig tTG assay is more sensitive but less specific than EMA, whereas the antihuman recombinant tTG assay is far more specific and just as sensitive as antiguinea pig tTG. Testing for EMA presents considerable interpretative problems and is difficult to standardise.

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.

The knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene (VGSCG), an important resistance mechanism against pyrethroids, was studied in Anopheles sacharovi Favre. It was found that the specific primers Agd1 and Agd2 used for polymerase chain reaction (PCR) amplification of Anopheles gambiae Giles VGSCG also amplified this genomic region in An. sacharovi. Comparison of the IIs4-IIs6 domain segments of the gene indicated 70% nucleotides common to both species and a genetic distance of 0.255 between them. Four different samples of pyrethroid-resistant An. sacharovi produced three types of amino acid, serine (TCG),leucine (TTG),and phenylalanine (TTT) at the kdr mutation point, whereas only two kdr mutations, leucine to phenylalanine and leucine to serine, occur in An. gambiae.