Celiac disease (CD) has been associated with non-alcoholic fatty liver disease (NAFLD) and other chronic liver diseases (CLD), such as primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC).

OBJECTIVE

To study the frequency of serological markers of CD in patients with NAFLD and CLD and their correlation with duodenal biopsy.

METHODS

In patients with NAFLD, PBC, AIH and PSC, we studied anti-endomysium (AE) IgA by indirect immunofluorescence and anti-gliadin IgA-IgG (AG) and human anti-tissue transglutaminase (tTG) IgA antibodies by an ELISA technique. Patients with positive serology for at least one marker underwent endoscopy with duodenal biopsies.

RESULTS

Positive CD markers were found in 9 of 101 patients (8.9%): 7 patients were positive for tTG alone, 1 for AE and AG, and 1 patient for 3 antibodies. Positivity was as follows: 3/38 (7.9%) in NAFLD, 3/44 (6.8%) in PBC, 2/16 (12.5%) in AIH and 1/3 in PSC. Endoscopy was performed in 8 patients, with normal duodenal biopsy in 7 and 1 patient with Marsh stage 1 CD with NAFLD, positive AE and AG. The only patient with 3 positive markers died during the study without undergoing endoscopy. None of the patients had symptoms suggestive of CD.

CONCLUSIONS

A high prevalence of positive tTG was found in patients with CLD and NAFLD. However, duodenal biopsy should be performed in these patients, given that the results of this procedure were normal in most patients in this study.

In the diagnostic work-up of celiac disease (CD) the simpler enzyme-linked immunosorbent assay (ELISA) for the identification of serum anti-transglutaminase (tTG) autoantibodies could substitute the immunofluorescence technique used for the detection of anti-endomysial antibodies (EmA). However, most of the studies on anti-tTG assay have considered pre-selected groups of patients and not consecutive subjects with suspected CD. The aim of this study was to compare the sensitivity, specificity and predictive value of anti-gliadin antibodies (AGA), EmA and two anti-tTG ELISAs, one based on guinea pig (gp)-tTG and the other on human (h)-tTG as antigens, in consecutive patients investigated for suspected CD. The study included 130 consecutive patients (age range 16-84 years), who underwent intestinal biopsy for suspected CD. They presented with one or more of the following symptoms: weight loss, anemia, chronic diarrhea, abdominal pain, dyspepsia, alternating bowel habits and constipation. At the time of admission in the study, an intestinal biopsy was performed and a serum sample was taken for immunoglobulin (Ig) G and IgA AGA, IgA EmA, anti-gp-tTG and anti-h-TG determination. Intestinal histology revealed that 15 patients had partial or total villous atrophy. In these patients the diagnosis of CD was confirmed at subsequent follow-up. The remaining 115 patients included in the study had an intestinal histology characterized by a normal villi/crypts ratio and were considered as controls. Serum EmA, anti-gp-tTG, and anti-h-tTG were positive in all the 15 CD subjects, whereas IgG and IgA AGA were positive in 10/15; in the control group, none were positive for serum EmA, but 11/115 (10%) were positive for anti-gp-tTG and 6/115 (5%) were positive for anti-h-tTG. The sensitivity was 100% for EmA, gp-tTG and h-tTG and 66% for IgA and IgG AGA. The specificity was 100% for EmA, 90% for anti-gp-tTG, 95% for anti-h-tTG, 74% for IgG AGA and 87% for IgA AGA. The negative predictive value was 100% for EmA, anti-h-tTG and anti-gp-tTG, 94% for IgG AGA and 95% for IgA AGA. The positive predictive value was 100% for EmA, 71% for anti-h-tTG (p = 0.03 vs EmA) and 58% for anti-gp-tTG (p = 0.003 vs EmA). Most of the patients who were false positive for anti-tTG had Crohn's disease or chronic liver disease. In conclusion, although both the anti-tTG ELISAs evaluated in the present study showed an optimum sensitivity, their low specificity determined positive predictive values which were significantly lower than those of EmA assay. Besides, the positive predictive value of gp-tTG was too low to warrant submitting a patient to intestinal biopsy for suspected CD.

