OBJECTIVE

This study was performed to evaluate the prevalence of celiac disease (CD) in Shiraz, southern Iran.

METHODS

Serum samples were collected from 1440 persons (age range = 20-83 years, mean age = 45.4 years) in 2004 and screened for endomysial and tissue transglutaminase antibodies. A questionnaire was completed for all subjects in relation to gastrointestinal (GI) symptoms and cases with positive serology were requested to undergo small-bowel biopsy.

RESULTS

Seven cases (0.5%) were positive for IgA anti-tissue transglutaminase (anti-tTG), and only two (0.14%) were positive for IgA anti-endomysial antibody (anti-EMA), both of whom had highly positive anti-tTg levels (40.4 and 48.0 IU/l). The major clinical symptoms of CD, such as recurrent abdominal pain and change in bowel habits were present in all patients with positive anti-tTG assays. Only five subjects with positive serology agreed to undergo upper GI endoscopy and duodenal biopsy. Three of these cases were reported with Marsh I histologic findings, while in the two cases with positive serologic anti-EMA, more advanced forms of CD were present.

CONCLUSIONS

The prevalence of CD in apparently healthy adults was lower than the reported series from northern parts of the country; therefore, we suggest a more long-term follow-up study in high-risk groups, especially in the apparently healthy subjects in our region.

BACKGROUND

The prevalence of symptomatic CD in Switzerland is thought to be 1 in 1,000 inhabitants. As in other countries, oligo- and asymptomatic CD is being diagnosed with increasing frequency in all age groups.

OBJECTIVE

To assess the prevalence of asymptomatic CD in adolescents in eastern Switzerland.

METHODS

Between September 1999 and July 2000 total serum IgA titres, anti-endomysium IgA (EMA) titres and anti-human tissue transglutaminase IgA (hTTG) titres were measured in the serum of healthy 11- to 18-year-old Swiss lower and upper secondary school students.

RESULTS

Of the 1,450 students (871 f = 60.1%, CI 95%) tested, 11 (10 f) had elevated levels of both EMA and TTG. The diagnosis of CD was confirmed in eight of these students by mucosal jejunal morphology (Marsh III); one exhibited normal histology. Two of the 11 students refused to undergo mucosal biopsy. None of the students, however, had symptoms suggestive of CD, nor were they stunted or underweight, and none of them had family members with known CD. All of the eight students with enteropathy went on a glutenfree diet and felt subjectively better than on a normal diet. Of the remaining students, 38 (2.6%) had family members with known CD. None of those with the relevant family history had elevated EMA or TTG levels.

CONCLUSIONS

Asymptomatic CD is common. It occurs in 1 in 132 (0.75%) Swiss adolescents. The absence of subjectively recognisable symptoms suggestive of family history or other risk factors makes it difficult to diagnose this type of CD.

A potential link between tissue-type transglutaminase (tTG) and cardiac hypertrophy was suggested recently. However, whether tTG is implicated in hypertrophic agonist-induced cardiac hypertrophy is not yet known. The purpose of this study was to investigate the effects of tTG on cardiomyocyte hypertrophy induced by endothelin (ET) 1. Real-time quantitative RT-PCR and Western blot analysis demonstrated that ET-1 increased the expression of tTG mRNA and protein in cardiomyocytes by activating ET(A) receptors. ET-1 failed to cause increases in cell size and [(3)H]leucine uptake, sarcomere reorganization, and gene induction of the atrial natriuretic factor when cardiomyocytes were treated with monodansylcadaverine, a competitive inhibitor of tTG. Furthermore, the effects of ET-1 on multifunctional activities of tTG were determined by evaluating the incorporation of [(3)H]putrescine into N,N'-dimethylated casein and charcoal absorption, respectively. The results showed that ET-1 did not influence the basal transglutaminase activity of cardiomyocytes but significantly inhibited the 0.1-mmol/L Ca(2+)-stimulated transglutaminase activity. Otherwise, ET-1 elevated the activity of GTPase in a concentration- and time-dependent manner. In vivo, right ventricular hypertrophy induced by 2 weeks of chronic hypoxia was depressed by the tTG inhibitor cystamine (10 to 30 mg/kg, 2 times per day, IP) in a dose-dependent manner. Taken together, our data strongly supported the notion that tTG may act as a positive regulator of the hypertrophic program in response to ET-1. This is probably attributable to the signaling activity of tTG rather than transglutaminase activity.

