OBJECTIVE

The optimal end point for randomized phase II trials of anticancer therapies remains controversial. We simulated phase II trials by resampling patients from N9741, a randomized phase III trial of chemotherapy regimens for metastatic colorectal cancer, and compared the power of various end points to detect the superior therapy (FOLFOX [infusional fluorouracil, leucovorin, and oxaliplatin] had longer overall survival than both IROX [irinotecan plus oxaliplatin] and IFL [irinotecan and bolus fluorouracil plus leucovorin]).

METHODS

Tumor measurements and progression-free survival (PFS) data were obtained for 1,471 patients; 1,002 had consistently measured tumors and were resampled (5,000 replicates) to simulate two-arm, randomized phase II trials with α = 0.10 (one sided) and 20 to 80 patients per arm. End points included log ratio of tumor size at 6, 12, and 18 weeks relative to baseline; time to tumor growth (TTG), estimated using a nonlinear mixed-effects model; and PFS. Arms were compared using rank sum tests for log ratio and TTG and a log-rank test for PFS.

RESULTS

For FOLFOX versus IFL, TTG and PFS had similar power, with both exceeding the power of log ratio at 18 weeks; for FOLFOX versus IROX, TTG and log ratio at 18 weeks had similar power, with both exceeding the power of PFS. The best end points exhibited>> 80% power with 60 to 80 patients per arm.

CONCLUSIONS

TTG is a powerful end point for randomized phase II trials of cytotoxic therapies in metastatic colorectal cancer; it was either comparable or superior to PFS and log ratio at 18 weeks. Additional studies will be needed to clarify the potential of TTG as a phase II end point.

BACKGROUND

The identification of specific serological algorithms allowing the diagnosis of celiac disease (CD) is a new challenge for both the clinic and the laboratory. We compared the diagnostic accuracy of three new tests proposed for CD screening with that of the well established IgA tTG, and ascertained whether any combination of these tools might enhance accuracy in diagnosing CD.

METHODS

In sera from 329 CD and 374 control children, the following were assayed: IgA tTG; IgA/IgG, which identify tTG-gliadin complexes (Aeskulisa Celi Check and CeliCheck IgGA); IgA/IgG, which identify deamidated gliadin peptides and tTG (QUANTA Lite(TM) h-tTG/DGP Screen).

RESULTS

When specificity was set at 100%, the most sensitive index of CD was IgA tTG (75.7%, cut-off=100U), followed by QUANTA Lite(TM) h-tTG/DGP Screen (65.3%, cut-off 145U), Aeskulisa Celi Check (62.6%, cut-off 909U/mL) and CeliCheck IgGA (59.6%, cut-off 977U/mL). Three algorithms were obtained by combining IgA tTG with each of the new tests. The algorithm obtained by measuring IgA tTG and QUANTA Lite(TM) h-tTG/DGP Screen allowed the correct identification of CD in 78.7% of cases (negative predictive value=97.3%).

CONCLUSIONS

The two-test based strategy could be used for the cost effective diagnosis of CD.

OBJECTIVE

Epidemiological studies of celiac disease (CD) among Saudi children have been performed only within some groups who are at a high risk of developing CD. The aim of this study was to determine the prevalence of CD among symptom-free children from the public schools of the military campus of National Guard in the Eastern Province of Saudi Arabia.

METHODS

Between 2012 and 2014, serum samples were collected from 1141 students (age 6-18 years) attending nine public schools of the military campus of National Guard in the Eastern Province of Saudi Arabia. Participants were screened for CD by testing for anti-tissue transglutaminase IgA (IgA-tTG) and IgG antibodies (IgG-tTG). Small intestinal biopsy was offered to all participants who tested positive for IgA-tTG [IgA-tTG >20 relative units (RU)/ml].

RESULTS

Of the 1141 participants, 32 were IgA-tTG positive. Thus, the estimated serology-positive prevalence was 3%. An intestinal biopsy was performed in 10 of the participants with antibody positivity. The biopsy findings of all 10 children were consistent with CD. Thus, the estimated biopsy-confirmed prevalence was about 1%.

CONCLUSIONS

The prevalence of CD was estimated to be about 1% among symptom-free children from the public schools of the military campus of National Guard in the Eastern Province of Saudi Arabia.