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.

BACKGROUND

Rheumatoid arthritis (RA) is frequently associated with organ or non-organ-specific autoantibodies or overt autoimmune disorders. Aim of our study was to assess the prevalence and concentration of a panel of organ-specific autoantibodies in patients with RA and to evaluate their relationship with clinical manifestations and treatment efficacy.

METHODS

Clinical and serological data from 20 patients with active RA (3M/17F), aged from 28 to 80 years and 50 healthy controls were analyzed. All patients fulfilled the 1987 American College of Rheumatology (ACR) classification criteria for RA and were treated with adalimumab and methotrexate. At baseline and after 6 months of therapy we tested anti-thyroid antibodies for thyroperoxidase (TPOAb) and thyroglobulin (TgAb) using an automated immunochemiluminescence assay (Immulite 2000, DPC, Los Angeles, CA), and anti-tissue transglutaminase (anti-tTG) using the ELISA assay (Phadia, Freiburg, Germany). Anti-smooth muscle (SMA), anti-liver kidney microsome (LKM), anti-parietal cells (APCA), anti-mitochondrial (AMA) and anti-liver cytosolic protein type 1 (LC1), anti-adrenal gland (ACA), anti-pancreatic islet (ICA) and anti-steroid-producing cell (stCA) antibodies were analyzed using a commercially available indirect immunofluorescence methods. Statistics were performed by the SPSS statistical software for Windows, using non parametric tests.

RESULTS

At baseline 6 out of 20 (30%) patients were positive for TPOAb and 8 (40%) for TgAb. After 6 months of treatment 5 (25%) patients had TPOAb and 8 (40%) TgAb. At baseline and after 6 months of treatment only 1 (5%) patient tested positive for IgA anti-tTG (celiac disease was confirmed by intestinal biopsy), and no patients had IgG anti-tTG. However, in RA patients IgG anti-tTG levels significantly increased during treatment (p = 0.017) and were higher than in healthy individuals both at baseline (p = 0.028) and after 6 months of treatment (p = 0.001). Only 1 (5%) patient was positive for APCA and no patient was positive for the other anti-organ-specific antibodies either at baseline or after 6 months of treatment.

CONCLUSIONS

The prevalence of organ-specific antibodies does not seem to change during anti-TNF treatment in RA patients. However, a slight and probably irrelevant increase of IgG anti-tTG antibody levels was observed.

The Schizosaccharomyces pombe DNA repair gene rhp51(+) encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51(+) transcript is increased by a variety of DNA-damaging agents. Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51(+) expression through the DRE of rhp51(+). The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51(+), indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51(+) enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1(+) is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51(+) was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51(+).

BACKGROUND

Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays.

METHODS

Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³⁵S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively.

RESULTS

Median incorporation of [³⁵S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively.

CONCLUSIONS

The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.

Phaneropteridae is a family of Orthoptera that displays an amazing amount of diversity in terms of both forms and species. We sequenced the mitochondrial genomes (mitogenomes) of two bush katydids: Ruidocollaris obscura and Kuwayamaea brachyptera (Phaneropterinae), and two true katydids: Orophyllus montanus and Phyllomimus detersus (Pseudophyllinae), to obtain further insight into the characteristics of the katydid mitogenomes and to investigate the taxonomic status of subfamily Pseudophyllinae and the diversity of gene arrangements among Phaneropteridae. The following general genomic characteristics were observed in the four katydids: a longer length of the mitogenomes (16,007bp-16,667bp) compared with Caelifera, abundant intergenic spacers, and accepted atypical initiation codons (GTG and TTG, found in cox1, nad1 and nad2). A new orientation of the gene arrangement "trnM-trnI-trnQ" was identified in P. detersus, which is the first representative of Polyneoptera found to carry this gene cluster. Large identical fragments (492bp) were detected in control region 1 (CR1) and control region 2 (CR2) of R. obscura. The high similarity of the duplicated CRs is likely due to a recent gene duplication or concerted evolution. Analyses of the duplicated CRs revealed one conserved stem-loop (on the N-strand) located in the identical sequences of both CRs that might be linked to replication initiation. Phylogenetic analyses based on 13 protein-coding genes and 2 ribosomal RNA genes from 20 Ensiferan species yielded the identical topologies between two different methods (maximum likelihood and bayesian inference). The newly sequenced Pseudophyllinae species was placed as the sister group of Phaneropterinae, and Mecopodinae clustered with Pseudophyllinae+Phaneropterinae. Additionally, we speculate that the species in Ruidocollaris and Sinochlora, as well as their closely related genera, may have undergone numerous rearrangement events.