OBJECTIVE

The European Society for Pediatric Gastroenterology and Nutrition proposed guidelines for the diagnosis of celiac disease, stating that duodenal biopsy is no longer needed if patients have symptoms and levels of immunoglobulin A anti-tissue transglutaminase (IgA anti-tTG) more than 10-fold the cut-off value. We evaluated the accuracy of this guideline in a well-characterized population using different commercial assays.

METHODS

We analyzed levels of IgA anti-tTG in serum samples from 104 consecutive pediatric and adult patients who were not deficient in IgA and were diagnosed with celiac disease from August 1, 2000, to December 31, 2009. We also analyzed serum samples from 537 consecutive patients without celiac disease (controls), collected from May 1, 2004, to October 12, 2006, who underwent intestinal biopsy analysis. Serum levels of antibodies were quantified using assays from Bio-Rad, INOVA, Genesis, and Thermo Fisher.

RESULTS

The likelihood ratio (probability of a specific result in patients divided by the probability of the same result in controls) for celiac disease increased with levels of IgA anti-tTG in all assays. Depending on the assay, the likelihood ratio for levels greater than 10-fold the cut-off value ranged from 111 to 294. The percentage of patients with celiac disease with levels of IgA anti-tTG greater than 10-fold the cut-off value ranged from 41% to 61%, depending on the assay. For levels of anti-tTG greater than 10-fold the cut-off value, the post-test probabilities for celiac disease (probability of disease, based on pretest probability and test result) were, depending on the assay, 89%-96% and 53%-75% for pretest probabilities (probability of disease depending on symptoms) of 7% and 1%, respectively.

CONCLUSIONS

To diagnose celiac disease based on serologic factors, it might be best to define thresholds for levels of IgA anti-tTG based on a predefined likelihood ratio or post-test probability, instead of a multiple of a cut-off value. Patients with a high pretest probability and levels of anti-tTG greater than 10-fold the cut-off value have a high probability for having celiac disease, aiding clinical decision making.

OBJECTIVE

Celiac disease (CD) is an autoimmune disease that can be complicated by impaired nutrition and growth. With the development of sensitive serologic tests, safe endoscopy, and efforts to educate primary care physicians, more children are diagnosed as having CD. The aim of this study is to evaluate the pattern of the presentation of pediatric CD in western New York.

METHODS

Chart review of pediatric patients with CD was undertaken. Patients' demographics, presenting features, disaccharidase assay (DA), celiac serology, and Marsh score were reviewed from patients seen at the Digestive Diseases and Nutrition Center, State University of New York at Buffalo from January 2003 through March 2013.

RESULTS

A total of 165 patients with CD were evaluated. Mean age was 10.7 ± 4.3 years, 76 male patients. The presenting features were abdominal pain (n = 87, 52.7%), constipation (n = 65, 38.9%), diarrhea (n = 52, 31.1%), family history of first-degree relative (n = 47, 28.1%), diabetes mellitus type 1 (n = 37, 22.2%), failure to thrive (n = 36, 21.8%), reflux (n = 25, 15.1%), vomiting (n = 24, 14.5%), fatigue (n = 15, 9%), short stature (n = 9, 5.4%), thyroid disease (n = 9, 5.4%), Down syndrome (n = 8, 4.8%). We found no correlation between Marsh score and serum tissue transglutaminase (tTG) immunoglobulin (Ig) A level at diagnosis and no correlation between DA and serum tTG IgA level, presenting feature and tTG IgA level, presenting feature and Marsh score, tTG IgA and DA, or between the age and the presenting feature.

CONCLUSIONS

Children newly diagnosed as having CD in western New York presented most frequently with abdominal pain and constipation and were older at the time of diagnosis than those described in the classical presentation of CD. We speculate that our patients may have a different long-term natural history and risk factors than originally described for patients with CD.

OBJECTIVE

Serum antiendomysial antibodies (EMAs), highly sensitive and specific serological markers of celiac disease (CD), are detectable in culture media of biopsy samples from CD patients. This finding can be considered an in vitro evidence that intestinal mucosa is a site of EMA production. To confirm this finding, we investigated the presence of EMAs and of anti-tissue transglutaminase (anti-tTG), recently identified as the autoantigen of the EMA, in fecal supernatants of CD patients.