The close association between celiac disease (CD) and autoimmune disorders is well documented in adult and pediatric patients. The aim of this study is to determine the prevalence of CD in Turkish children with autoimmune thyroiditis (AT). Sera from 101 children with AT (11 boys and 90 girls, from 2 to 18 years of age; mean age 12.28 +/- 3.26 years) and 103 healthy children (46 boys and 57 girls, from 3.5 to 17 years of age; mean age 12.18 +/- 3.11 years) were screened for CD using the IgA anti-tissue transglutaminase (IgA anti-tTG) antibody and total serum IgA. Small intestinal biopsy was offered to all antibody-positive patients. IgA anti-tTG was positive in eight children (7.9%) with AT. None of the serum samples of healthy children were positive for IgA anti-tTG antibody. Selective IgA deficiency was not detected in patients or controls. Intestinal biopsy was accepted by seven patients. In five patients (4.9%), subtotal villous atrophy was found. These findings indicate that the prevalence of CD is higher in Turkish children with AT than in healthy controls. Routine screening for CD should be performed in children with AT.

OBJECTIVE

Here we compared analytical and clinical performance characteristics of two novel automated assay systems for the detection of celiac disease (CD) specific antibodies: QUANTA Flash (INOVA Diagnostics, Inc.) and EliA (Thermo Scientific).

METHODS

A total of 74 biopsy-proven CD patients (2 with IgA deficiency) and 138 controls were tested by both methods.

RESULTS

Sensitivities of QUANTA Flash assays ranged from 35.1% to 90.5% and specificities from 96.4% to 99.3%, while sensitivities for EliA assays ranged from 37.8% to 90.5% (equivocal considered positive) and specificities from 97.1% to 100.0%. Good qualitative agreement was found between all assays. Thirty-four (50.0%) of the 68 QUANTA Flash h-tTG IgA positive results were higher than 10 times the upper limit of normal (ULN). In contrast, only 22.8% of the EliA tTG IgA positive samples were >10x ULN. Seventy-three (98.6%) biopsy-proven CD patients were correctly identified with the QUANTA Flash h-tTG IgA+DGP IgG combination, while 64 (86.5%) and 72 (97.3%) (depending on equivocal range) were identified with the same combination of EliA assays.

CONCLUSIONS

The QUANTA Flash CD assays have outstanding clinical performance. Of particular clinical significance, in light of proposals to decrease the absolute necessity of biopsy, was the demonstration that 50% of the QUANTA Flash h-tTG IgA results were >10x ULN.

BACKGROUND

Iron deficiency anemia (IDA) is one of the well recognized presentations of celiac disease (CD). According to the lack of data from our population in this regard, we determined the prevalence of CD in patients presenting with IDA to see if it is worthwhile to do a precise screening for CD in such patients.

METHODS

This cross-sectional study was conducted on patients referred with IDA to Poursina Hakim Gastroenterology Clinic, Isfahan (IRAN). All included patients underwent upper gastrointestinal endoscopy and duodenal biopsy. Histopathological changes were assessed according to the Marsh classification. Also, patients were evaluated for IgA anti-tissue transglutaminase (t-TG) antibody with enzyme-linked immunosorbent assay (ELISA) technique. CD was defined as having Marsh II or above histopathology or being seropositive with Marsh I histopathology and having a good response to gluten free diet (GFD).

RESULTS

During the study, 130 patients with the mean age of 35.5±13.7 (67.7% female [20.4% post-menopausal]) were undergone seropathological studies. According to histopathological study and a clinical response to GFD, 13 patients (10%) were ultimately diagnosed with CD. Nine patients (6.9%) were seropositive, from which, five patients (3.8%) were ultimately diagnosed as CD cases. IgA anti-tTG became negative in all of these patients after six months of GFD.

CONCLUSIONS

CD should be considered in any adult patient presenting with unexplained IDA, even if not accompanied with gastrointestinal symptoms. Routine duodenal biopsy performed during diagnostic upper gastrointestinal endoscopy is worthwhile in order to investigate for CD as an underlying cause of IDA in adult patients.