BACKGROUND

Organ function after liver transplantation is determined by ischemia-reperfusion injury. Destruction of Kupffer cells with gadolinium chloride (GdCl3) has been shown to have a possible preventive effect on the extent of this injury, which can be extrapolated by analyzing the distribution of hepatic microperfusion. The aim of this study was to evaluate the protective effect of GdCl3 on disturbances of microperfusion in the transplanted liver.

METHODS

Landrace pigs were randomly divided into three groups. In the control group (CG; n=6) a mapping of the native liver was conducted. For mapping, the four hepatic liver lobes were named from right to left with A to D and every lobe was divided into three vertical segments (cranial, medial, and caudal). In each of these 12 areas, microperfusion was quantified using a thermodiffusion probe (TD [mL/100 g/min]). The other two groups were considered as transplanted treated group (TTG; n=10) and transplanted nontreated group (TnTG; n=10). The TTG received an infusion of 20 mg/kg GdCl3 intravenously 24 hours before organ harvesting. Then standardized orthotopic liver transplantation was performed. In TnTG, standardized orthotopic liver transplantation was carried out without prior GdCl3 injection. In the recipients, the microperfusion of transplanted livers were mapped in both TnTG and TTG, in two different time points (1 hour [n=5] and 24 hours (n=5]) after reperfusion.

RESULTS

A significant reduction of macrophages in the TTG livers in comparison to the CG and TnTG livers was observed (P<.05). However, the number of macrophages in CG and TnTG livers showed no significant difference (P>.05). Regarding liver microperfusion, in TnTG, a marked heterogeneity was detected in the livers after reperfusion. Significant differences between liver lobes (horizontal planes; P=.032) and vertical layers of intralobar liver parenchyma (P=.029) were observed. The same pattern was seen in TTG livers after reperfusion and a significant difference between horizontal (P=.024) and vertical layers (P=.018) of liver tissue were observed. Comparing intralobar regional flow data between vertical planes 24 hours after reperfusion still showed a prominent variation of hepatic tissue perfusion in TnTG livers (P=.028). Within the same horizontal layers, no significant differences between lobes were measured anymore (P=.16). Contrary to TnTG, in TTG, a homogenous pattern of regional liver tissue perfusion was recorded 24 hours after reperfusion. Comparison of TD data on the liver regions showed no significant microperfusion differences in either horizontal (P=.888) or vertical (P=.841) layers.

CONCLUSIONS

Application of GdCl3 resulted in a significant reduction of Kupffer cells. Twenty four hours after transplantation microperfusion showed a homogeneous pattern, which constituted an earlier and better recovery of the transplanted liver. Therefore, destruction of Kupffer cells reduced ischemia-reperfusion injury and seemed to be responsible for the early recovery of microperfusion disturbances and thus for an improvement of graft function.