METHODS

Twenty-one newly diagnosed CD patients, 10 treated CD patients on a gluten-free diet, and 14 control disease patients on a gluten-containing diet were enrolled. Twenty-four-hour stool collections and fecal supernatants were obtained from all patients in the study. Biopsy cultures were also performed. IgA EMAs were detected in sera, culture media, and fecal supernatants. IgA, IgG, IgM, and IgE anti-gliadin antibodies (AGAs) and IgA anti-tTG antibodies were measured in fecal supernatants. The weights, water content, and pHs of the 24-h stool collections were also measured.

RESULTS

In all untreated CD patients EMAs were detectable in sera, culture media, and fecal supernatants. In treated CD patients, EMAs were detected only in culture media after in vitro gliadin challenge. No EMAs were detected in controls. Anti-tTG levels were higher in untreated CD patients than in treated CD patients and controls. IgA AGA levels were higher in untreated CD patients than in treated CD and control patients, whereas IgM AGAs were higher in both untreated and treated CD patients than in controls. No statistically significant differences were observed for IgG and IgE AGAs among the above-mentioned populations. Fecal weights, water content, and pHs were higher in untreated CD than in control patients.

CONCLUSIONS

The presence of EMAs in fecal supernatants represents the in vivo proof that intestinal mucosa is a site of EMA production. Furthermore, EMA detection in the stools could be a simple and useful additional tool to clarify diagnosis in the patchy conditions of CD.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5'OH-CCT ACC CCT TCC TTG TA-3'OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5'OH-CCT ACC CCT TCC TTG TA-3'OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5'OH-ATGAACCGGAGGCCC-3'OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides.

OBJECTIVE

Celiac disease has characteristics of an autoimmune disease, such as increased antibody levels to tissue transglutaminase (tTG). However, assays to measure these biomarkers in blood samples do not identify patients with sufficient accuracy for diagnosis or monitoring of celiac disease. We aimed to discover biomarkers of celiac disease derived from neoepitopes of deamidated gliadin peptides (DGP) and tTG fragments and to determine if immune reactivity against these epitopes can identify patients with celiac disease with mucosal healing.

METHODS

We analyzed serum samples from 90 patients with biopsy-proven celiac disease and 79 health individuals (controls) for immune reactivity against the tTG-DGP complex (discovery cohort). A fluorescent peptide microarray platform was used to estimate the antibody binding intensity of each synthesized tTG-DGP epitope. We validated out findings in 82 patients with newly diagnosed celiac disease and 217 controls. We tested the ability of our peptide panel to identify patients with mucosal healing (based on the histologic analysis) using serum samples from patients with treated and healed celiac disease (n=85), patients with treated but unhealed celiac disease (n=81; villous atrophy despite a adhering a gluten-free diet), patients with untreated celiac disease (n=82), and disease controls (n=27), villous atrophy without celiac disease), and healthy controls (n=217). Data was analyzed using principal component analysis followed by machine learning and support vector machine modeling.

RESULTS

We identified 172 immunogenic epitopes of the tTG-DGP complex. We found significantly increased immune reactivity against these epitopes vs. controls. In the training cohort, the set of neoepitopes derived from the tTG-DGP complex identified patients with celiac disease with 99% sensitivity and 100% specificity. Serum samples from patients with untreated celiac disease had the greatest mean antibody-binding intensity against the tTG-DGP complex (32.5 ±16.4). The average antibody-binding intensity was significantly higher in serum from patients with treated but unhealed CeD mucosa (15.1 ± 7.5) than in patients with treated and healed CeD mucosa (5.5±3.4) (P<.001). The assay identified patients with mucosa healing status with 84% sensitivity and 95% specificity.

CONCLUSIONS

We identified immunogenic epitopes of the tTG-DGP complex, and found that an assay to measure the immune response to epitopes accurately identified patients with celiac disease, as well as patients with mucosal healing. This biomarker assay might be used in detection and monitoring of patients with celiac disease.