Celiac disease (CD) is sustained by abnormal intestinal mucosal T-cell response to gluten and it is strongly associated with HLA class II molecules encoded by DQA1*0501/DQB1*02 (DQ2) or DQA1*03/DQB1*0302 (DQ8). The in vitro stimulatory activity of gliadin increases after treatment with tissue transglutaminase (tTG) which catalyses the deamidation of specific residues of glutamine to glutamate that can serve as anchors for binding to DQ2 as well as to DQ8 molecules. We modelled the three-dimensional structure of the DQ2 dimer protein, the most frequent in celiac patients, by using a homology modelling strategy, and deposited the model in the Protein Data Bank (PDB). Then, we simulated the interactions of DQ2 with different gluten peptides and the deamidation of specific peptide glutamines in the known p4, p6, p7 and p9 anchor positions, as well as in p1 and p5 positions, and other substitutions for which experimental effects on binding are available by previous experimental studies. By evaluating the energy of interaction and the H-bond interactions, we were able to distinguish what substitutions improve the interaction peptide-DQ2, in agreement with previously published experimental data. By analysing the peptide-DQ2 complex at the atom level, we observed that these glutamate side chains can interact with specific positively charged amino acids of DQ2, absent in other HLA alleles not related to celiac disease. The simulation was also extended to other peptides, related to celiac disease but for which no experimental data exists about the effects of glutamine deamidation. Our results give an interpretation at the molecular level of previously reported binding experimental data and open a new window to gain further insights about peptide recognition in celiac disease.

OBJECTIVE

Children with celiac disease (CD) may experience deficiencies of several micronutrients. The objectives of the present study were to determine the prevalence of micronutrient deficiencies in children with CD at diagnosis, 6 months, and 18 months after the start of a gluten-free diet (GFD), and examine any correlation between micronutrient deficiencies, serum tissue transglutaminase (TtG) immunoglobulin A (IgA) antibody titers, and the degree of mucosal damage at diagnosis.

METHODS

Children (<17 years) with CD had their serum vitamins, minerals, and anti-TtG IgA antibodies measured at diagnosis, 6 and 18 months after starting a GFD. Histopathological changes of duodenal biopsies at diagnosis were documented using modified MARSH classification.

RESULTS

The medical records of 140 children (mean age at diagnosis 7.8 ± 4.01 years, 87 girls [621%]) with CD were examined. At diagnosis, serum vitamin D was the most commonly deficient vitamin in 70% of children. Serum ferritin was subnormal in 34.5% with zinc in 18.6% children but only 12 (10.9%) children had iron deficiency anemia. There was no correlation between micronutrient deficiencies at diagnosis and serum TtG IgA antibody titers or the degree of villous atrophy. The majority of serum levels of measured micronutrients had normalized after 6 months of starting GFD except for vitamin D, which improved but remained subnormal.

CONCLUSIONS

At diagnosis, most children with CD have vitamin D deficiency. The degree of micronutrient deficiencies does not correlate with the degree of villous atrophy or serum titers of anti-TtG IgA antibodies.

The cytopathic effect of HIV has been shown to be associated with the induction of apoptosis and the inhibition of proliferation of T cells. However, the cellular and molecular mechanisms at the basis of the dramatic immune cell loss caused by HIV in patients suffering from acquired immunodeficient syndrome (AIDS), are not yet fully established. We demonstrated that "tissue" transglutaminase (tTG) gene expression is induced in the immune system of seropositive individuals (peripheral blood mononuclear cells and lymph nodes). tTG is a multifunctional protein involved in a variety of fundamentally important cellular functions, in addition to cell death by apoptosis. The presence of high tTG levels in immune-competent cells of HIV+ persons might exert an important role in HIV-infection by influencing viral production. We propose that, in addition to its multiple functions, tTG might interfere with HIV replication by altering the viral mRNA trafficking between the nucleus and the cytoplasm. This effect might be due to its specific interaction with eIF5A, a cellular partner of HIV Rev protein, which is essential for HIV replication in immune-competent cells. Given the presence of high tTG levels in HIV+ individuals, it would be of interest to pursue the potential role of this multifunctional protein in the development of strategies aimed at the pharmacologic regulation of HIV production.

We demonstrated a germline p53 replication error in two generations of a Li-Fraumeni family affected with liposarcoma, adrenocortical carcinoma, and osteosarcoma. The trinucleotide repeat mutation changed 5'-AGT GTG GTG GTG-3' at codons 215-218 to 5'-AGT TGG TTG GTG GTG-3'. The predicted protein would be elongated by one amino acid (val216->>trp leu) without a change in charge. Detection of p53 in the adrenal tumor by immunostaining suggested that the mutant protein was expressed. Persistence of the mutation in the germline may suggest a defect in DNA repair in the family member first affected. This is the first report where germline transmission of replication-damaged p53 trinucleotide repeats is associated with the Li-Fraumeni syndrome.