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases (KdtA) are multifunctional glycosyltransferases with primary structures of low similarity. Totally degenerated primers were deduced from two stretches of identical amino acids between known KdtA sequences and used to amplify by PCR a kdtA-specific fragment from Acinetobacter baumannii ATCC 15308 DNA which was then applied as a probe for the cloning and sequencing of the complete Kdo transferase gene. With conserved PCR primers for this structural gene from A. baumannii ATCC 15308, also kdtA genes of A. baumannii ATCC 19606 and A. haemolyticus ATCC 17906 were obtained, cloned from the chromosome and sequenced. The genes coded for proteins with similarities to known Kdo transferases. Within the genus Acinetobacter, the identity and similarity of the deduced amino acid sequences were 71% and 84.5%, respectively. The kdtA sequences of both A. baumannii strains were identical and possessed a TTG start codon, whereas ATG was found in the case of A. haemolyticus. The genes from Acinetobacter and kdtA from Escherichia coli K-12 were expressed in the Gram-positive bacterium Corynebacterium glutamicum. In vitro tests confirmed the function of the gene products as Kdo transferases, which transferred mainly two Kdo residues to a synthetic lipid A precursor of E. coli. Also, no differences between the cloned kdtA genes from A. baumanniii, A. haemnolyticus and E. coli were observed when tetraacyl or hexaacyl lipid A were tested, since all transferases acted more efficiently on the former. With limiting amounts of acceptor, all Kdo transferases were able to transfer a third Kdo residue with varying efficiency.

Previous studies indicated that the human X-ray repair complementing group 3 gene (XRCC3) plays an important role in hepatocellular carcinoma (HCC) susceptibility. We aimed to investigate the association of XRCC3 genetic polymorphism with HCC risk. This study was conducted in a Chinese Han population consisting of 300 HCC cases and 300 sex- and age-matched cancer-free controls. Three genetic variants (rs861539, rs12432907, and rs861537) were genotyped by the TaqMan® SNP Genotyping Assay. Our findings suggested that the TT genotype and T allele from rs861539 genetic variants were statistically associated with HCC risk. The TT genotype was statistically associated with the increased risk of HCC compared to CC wild genotype (P < 0.001). And the T allele was more common in the HCC patients than that in the control subjects. (OR = 1.97, 95% confidence interval (CI) 1.457 ~ 2.659, P < 0.001). Haplotype-based case-control study analysis indicated that TTG haplotype was more frequent in HCC groups than in the control group (odds ratio (OR) = 1.967, 95% CI 1.456 ~ 2.658); however, the CTG haplotype is more common in the control group than that in the HCC group (OR = 0.550, 95 % CI 0.430 ~ 0.703; P < 0.001). Our data indicated that genetic variants of the XRCC3 gene were statistically associated with HCC risk in a Chinese population.

BACKGROUND

Evidence indicates that patients with familial achalasia associated with Allgrove or triple-A syndrome (i.e. alacrima, achalasia and adrenocorticotropin-resistant adrenal insufficiency with neurological impairment) have mutations of the alacrima achalasia adrenal insufficiency syndrome (AAAS) gene.

OBJECTIVE

The present study was aimed at identifying possible AAAS gene mutations in patients with established idiopathic non-familial achalasia.

METHODS

Genomic DNA of 41 patients was isolated from peripheral blood cells using standard methods. The 16 exons of the AAAS gene (or ALADIN) were screened for mutations using the denaturing high-performance liquid chromatography method.

RESULTS

Four heterozygous nucleotidic variations have been identified in patients with idiopathic achalasia, among which three were exonic conservative polymorphisms [i.e. D138D (GAT->>GAC), L227L (TTG->>CTG) and F285F (TTC->>TTT) in exons 5, 7 and 9, respectively]. The fourth nucleotidic variation was located in intron 13 (IVS14-23delT). All variants have been regarded as polymorphisms resulting in a normal ALADIN protein since they are either conservative or lying outside the consensus splice sites.

CONCLUSIONS

Our data do not support a pathogenetic role for common AAAS gene mutations in patients with idiopathic achalasia as seen in Allgrove syndrome. These findings suggest the participation of different mechanisms in the pathogenesis of idiopathic achalasia.