The fuzzless gene GaFzl was fine mapped to a 70-kb region containing a GIR1 gene, Cotton_A_11941, responsible for the fuzzless trait in Gossypium arboreum DPL972. Cotton fiber is the most important natural textile resource. The fuzzless mutant DPL972 (Gossypium arboreum) provides a useful germplasm resource to explore the molecular mechanism underlying fiber and fuzz initiation and development. In our previous research, the fuzzless gene in DPL972 was identified as a single dominant gene and named GaFzl. In the present study, we fine mapped this gene using F₂ and BC₁ populations. By combining traditional map-based cloning and next-generation sequencing, we mapped GaFzl to a 70-kb region containing seven annotated genes. RNA-Sequencing and re-sequencing analysis narrowed these candidates to two differentially expressed genes, Cotton_A_11941 and Cotton_A_11942. Sequence alignment uncovered no variation in coding or promoter regions of Cotton_A_11942 between DPL971 and DPL972, whereas two single-base mutations in the promoter region and a TTG insertion in the coding region were detected in Cotton_A_11941 in DPL972. Cotton_A_11941 encoding a homologous gene of GIR1 (GLABRA2-interacting repressor) in Arabidopsis thaliana is thus the candidate gene most likely responsible for the fuzzless trait in DPL972. Our findings should lead to a better understanding of cotton fuzz formation, thereby accelerating marker-assisted selection during cotton breeding.

OBJECTIVE

To assess the possible role of genetic polymorphisms in DNA repair gene XRCC1 (X-ray repair cross-complementing group 1) during spermatogenesis by investigating the associations of one promoter polymorphism (T-77C) and two exonic polymorphisms (Arg194Trp and Arg399Gln) in XRCC1 gene with risk of idiopathic azoospermia in a Chinese population.

METHODS

The genotype and allele frequencies of three observed polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism based on a Chinese population consisting of 171 idiopathic azoospermia subjects and 247 normal-spermatogenesis controls.

RESULTS

In our study, all the observed genotype frequencies were in agreement with Hardy-Weinberg equilibrium. The 399A (GA+AA) allele frequency for idiopathic azoospermia subjects and controls was 0.216 and 0.269, respectively. Compared with GG genotype, the AA genotype of Arg399Gln showed a significant association with a decreased risk of idiopathic azoospermia (odds ratio = 0.315; 95% confidence interval = 0.12-0.86). However, no significant differences were found between the cases and controls for T-77C and Arg194Trp polymorphisms. The major haplotypes of XRCC1 gene were TCG, TTG and TCA, whereas no haplotypes appeared to be significantly associated with idiopathic azoospermia based on the cutoff of P < 0.05.

CONCLUSIONS

In a selected Chinese population, AA genotype of Arg399Gln appears to contribute to a decreased risk of idiopathic azoospermia, while we have not any evidence of involvement of XRCC1 T-77C and Arg194Trp polymorphisms in idiopathic azoospermia.

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q31 and the C-terminal K162 are involved in inter- and intramolecular crosslinking (transamidation). Q31 is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q66, which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.

Protein misfolding and the formation of stable insoluble protein complexes by self-interacting proteins, in particular amyloid-β and tau protein, play a central role in the pathogenesis of Alzheimer's disease (AD). Unfortunately, the underlying mechanisms that trigger the misfolding of self-interacting proteins that eventually results in formation of neurotoxic dimers, oligomers, and aggregates remain unclear. Elucidation of the driving forces of protein complex formation in AD is of crucial importance for the development of disease-modifying therapies. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that induces the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds, which results in both intra- and intermolecular protein cross-links. These tTG-catalyzed intermolecular cross-links induce stable, rigid, and insoluble protein complexes, whereas intramolecular cross-links change the conformation of proteins. Inhibition of tTG-catalyzed cross-linking counteracts the formation of protein aggregates, as observed in disease-models of other protein misfolding diseases, in particular Parkinson's and Huntington's diseases. Although data of tTG activity in AD models is limited, there is compelling evidence from both in vitro and postmortem human brain tissue of AD patients that point toward a crucial role for tTG in the pathogenesis of AD. Here, we review these data on the role of tTG in the initiation and development of protein aggregates in AD, and discuss the possibility to use inhibitors of the cross-linking activity of tTG as a new therapeutic approach for AD.

Atherosclerosis is characterized by thickening of the vessel wall, smooth muscle cell proliferation, macrophage infiltration, and deposition of a fibrin network. Transglutaminases are a family of enzymes catalyzing the formation of stable covalent cross-links between proteins. Here, we show that tissue transglutaminase (tTG) synthesis by human umbilical vein endothelial cells is upregulated by thrombin, the serine protease that causes fibrin formation and many cellular inflammatory effects. Thrombin upregulated tTG 2-fold at the mRNA and protein level. Cellular cross-linking activity was increased to an even greater extent; antibody to tTG neutralized the increased activity. The effect on tTG expression required active thrombin and was mediated mainly through protease-activated receptor-1, a thrombin receptor. Increased tTG antigen and activity were evident in human umbilical vein endothelial cells and extracellular matrix in situ. Thrombin treatment also led to a cellular redistribution of tTG. Normal vessel wall stained positively for tTG in the smooth muscle cells and in the subendothelium. The intensity of staining increased in vessel walls with plaque, where there was a striking increase in tTG in the smooth muscle cells immediately below the plaque. These studies indicate a role for tTG in the stabilization of atherosclerotic plaques and suggest that its local expression can be controlled by thrombin.