The root epidermis of Arabidopsis thaliana is formed by alternate files of hair and non-hair cells. Epidermal cells overlying two cortex cells eventually develop a hair, while those overlying only one cortex cell do not. Here we propose a network model that integrates most of the available genetic and molecular data on the regulatory and signaling pathways underlying root epidermal differentiation. The network architecture includes two pathways; one formed by the genes TTG, R homolog, GL2 and CPC, and the other one by the signal transduction proteins ETR1 and CTR1. Both parallel pathways regulate the activity of AXR2 and RHD6, which in turn control the development of root hairs. The regulatory network was simulated as a dynamical system of eight discrete state variables. The distinction between epidermal cells contacting one or two cortical cells was accounted for by fixing the initial states of CPC and ETR1 proteins. The model allows for predictions of mutants and pharmacological effects because it includes the ethylene receptor. The dynamical system reaches one of the six stable states depending upon the initial state of the CPC variable and the ethylene receptor. Two of the stable states describe the activation patterns observed in mature trichoblasts (hair cells) and atrichoblasts (non-hair cells) in the wild-type phenotype and under normal ethylene availability. The other four states correspond to changes in the number of hair cells due to experimentally induced changes in ethylene availability. This model provides a hypothesis on the interactions among genes that encode transcription factors that regulate root hair development and the proteins involved in the ethylene transduction pathway. This is the first effort to use a dynamical system to understand the complex genetic regulatory interactions that rule Arabidopsis primary root development. The advantages of this type of models over static schematic representations are discussed.

The melon thrips, Thrips palmi is a serious pest and vector for plant viruses on a wide range of economically important crops. DNA barcoding evidenced the presence of cryptic diversity in T. palmi and that warrants exhaustive molecular studies. Our present study is on decoding the first complete mitochondrial genome of T. palmi (15,333 bp) through next-generation sequencing (NGS). The T. palmi mt genome contains 37 genes, including 13 Protein coding genes (PCGs), two ribosomal RNA (rRNAs), 22 transfer RNA (tRNAs), and two control regions (CRs). The majority strand of T. palmi revealed 78.29% A+T content, and 21.72% G+C content with positive AT skew (0.09) and negative GC skew (-0.06). The ATN initiation codons were observed in 12 PCGs except for cox1 which have unique start codon (TTG). The relative synonymous codon usage (RSCU) analysis revealed Phe, Leu, Ile, Tyr, Asn, Lys and Met were the most frequently used amino acids in all PCGs. The codon (CGG) which is assigned to Arginine in most insects but absent in T. palmi. The Ka/Ks ratio ranges from 0.078 in cox1 to 0.913 in atp8. We observed the typical cloverleaf secondary structure in most of the tRNA genes with a few exceptions; absence of DHU stem and loop in trnV and trnS, absence of DHU loop in trnE, lack of T-arm and loop in trnN. The T. palmi gene order (GO) was compared with ancestral GO and observed an extensive gene arrangement in PCGs, tRNAs and rRNAs. The cox2 gene was separated from the gene block 'cox2-trnL2' in T. palmi as compared with the other thrips mt genomes, including ancestor GO. Further, the nad1, trnQ, trnC, trnL1, trnV, trnF, rrnS, and rrnL were inversely transpositioned in T. palmi GO. The gene blocks 'trnQ-trnS2-trnD' and 'trnN-trnE-trnS1-trnL1' seems to be genus specific. The T. palmi mt genome contained 24 intergenic spacer regions and 12 overlapping regions. The 62 bp of CR2 shows the similarity with CR1 indicating a possible duplication. The occurrence of multiple CRs in thrips mt genomes seems to be a derived trait which needs further investigation. Although, the study depicted extensive gene rearrangements in T. palmi mt genome, but the negative GC skew reflects only strand asymmetry. Both the ML and BI phylogenetic trees revealed the close relationships of Thrips with Scirtothrips as compared to Frankliniella. Thus, more mt genomes of the diverse thrips species are required to understand the in-depth phylogenetic and evolutionary relationships.

A heterotrimeric flavoprotein-cytochrome c complex fructose dehydrogenase (FDH) of Gluconobacter japonicus NBRC3260 catalyzes the oxidation of d-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The fdhSCL genes encoding the FDH complex of G. japonicus NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for fdhSCL encoding the small, cytochrome c, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of fdhSCL was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of Gluconobacter oxydans and a G. oxydans strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated (TTG)FDH and (ATG)FDH. Membranes of the cells producing recombinant (TTG)FDH and (ATG)FDH showed approximately 20 times and 100 times higher specific activity than those of G. japonicus NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high d-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome c subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.

OBJECTIVE

To investigate the anti-fibrosis effect of the tissue transglutaminase (tTG) specific inhibitor cystamine on liver fibrosis.