OBJECTIVE

Celiac disease (CD) is an autoimmune disease triggered by exposure to gluten-containing foods. IgA autoantibodies to tissue transglutaminase (TTG) are elevated in CD, but little is known about the gastrointestinal state before the appearance of TTG. Antibodies to wheat storage globulin Glo-3A have been studied in type 1 diabetes, and may be a marker of altered mucosal barrier and/or immune function. In the present study, we investigated antibody responses to Glo-3A in CD.

METHODS

In the Diabetes Autoimmunity Study in the Young, children were studied prospectively from birth for the appearance of TTG and CD. Fifty cases of CD were frequency matched with 50 controls on age (of TTG seroconversion in the case), sex, ethnicity, presence of a first-degree relative with type 1 diabetes mellitus, and human leukocyte antigen -DR3 genotype. In cases and controls, IgG antibodies to Glo-3A were analyzed in a blinded manner in the sample collected at the time of seroconversion to TTG positivity (or the matched sample in controls) and in all of the previous samples since birth (mean 4.5 samples). The association between Glo-3A antibody levels and CD case status was explored using t tests at the TTG-positive visit and when Glo-3A levels were highest, and mixed modeling to describe Glo-3A over time.

RESULTS

At the time of first elevated TTG (mean 4.9 years), patients with CD had higher Glo-3A antibody levels than controls (13.3 ± 17.2 vs 7.6 ± 11.7, P = 0.005). In both cases and controls, Glo-3A antibodies appear to peak at a mean age of 2.9 years, before mean age of initial TTG seroconversion. The peak Glo-3A antibody levels were higher in cases than controls (25.5 ± 21.8 vs 14.9 ± 18.3 P = 0.0007). Using mixed modeling to account for multiple visits per person, cases had higher levels of Glo-3A antibodies than controls at all ages from birth to TTG seroconversion (β = 0.53, P = 0.002).

CONCLUSIONS

Compared with controls, CD cases have higher Glo-3A antibody responses in the beginning years, before initial detection of TTG.

CONCLUSIONS

These results demonstrate that functional magnetic resonance imaging (fMRI) is an optimal tool to investigate the auditory cortex. The study suggests that there is a medio-lateral gradient of responsiveness to high frequencies medially and low frequencies laterally. The contralateral auditory cortex is more responsive than the ipsilateral cortex to tones presented monaurally.

OBJECTIVE

To demonstrate the activation of the primary auditory cortex in normal-hearing subjects using fMRI and to examine the response and topographic location of activation in the human auditory brain to stimulation with two different frequencies in a large group of volunteers.

METHODS

Scanning was performed on a 1.5 Tesla MR with head gradient coils and a birdcage radiofrequency coil. Multiplanar echo-planar images were acquired in 32 subjects aged between 18 and 49 years. Two groups were defined, according to age (group A, 18 to <35 years old; group B, 35 to <50 years old). We studied normal-hearing subjects scanned while listening to auditory stimuli: narrative text in one volunteer and non-speech noise (pure tones 750 Hz and pure tones 2 KHz) in all subjects.

RESULTS

For both tone frequencies, auditory activation was observed bilaterally across the supratemporal plane in 29 of the 32 subjects (90.62%) with a probability level of p<0.001. In Heschl's gyrus (HG) contralateral to the stimulated ear, the extent of activation was generally greater than in homolateral HG. There were no statistical differences in HG activation according to age or sex. The 750 Hz tone activated more voxels in the medial area of the transverse temporal gyrus (TTG) whereas the 2000 Hz tone activated more voxels in the lateral TTG.