The longhorn beetle Dorysthenes paradoxus (Faldermann, 1833) (Coleoptera: Cerambycidae) is not only a serious agricultural pest but also a traditionally edible insect in China. However, no genetic information on this species has been acquired. In the present study, we report the mitochondrial genome (mitogenome) of Do. paradoxus, as the first complete mitogenome of Prioninae. The circular mitogenome of 15,922 bp encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), and it contains an A+T-rich region. This mitogenome exhibits the lowest A+T content (71.13%) but harbors the largest AT skew (0.116) among the completely sequenced Cerambycidae species. Eleven of the 13 PCGs have a typical ATN start codon, whereas COI and ND1 are tentatively designated by AAT and TTG, respectively. Only 4 of the 13 PCGs harbor a complete termination codon, and the remaining 9 possess incomplete termination codons (T or TA). Apart from tRNASer(AGN), the other 21 tRNAs can fold into a typical clover-leaf secondary structures. The Do. paradoxus A+T-rich region contains two poly-T stretches and a tandem repeat that comprises two 47-bp-long copies. Both Bayesian inference and Maximum likelihood analyses confirmed the subfamily ranks of Cerambycidae ([Prioninae + Cerambycinae] + Lamiinae) and the close relationship between Philinae and Prioninae/Cerambycinae. However, the data did not support the monophyly of Prioninae and Cerambycinae. The mitogenome presented here provides basic genetic information for this economically important species.

BACKGROUND

Celiac disease (CD) is an immune-mediated disorder induced by the ingestion of gluten in genetically susceptible persons. The prevalence of CD in Malaysia is unknown. We aim to determine the seroprevalence of CD antibodies and also investigate the correlation between H. pylori infection and CD in the young and healthy multiracial Malaysian population.

METHODS

Healthy young adult volunteers between the ages of 18-30 years were consecutively recruited from June 2012 to May 2014 at the University of Malaya Medical Centre (UMMC), Kuala Lumpur. Serum samples from all the participants were tested for anti-gliadin antibody immunoglobulin A/immunoglobulin G (IgA/IgG) and anti-tissue transglutaminase antibody (tTG) IgA/IgG. Samples positive for both anti-gliadin and anti-tTG were further validated for anti-human endomysial IgA antibodies (EmA). Serological diagnosis of CD was made when anti-gliadin, anti-tTG and anti-EmA were positive.

RESULTS

562 qualified participants with mean age 24 ± 2.4 years old were recruited into our study. CD was found in 7 participants where most of them were asymptomatic and unaware of their CD status. The median of anti-gliadin and anti-tTG IgA/IgG value was 38.2 U/ml (interquartile range, 28.3-60.4 U/ml) and 49.2 U/ml (interquartile range, 41.1-65.9 U/ml), respectively. Seroprevalence of CD antibodies was 1.9% (6 out of 324) in female while only 0.4% (1 out of 238) in male. Seroprevalence among Malay was 0.8% (2 of 236), Chinese was 1.7% (3 of 177) and Indian was 1.3% (2 of 149). Overall, seroprevalence of CD antibodies in healthy asymptomatic adults in the Malaysian population was 1.25% (95% CI, 0.78%-1.72%). No significant relationship was discovered between CD and H. pylori infection.

CONCLUSIONS

The seroprevalence of CD antibodies in healthy young adults in the Malaysian population was 1.25% (1 in 100). CD is underdiagnosed and it could be a much greater problem in Malaysia than previously thought.

OBJECTIVE

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by degeneration of both upper and lower motor neurons. In this study, we aimed to measure both plasma and cerebrospinal fluid (CSF) phosphorylated neurofilament heavy chain (pNF-H) levels in patients with ALS, and determine if there is a correlation. We also evaluated plasma and CSF pNF-H levels to investigate if this biomarker could predict the time to generalization (TTG) in ALS better.