METHODS

Sixty-eight male Sprague Dawley rats were divided into three groups: normal control, liver fibrosis control and cystamine-treated group. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl(4)), and Cystamine was administrated by intraperitoneal injection starting 2 d before the first administration of CCl(4). Animals in each group were further divided into 2 subgroups according to two time points of 4 wk and 8 wk after treatment. Hepatic function, pathological evaluation (semi-quantitative scoring system, SSS) and liver hydroxyproline (Hyp) content were examined. Real-time PCR was used to detect the expression of tTG, smooth muscle alpha actin (alpha-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1) and collagen-1 mRNA. The expressions of tTG and alpha-SMA protein were detected by Western Blotting.

RESULTS

Eight weeks after treatment, the SSS score of liver was significantly less in the cystamine group than that in the fibrosis control group (P < 0.01). The levels of alanine aminotransferase (ALT) and total bile acid (TBA) at the 4 wk and 8 wk time points were decreased in the cystamine group compared with those in fibrosis controls (P < 0.01). Liver hydroxyproline content at the 4 wk and 8 wk time points showed a substantial reduction in the cystamine group compared to fibrosis controls (P < 0.01). The expression of tTG, alpha-SMA, collagen-1, TIMP-1 mRNA and tTG, as well as alpha-SMA protein was downregulated in the cystamine group compared to fibrosis controls.

CONCLUSIONS

Cystamine can ameliorate CCl(4) induced liver fibrosis and protect hepatic function. The possible mechanism is related to the reduced synthesis of the extracellular matrix (ECM) caused by the inhibition of hepatic stellate cell activation and decreased expression of TIMP-1.

BACKGROUND

Celiac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten. Serology and organ culture system can support CD diagnosis, despite histology being the gold standard.

OBJECTIVE

We wanted to test the uniformity of application of Marsh-Oberhuber criteria by five different histologists. We also compared histological and serological data with cultural results to consider new possible strategies in CD diagnosis.

METHODS

We studied 114 patients, who were divided in two groups. Group A was composed of 66 patients on a gluten-containing diet, with gluten-related signs and symptoms, showing positive serological anti-endomysial antibodies (EMA) and anti-tissue transglutaminase (anti- tTG). Group B was composed of 48 disease-control patients, presenting serological EMA and anti-tTG negative results. All patients studied underwent esophagogastroduodenoscopy with duodenal biopsy and duodenal mucosa organ culture. All histological samples were evaluated by five different histologists according to an appropriate questionnaire following Marsh-Oberhuber classification. Cohen κ inter-test was used for evaluating the agreement between histologists regarding group A.

RESULTS

Strength of agreement was fair/moderate for villous:crypt ratio, moderate/good for villous height and crypt depth, and poor for intraepithelial lymphocytosis. Patients belonging to group A presented positive serological as well as cultural results in 100% of cases. None of the patients belonging to group B presented serological or cultural positive results.

CONCLUSIONS

Our study stresses the limits of histological interpretation due to the lack of uniformity in the use of Marsh-Oberhuber classification. These findings could cast doubt on the role of histology as CD gold standard and could open a debate on the most appropriate CD diagnostic procedure.

UNASSIGNED

Tissue transglutaminase (tTG) is induced in injured and remodelling tissues, and modulates cellular phenotype, while contributing to matrix cross-linking. Our study tested the hypothesis that tTG may be expressed in the pressure-overloaded myocardium, and may regulate cardiac function, myocardial fibrosis and chamber remodelling.

UNASSIGNED

In order to test the hypothesis, wild-type and tTG null mice were subjected to pressure overload induced through transverse aortic constriction. Moreover, we used isolated cardiac fibroblasts and macrophages to dissect the mechanisms of tTG-mediated actions. tTG expression was upregulated in the pressure-overloaded mouse heart and was localized in cardiomyocytes, interstitial cells, and in the extracellular matrix. In contrast, expression of transglutaminases 1, 3, 4, 5, 6, 7 and FXIII was not induced in the remodelling myocardium. In vitro, transforming growth factor (TGF)-β1 stimulated tTG synthesis in cardiac fibroblasts and in macrophages through distinct signalling pathways. tTG null mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross-linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure-overloaded myocardium. In vitro, tTG did not modulate TGF-β-mediated responses in cardiac fibroblasts; however, tTG loss was associated with accentuated proliferative activity. Moreover, when bound to the matrix, recombinant tTG induced synthesis of tissue inhibitor of metalloproteinases (TIMP)-1 through transamidase-independent actions.