The sequence structure of the ribosomal internal transcribed spacer 2 (ITS2) was determined for six species of Khawia (Cestoda: Caryophyllidea), parasites of cyprinid fish in the Holarctic Region. Homologous intragenomic ITS2 structure was found in Khawia armeniaca, Khawia baltica, and Khawia rossittensis; whereas divergent intragenomic ITS2 copies were detected in Chinese, Japanese, and Slovak isolates of Khawia sinensis and in Khawia japonensis, both parasitic in common carp, and in Khawia saurogobii, recently described from Chinese lizard gudgeon in China. Despite distinct morphological differences between K. saurogobii and K. sinensis, both species display very high level of molecular homogeneity. Variation in number of short repetitive motifs [(GCCT)(n) (GCCC)(n)], [(GTG)(n)], [(ATAC)(n)], [ACGTGT (TCGTGT)(n)], [(GT)(n)], [(GT)(n)], and [(ACCT)(n) (GCCT)(n)] resulted in assortment of ITS2 sequences in four ITS2 variants in K. saurogobii from China, three in Chinese and Japanese isolates of K. sinensis, and five ITS2 variants in K. sinensis from Slovakia. In K. japonensis, the structure and arrangement of microsatellites was different from those of K. sinensis and K. saurogobii. The heterogeneity in the number of two microsatellite regions [(TG)(n); (TTG)(n)] divided ITS2 clones into two variants-first ITS2 variant (472 bp) with (TG)(5) and (TTG)(6), and second variant with (TG)(7) and (TTG)(2) (465 bp). Sequence identity of K. saurogobii with all but one (K. sinensis) congeneric species ranged between 49.5 and 69.2%, which corresponds to the interspecific differences. In contrast, sequence identity of K. saurogobii and K. sinensis (87.6-95.0%) failed into the range of intraspecific variation determined for K. sinensis samples. This close genetic similarity indicates that recently described K. saurogobii may have undergone morphological divergence as a result of ongoing sympatric speciation by host switching.

BACKGROUND

Serum IgA antibodies against tissue transglutaminase (tTG) are reliable markers for celiac disease (CD), still the diagnostic performance of anti-tTG immunoassays can be improved. A novel ELISA, using fibronectin (FN) as tTG binding protein, was evaluated for the detection of anti-tTG antibodies in CD.

METHODS

Sera from 173 children with untreated CD and 97 controls were analyzed for IgA, IgG, and IgM anti-tTG antibodies with ELISA using human recombinant tTG with or without FN.

RESULTS

The areas under the ROC (receiver operating characteristic) curves were significantly higher for FN/tTG ELISA compared to tTG ELISA for IgG (0.930 versus 0.809; p < 0.001) and IgM (0.850 versus 0.811; p = 0.019), but not for IgA (0.977 versus 0.970; p = 0.356), respectively. At the fixed diagnostic specificity (100% for IgA and IgM, 99% for IgG), the sensitivity of all three FN/tTG ELISA (92.5% for IgA, 60.7% for IgG, 50.3% for IgM) exceeded those obtained in tTG ELISA (89.0% for IgA, 48.6% for IgG, 38.7% for IgM; p < 0.05). The combined use of IgA- and IgG-FN/tTG ELISA resulted in 95.4% sensitivity and 99.0% specificity for CD.

CONCLUSIONS

Using FN to bind tTG improves the diagnostic performance of solid phase anti-tTG antibody assays for childhood CD.

Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9 kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at -874 site (a 200 bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I(+) was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (P < 0.05) and its effect on egg number at 37 weeks of age (E37) and egg number at 57 weeks of age (E57) was not significantly different (P>> 0.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I(+) tends to increase cFSHR transcription.

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.