METHODS

Paired plasma and CSF pNF-H levels of patients with ALS (n=51), multiple system atrophy (n=12) and controls (n=30) were measured by monoclonal sandwich ELISA. TTG, which also indicates the time of symptoms spreading from spinal or bulbar localization to both, was evaluated in all ALS patients.

RESULTS

We found a strong correlation between plasma and CSF pNF-H levels within individual patients (r=0.712, p<0.001). The mean TTG in ALS patients was 22.9months (range 1-84months), and there was an inverse correlation between plasma (r=-0.661; p<0.001)/CSF (r=-0.869; p<0.001) pNF-H levels and TTG.

CONCLUSIONS

The correlation of plasma and CSF pNF-H levels would bring insight into the pathogenesis of ALS. TTG might play an important role in the future study of ALS, as an early indicator of survival.

OBJECTIVE

Seroreactivity against the Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (I2), and Bacteroides caccae TonB-linked outer membrane protein (OmpW) has been detected in celiac disease patients with small-bowel mucosal atrophy. Levels of these antibodies decrease during a gluten-free diet, but their functions and time of appearance in celiac disease are not known. We aimed to search for evidence of possible microbial targets of the immune responses in the early-stage celiac disease patients who showed normal small-bowel mucosal architecture at the time of the first investigations, but later on a gluten-containing diet developed mucosal atrophy.

METHODS

Forty-four cases with proven early-stage celiac disease and normal mucosal morphology were enrolled. Patients' sera were tested for celiac disease antibodies against tissue transglutaminase (tTG-ab), endomysium, and for microbial antibodies against I2, OmpW, and ASCA IgG and IgA isotypes in both at the time of diagnosis and while on a gluten-free diet.

RESULTS

Thirty-four (77%) of 44 patients with early-stage celiac disease had elevated serum antibodies to one or more of the antibodies ASCA, I2, and OmpW. Furthermore, 5 of 6 cases negative for both tTG-ab and endomysium showed positivity for the microbial markers. Seroreactivity to ASCA IgA, ASCA IgG, and OmpW decreased significantly during gluten-free diet.

CONCLUSIONS

Seroreactivity to different microbial antigens is evident already in patients with early-stage celiac disease. ASCA antibodies seem to be gluten-dependent. The results indicate that the microbial targets might have a role in the early development of celiac disease.

The interleukin-20-receptor I complex (IL-20-RI) is composed of two chains, IL20RA and IL20RB. Its ligands are the three members of the IL19 subfamily of cytokines, IL-19, IL-20 and IL-24. These cytokines are important in the manifestation of psoriatic lesions and, recently, an association of polymorphisms of IL20 with psoriasis has been described. In the present study we tested the hypotheses that genetic variations of the IL-20-RI influence susceptibility to psoriasis and investigated single nucleotide polymorphisms (SNPs) in the IL20RA and IL20RB genes in psoriasis patients (n=254) and healthy controls (n=224). We found no association of any of the investigated SNPs with the disease. Analysis of pairwise linkage disequilibrium (LD) across studied markers revealed a strong level of LD between SNPs within the IL20RA gene and SNPs within the IL20RB gene, and, for both genes six common haplotypes were identified with an estimated frequency>>or=1%. Haplotype analyses suggested that the IL20RA haplotype CCG (rs1184860, rs1167846, rs1167849) is significantly associated with psoriasis (OR 3.14, 95% CI 1.61-6.14), whereas the TTG haplotype had a protective effect (OR 0.20, 95% CI 0.07-0.55). The risk haplotype defining SNPs 1167846 and 1184860 were found to modify paired box 5 and homeobox A9 sites, respectively, two transcription factors related to the differentiation of immune cells. Further studies are needed to confirm the genetic association and to investigate the functional relevance of IL20RA haplotypes in psoriasis.

OBJECTIVE

To compare strategies for diagnosing celiac disease (CD).

METHODS

A decision analytic model was used to compare five strategies on diagnostic performance and costs.

RESULTS

First, tTG screening alone is the least costly strategy ($22/individual). While the NPV is high (99.8%), the PPV is low (63.4%). Second, if tTG-positive patients undergo esophagogastroduodenoscopy (EGD) to confirm CD, the PPV increases to 100% ($2,237/false-positive diagnosis avoided). Third, if EGDs are restricted to only those who are both tTG and HLA DQ2/8 positive, costs are slightly reduced ($59 vs. $63/individual), while PPV and NPV remain unchanged. Fourth, screening tTG-negative patients for IgA deficiency increases the NPV to 99.9% ($32,605/false-negative diagnosis avoided). Sensitivity analyses revealed that as the prevalence of CD increases, the cost of avoiding a false-positive diagnosis by adding EGD to the tTG alone strategy increases considerably.