UNASSIGNED

Following pressure overload, endogenous tTG mediates matrix cross-linking, while protecting the remodelling myocardium from dilation by exerting matrix-preserving actions.

OBJECTIVE

Assessment of treatment response in children with celiac disease (CD) after commencing a strict gluten-free diet (GFD) is generally based on the resolution of clinical features and normalization of serology. Recent adult studies have shown that serologic markers do not correlate with mucosal recovery. We aimed (i) to determine whether anti-tissue transglutaminase immunoglobulin (Ig)A (tTG) and anti-deamidated gliadin peptide IgG (DGP) antibodies are sensitive and specific markers of mucosal recovery in children with CD on a GFD for at least 12 months, and (ii) to determine whether a validated dietary questionnaire of compliance can identify patients with mucosal recovery.

METHODS

A total of 150 children with biopsy-proven CD were prospectively evaluated with duodenal biopsies at ≥12 months on GFD, paired with repeat tTG and DGP serology. The biopsies were reviewed in a blinded manner by two histopathologists and graded by Marsh criteria. A validated questionnaire of dietary compliance was also administered.

RESULTS

Of 150 children recruited, 27 (18%) had positive serology, 97 (65%) had negative serology, and 26 (17%) had equivocal serology. Of the 97 children with negative serology, none had Marsh type 3 enteropathy. Of the 27 patients with positive serology, only 6 had Marsh type 3 changes. The sensitivity and specificity of serology as a marker of significant mucosal pathology was 75 and 85%, respectively, with a positive predictive value of 22% but a negative predictive value of 98%. Of the 129 (86%) questionnaires completed, 88% reported good or excellent compliance with a GFD (negative predictive value 97%).

CONCLUSIONS

This study suggests that follow-up using two serological tests in children with CD on a GFD may obviate the need for repeat mucosal biopsy in the majority of patients. A standardized dietary questionnaire may be useful in identifying patients who require further evaluation.

We present the complete mitochondrial (mt) genome sequence of the stonefly, Styloperla spinicercia Wu, 1935 (Plecoptera: Styloperlidae), the type species of the genus Styloperla and the first complete mt genome for the family Styloperlidae. The genome is circular, 16,129 base pairs long, has an A+T content of 70.7%, and contains 37 genes including the large and small ribosomal RNA (rRNA) subunits, 13 protein coding genes (PCGs), 22 tRNA genes and a large non-coding region (CR). All of the PCGs use the standard initiation codon ATN except ND1 and ND5, which start with TTG and GTG. Twelve of the PCGs stop with conventional terminal codons TAA and TAG, except ND5 which shows an incomplete terminator signal T. All tRNAs have the classic clover-leaf structures with the dihydrouridine (DHU) arm of tRNASer(AGN) forming a simple loop. Secondary structures of the two ribosomal RNAs are presented with reference to previous models. The structural elements and the variable numbers of tandem repeats are described within the control region. Phylogenetic analyses using both Bayesian (BI) and Maximum Likelihood (ML) methods support the previous hypotheses regarding family level relationships within the Pteronarcyoidea. The genetic distance calculated based on 13 PCGs and two rRNAs between Styloperla sp. and S. spinicercia is provided and interspecific divergence is discussed.

OBJECTIVE

To evaluate mucosal baseline mRNA expression of tissue transglutaminase 2 (<em>tTG</em>2), interferon gamma (IFNγ), toll-like receptor 2 (TLR2) and Myeloid Differentiation factor 88 (MyD88) in patients with microscopic enteritis (ME).

METHODS

We retrospectively enrolled 89 patients with ME of different etiology, which was defined within a 2-year mean period of follow-up. Baseline histological examination was performed on Hematoxylin-Eosin stained sections and CD3 lymphocyte immunohistochemistry was used for intraepithelial lymphocyte count (IELs). ME was defined according to the criteria of Bucharest Consensus Conference. For each patient, formalin embedded biopsy samples of the duodenum referred to the period of ME diagnosis were retrieved. Real-time polymerase chain reaction (RT-PCR) was used to detect the amount of mRNA coding for <em>tTG</em>2, IFNγ, TLR2 and MyD88, and the quantity was expressed as fold change compared to controls. Control group was represented by duodenal normal specimens from 15 healthy subjects undergoing endoscopy for functional symptoms. Comparisons among continuous variables were performed by One way analysis of variance (ANOVA) and Bonferroni's test. The χ(2) test was used for categorical variables. Pearson's test was used to evaluate correlations. Receiver operating curves were drawn for all four markers to estimate sensitivity and specificity in discriminating the development of CD and GS.