Fluoroquinolone resistance is an emerging problem in companion animal practice. The present study aimed to determine comparative fluoroquinolone minimum inhibitory concentrations (MICs) for enrofloxacin, marbofloxacin and pradofloxacin and identify plasmid-mediated quinolone resistance (PMQR) mechanisms in 41 multidrug-resistant (MDR) Escherichia coli isolates representing three main clonal groups (CGs) cultured from extraintestinal infections in dogs. All isolates were resistant to fluoroquinolones and the PMQR genes qnrA1, qnrB2, qnrS1 and qepA were identified in isolates from each CG. For a subset of 13 representative isolates, fluoroquinolone chromosomal resistance mechanisms were characterized. CG1 isolates had three mutations in the quinolone resistance determining region (QRDR), two in gyrA (Ser TCG-83→Leu TTG and Asp GAC-87→Asn AAC) and one in parC (Ser AGC-80→Ile ATT), whilst CG2 and CG3 isolates also possessed an additional mutation in parC (Glu GAA-84→Gly GGA) which was reflected in higher fluoroquinolone MICs compared to CG1. Organic solvent tolerance was demonstrated in 8 of the 13 isolates, and all 13 isolates demonstrated enhanced efflux on the basis of a 4-fold decrease or greater in the MIC of enrofloxacin when incubated with an efflux pump inhibitor. A mutation in acrR which can cause overexpression of the AcrAB multidrug efflux pump was detected in CG1 strains. These findings indicate that fluoroquinolone resistance in MDR E. coli isolated from extraintestinal infections in dogs is associated with a combination of target mutations in the QRDRs, transferable PMQR mechanisms and enhanced efflux.

Human lactate dehydrogenase (LDH)--B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue "35" (GCG, Ala->>GAG, Glu), a T to G at residue "172" (TTT, Phe->>GTT, Val), and an A to T at residue "176" (ATG, Met->>TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.

Newborns at high risk of celiac disease (CD) were recruited in Italy in the context of the PreventCD study and closely monitored for CD, from 4 months up to a mean age of 8 years at follow-up. The aim of our study was to investigate intestinal T-cell reactivity to gliadin at the first clinical and/or serological signs of CD.

Gliadin-reactive T-cell lines were generated from intestinal biopsies of 19 HLA-DQ2-or HLA-DQ8-positive children. At biopsy, 11 children had a diagnosis of acute CD, two of potential CD, and six were non-celiac controls. Immune reactivity was evaluated against gliadin and known immunogenic peptides from α-, γ-, or ω-gliadins. The role of deamidation by transglutaminase (tTG) in determining the immunogenicity of gliadin was also investigated.

Most of the children with CD (either acute or potential) had an inflammatory response to gliadin. Notably, signs of T-cell reactivity to gliadin were also found in some non-celiac subjects, in which IFN-γ responses occurred mainly when regulatory IL-10 and TGF-β cytokines were blocked. Interestingly, PreventCD children reacted to gliadin peptides found active in adult CD patients, and tTG deamidation markedly enhanced gliadin recognition.

T cells reactive to gliadin can be detected in the intestine of children at high risk of developing CD, in some cases also in the presence of a normal mucosa and negative CD-associated antibodies. Furthermore, children at a very early stage of CD recognize the same gliadin epitopes that are active in adult CD patients. Tissue transglutaminase strongly enhances gluten T-cell immunogenicity in early CD.

OBJECTIVE

Isotretinoin is a vitamin A-derived medication that is associated with significant adverse effects including arthralgia/ myalgia, nose bleeds, headache, dyslipidemia, liver dysfunction and depression. The mechanism for development of such adverse effects remains elusive, and it is not known why adverse effects develop only in some patients. In this study, we examined the association between rs9303285, rs2715554 and rs4890109 genetic polymorphisms in the retinoic acid receptor alpha (RARA), one of the main targets of isotretinoin, and the adverse effects of oral isotretinoin therapy.

METHODS

Clinical adverse effects data were collected from patient file and by patient interview. Lipids and liver enzymes were measured in blood samples collected from acne patients (n = 300) at baseline and during oral isotretinoin treatment. RARA polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTS

Three-locus haplotype (Rs2715554 C/T - Rs4890109 G/T - Rs9303285 T/C) analysis showed that frequencies of CTG and TTG haplotypes are significantly associated with occurrence of arthralgia, myalgia, nose bleeds and headache in patients treated with isotretinoin. In addition, TCG haplotype was associated with nose bleeds and headache, whereas TTT haplotype was associated with arthralgia and myalgia. Furthermore, levels of AST were increased, and AST% change was more, after 1 month of treatment in patients with the TC genotype of rs2715554 polymorphism. Finally, allele T of rs9303285 was found to be protective against developing depression in the patients treated with isotretinoin.