CONCLUSIONS

When the pre-test probability of CD is low, patients with positive tTG serology should undergo EGD with biopsy-either directly or after positive screening for HLA DQ2/8-to confirm CD. As the pre-test probability of CD increases, the added cost of EGD should be weighed against the consequences of a false-positive diagnosis. Routinely screening for IgA deficiency in order to avoid a false-negative diagnosis is quite costly.

The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

Tissue transglutaminase (tTG) expression, distribution and activity were examined in human placenta and derived cells. Immunochemical techniques and RT-PCR were used to demonstrate tTG protein and mRNA in stromal cells and trophoblast in first trimester and at term, with higher levels later in pregnancy. Decidual cells also produce tTG. The data were confirmed using primary cultures of trophoblast, fibroblasts and decidual stromal cells. Substrate incorporation studies indicated tTG activity in association with fibroblast extracellular matrix and the syncytial microvillous membrane (MVM), where several target polypeptides could be observed. tTG is a major autoantigen in Coeliac disease (CoD) which is associated with poor pregnancy outcome. tTG at the placental MVM is a plausible target of maternal autoantibody action.

OBJECTIVE

Tissue transglutaminase was identified as the main autoantigen in coeliac disease (CD) but enzyme immunoassays applying the commercially available antigen from guinea pig liver show insufficient specificity and sensitivity for diagnosis as compared with endomysium antibodies (EmA). The aim of this present study was to develop a new method for the cloning and expression of human tissue transglutaminase (hu-tTG) and to test hu-tTG in the serological diagnosis of CD.

METHODS

Hu-tTG was cloned and expressed using a baculovirus system and SF9 insect cells. The enzyme carried a C-terminal His tag allowing efficient affinity purification from cell lysates. The recognition of hu-tTG by human sera was checked by using an enzyme linked immunosorbent assay (ELISA). For this, 35 patients with active CD were compared with 144 controls (18 patients with bioptically excluded CD, 89 blood donors, 30 patients with inflammatory bowel disease, and seven patients with cystic fibrosis).

RESULTS

The ELISA using hu-tTG showed a sensitivity of 100% and a specificity of 98.6%. Titres of antibodies against hu-tTG (anti-hu-tTG) were positively correlated with EmA titres. All results negative for EmA were also negative for anti-hu-tTG. There were, however, EmA positive results up to a titre of 1 : 80 below the cut-off for anti-hu-tTG. For comparison, antibodies against guinea pig tissue transglutaminase (anti-gp-tTG) were determined in parallel. All patients with anti-hu-tTG below the cut-off were also negative for anti-gp-tTG. However, there were eight patients positive for anti-hu-tTG but negative for anti-gp-tTG.

CONCLUSIONS

The new test reaches and even exceeds diagnostic efficiency of EmA for coeliac diagnosis.

Anti-tissue transglutaminase (tTG) antibodies (AtTGA) are typically found in serum of patients with untreated coeliac disease (CD). tTG catalyses crosslinking of peptides an activity supposed to be important in neurological disorders. tTG occurs in cerebrospinal fluid (CSF) and its assay in CSF was suggested to be diagnostically useful. However, nothing is known about AtTGA in CSF. Therefore, in 129 unselected CSF-serum pairs IgA- and IgG-AtTGA were assayed by ELISA using human recombinant tTG. For comparison, IgA- and IgG-anti-gliadin antibodies (AGA), typically coexisting with AtTGA were measured. Albumin, total IgA and IgG and further parameters were determined according to routine programme recommended by the European CSF consensus group. AtTGA were detected in 27 (IgA) and in 63 (IgG) CSF samples. Antibody indices (AI) could be calculated for AtTGA from 21 (IgA) and from 61 (IgG) sample pairs. AI for AtTGA was >2 in 11 (IgA) and in 22 (IgG) sample pairs, hinting to intrathecal antibody synthesis. AI for AGA was >2 only for 1 (IgA) and 2 (IgG) sample pairs. Patients with normal routine findings had significantly higher AI for IgA-AtTGA than patients with abnormal findings. This is the first demonstration of AtTGA in CSF and their intrathecal synthesis. The pathogenetic relevance of this new autoantibody species remains to be clarified.