RESULTS

After a period of follow up of 21.7 ± 11.7 mo, the following diagnoses were achieved: gluten related disorders in 48 subjects (31 CD; 17 GS) and non-gluten related ones in 41 (29 Irritable Bowel Syndrome - IBS; 12 Others). CD patients had the highest tTG> Other ME>> GS = IBS>> negative controls. A cut off value of 2.258 was able to discriminate between CD and GS with a sensitivity of 52.94% and a specificity of 87.1%. Additionally, CD patients had the highest IFNγ levels (8.5 ± 4.1). ANOVA plus Bonferroni demonstrated CD>> Other ME>> GS = IBS>> negative controls. A cut off of 1.853 was able to differentiate CD and GS with a sensitivity of 47.06% and a specificity of 96.77%. Patients with non gluten-related causes of ME exhibited the highest TLR2 levels (6.1 ± 1.9) as follows: Other ME>> CD = GS = IBS>> negative controls. TLR2 was unable to discriminate CD from GS. Patients with CD overexpressed MyD88 levels similarly to non gluten-related causes of DL (7.8 ± 4.9 and 6.7 ± 2.9), thus CD = Other ME>> GS = IBS>> negative controls. A cut off of 3.722 was able to differentiate CD from GS with a sensitivity of 52.94% and a specificity of 74.19%. IELs count (15-25 and more than 25/100 enterocytes) strongly correlated with mRNA levels of all tested molecules (P < 0.0001).

CONCLUSIONS

Our results confirm that a single marker is unable to predict a discrimination among ME underlying conditions as well as between CD and GS. Mucosal high levels of tTG and IFNγ mRNA may predict the development of CD more than GS with high specificity, despite an expected low sensitivity. TLR2 does not discriminate the development of CD from GS. MyD88 levels indicate that intestinal permeability is more increased when a severe intestinal damage underlies ME in both gluten related and unrelated conditions. Therefore, the results of the present paper do not seem to show a clear translational value.

BACKGROUND

Upstream open reading frames (uORFs) and upstream AUGs (uAUGs) can regulate the translation of downstream ORFs. The AT rich genome of Plasmodium falciparum, due to the higher AT content of start and stop codons, has the potential to give rise to a large number of uORFs and uAUGs that may affect expression of their flanking ORFs.

METHODS

A bioinformatics approach was used to detect uATGs associated with different genes in the parasite. To study the effect of some of these uAUGs on the expression of the downstream ORF, promoters and 5' leaders containing uAUGs and uORFs were cloned upstream of a luciferase reporter gene. Luciferase assays were carried out in transient transfection experiments to assess the effects of uAUGs and mutations on reporter expression.

RESULTS

The average number of uATGs and uORFs seen in P. falciparum coding sequences (CDS) is expectedly high compared to other less biased genomes. Certain genes, including the var gene family contain the maximum number of uATGs and uORFs in the parasite. They possess ~5 times more uORFs and ~4.5 times more uAUGs within 100 bases upstream of the start codons than other CDS of the parasite. A 60 bp upstream region containing three ORFs and five ATGs from var gene PF3D7_0400100 and a gene of unknown function (PF3D7_0517100) when cloned upstream of the luciferase start codon, driven by the hsp86 promoter, resulted in loss of luciferase activity. This was restored when all the ATGs present in the -60 bp were mutated to TTGs. Point mutations in the ATGs showed that even one AUG was sufficient to repress the luciferase gene.

CONCLUSIONS

Overall, this work indicates that the P. falciparum genome has a large number of uATGs and uORFs that can repress the expression of flanking ORFs. The role of AUGs in translation initiation suggests that this repression is mediated by preventing the translation initiation complex from reaching the main AUG of the downstream ORF. How the P. falciparum ribosome is able to bypass these uAUGs and uORFs for highly expressed genes remains a question for future research.