CONCLUSIONS

Our findings suggest an association between polymorphisms of RARA gene and some of some common adverse effects of oral isotretinoin.

BACKGROUND

Mutations in the SOD1 gene are responsible for approximately 25% of all familial amyotrophic lateral sclerosis (ALS) cases. However, the correlation between the clinical and pathological features and the various SOD1 gene mutations has not been well characterized.

OBJECTIVE

To screen the SOD1 gene in search of potential mutations and to obtain clinical and pathological data for 2 Japanese families with ALS.

METHODS

Clinical histories and neurological findings, gross and microscopic pathological features, and DNA analysis of the SOD1 gene.

RESULTS

The 2 families with ALS showed a novel missense mutation in the SOD1 gene, which was heterozygous for point mutation TTG to TCG, causing substitution of leucine for serine at codon 126 (Leu126Ser) in exon 5. Clinically, patients showed slower disease progression and lack of upper motor neuron signs. Neuropathologically, the autopsied patient showed the form of familial ALS with posterior column involvement, and the pontocerebellar tract and the dentate nuclei of the cerebellum were also involved. Furthermore, abundant Lewy body-like hyaline inclusions were observed in the affected motor and nonmotor neurons.

CONCLUSIONS

Familial ALS with a novel Leu126Ser mutation in the SOD1 gene showed mild clinical features and lack of upper motor neuron signs. We believe that Leu126Ser might be associated with the clinical features and that the mutation site in the SOD1 gene and disease duration might be associated with the formation of Lewy body-like hyaline inclusions.

The role of tissue transglutaminase (tTG), a calcium-dependent and GTP-modulated enzyme, in apoptotic death induced by GTP depletion in islet beta-cells was investigated. GTP depletion and apoptosis were induced by mycophenolic acid (MPA) in insulin-secreting HIT-T15 cells. MPA treatment increased in situ tTG activity (but not protein levels) in a dose- and time-dependent manner in parallel with the induction of apoptosis. MPA-induced increases of both tTG activity and apoptosis were entirely blocked by co-provision of guanosine but not adenosine. MPA-enhanced tTG activity could be substantially reduced by co-exposure to monodansylcadaverine or putrescine (tTG inhibitors), and largely blocked by lowering free Ca(2+) concentrations in the culture medium. However, MPA-induced cell death was either not changed or was only slightly reduced under these conditions. By contrast, a pan-caspase inhibitor (Z-VAD-FMK) entirely prevented apoptosis induced by MPA, but did not block the enhanced tTG activity, indicating that GTP depletion can induce apoptosis and activate tTG either independently or as part of a cascade of events involving caspases. Importantly, the morphological changes accompanying apoptosis could be markedly prevented by tTG inhibitors. These findings suggest that the effect of the marked increase in tTG activity in GTP depletion-induced apoptosis of insulin-secreting cells may be restricted to some terminal morphological changes.

In this study, we show that sex hormones (testosterone, estradiol, and progesterone) act as physiological modulators of programmed cell death (PCD) during the frog liver involution observed postvitellogenesis. PCD in parenchymal cells is paralleled by the specific induction of the "tissue" transglutaminase (tTG) gene. tTG protein specifically accumulates in hepatocytes showing the morphological features of apoptosis. The hormone-dependent increase of both PCD and tTG was reproduced in ovariectomized frogs. Treatment of castrated animals with testosterone, estradiol, and progesterone inhibited the induction of both tTG and PCD, thus indicating that in vivo the drop in the circulating sex hormone is the signal favoring the involution phase of the maternal frog liver after mating. Although an affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts in frog liver with a 55- to 60-kDa protein, concomitant with the onset of PCD, tTG cleavage products were detected, suggesting a proteolytic processing of the enzyme protein. These results represent the first evidence indicating that the physiological involution occurring postvitellogenesis of frog liver takes place by programmed cell death and that this, together with the concomitant induction of tTG gene expression, is regulated by sex hormones.