Active anti-tumour necrosis factor (TNF)-α immunization with the kinoid of TNF-α (TNF-K) induces polyclonal anti-TNF-α antibodies and ameliorates arthritis in human TNF-α (hTNF-α) transgenic mice (TTg). We compared the efficacy of TNF-K to that of infliximab (IFX) and of TNF-K and IFX co-administration, and evaluated whether the titres of anti-hTNF-α antibodies induced by immunization were a determinant of TNF-K efficacy. Forty-eight TTg mice received one of the following treatments: TNF-K immunization (TNF-K group); weekly IFX throughout the study duration (IFXw0-15); TNF-K plus weekly IFX for 4 weeks (TNF-K + IFX); and weekly IFX for 4 weeks (IFXw0-4); PBS. Animals were killed at week 16. Anti-hTNF-α antibody titres and clinical and histological scores were compared. All TNF-K immunized mice (TNF-K and TNF-K + IFX) produced anti-hTNF-α antibodies. Titres were higher in TNF-K versus TNF-K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = -0·78; P = 0·0001) and destruction (R = -0·67; P = 0·001). TNF-K + IFX had higher histological inflammation and destruction versus TNF-K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti-hTNF-α antibody titres identified the criterion cut-off value to discriminate most effectively between the TNF-K and TNF-K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF-α-dependent arthritis, protection from articular damage by TNF-K correlates with the titres of induced anti-hTNF-α antibodies. The co-administration of TNF-K and a short course of infliximab does not result in less articular damage versus solely TNF-K, due probably to lower anti-hTNF-α antibody production. These results are relevant for future development of active anti-TNF-α immunization in human disease.

The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.

Celiac disease (CD) can only be treated by rigorous life-long gluten-free diet (GFD). The study included 102 mothers and their CD children treated with GFD for at least two years. Frequency and cause of diet failure in children treated at present (54 children) and 10 years ago (48 children) were compared. Dietary adherence was evaluated serologically (tTG), while diet management difficulties were examined by means of a questionnaire. The study shows that one-third of patients fail to follow GFD, more often 10 years ago than now (40% vs. 26%; p < 0.05), mainly children aged 13⁻18 (54% vs. 40% now; p < 0.05). Younger children (up to 12) are less likely to abandon the diet (27% vs. 8%; p < 0.05). In this age group non-intentional diet failure prevails, while teenagers interrupt their diet intentionally (45% vs. 33%; p = ns (small population of children in this groups)). Currently, the most common causes of teenage diet failure are the absence of symptoms after consuming a small amount of gluten and, even more often, troublesome diet administration. Previously, the absence of peer acceptance prevailed. With this study we found that: 1. In West Pomerania, every fourth CD child does not follow GFD. 2. For years, teenagers have failed to follow GFD due to the absence of symptoms after consuming small amounts of gluten. 3. The incidence of non-intentional failure to follow GFD has significantly decreased over years, which indicates better dietary care.

BACKGROUND

Atopic allergy is effected by a number of environmental exposures, such as dry air and time spent outdoors, but there are few estimates of the prevalence in populations from sub-arctic areas.

OBJECTIVE

To determine the prevalence and severity of symptoms of food, inhalation and skin-related allergens and coeliac disease (CD) in the sub-arctic region of Sweden. To study the correlation between self-reported allergy and allergy test results. To estimate the heritability of these estimates.

METHODS

The study was conducted in Karesuando and Soppero in Northern Sweden as part of the Northern Sweden Population Health Study (n=1,068). We used a questionnaire for self-reported allergy and CD status and measured inhalation-related allergens using Phadiatop, food-related allergens using the F × 5 assay and IgA and IgG antibodies against tissue transglutaminase (anti-tTG) to indicate prevalence of CD.

RESULTS

The prevalence of self-reported allergy was very high, with 42.3% reporting mild to severe allergy. Inhalation-related allergy was reported in 26.7%, food-related allergy in 24.9% and skin-related allergy in 2.4% of the participants. Of inhalation-related allergy, 11.0% reported reactions against fur and 14.6% against pollen/grass. Among food-related reactions, 14.9% reported milk (protein and lactose) as the cause. The IgE measurements showed that 18.4% had elevated values for inhalation allergens and 11.7% for food allergens. Self-reported allergies and symptoms were positively correlated (p<0.01) with age- and sex-corrected inhalation allergens. Allergy prevalence was inversely correlated with age and number of hours spent outdoors. High levels of IgA and IgG anti-tTG antibodies, CD-related allergens, were found in 1.4 and 0.6% of participants, respectively. All allergens were found to be significantly (p<3 e-10) heritable, with estimated heritabilities ranging from 0.34 (F × 5) to 0.65 (IgA).

CONCLUSIONS

Self-reported allergy correlated well with the antibody measurements. The prevalence of allergy was highest in the young and those working inside. Heritability of atopy and sensitization was high. The prevalence of CD-related autoantibodies was high and did not coincide with the self-reported allergy.