Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis

Abstract. Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1–16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Serological classification and epitope specificity of <Emphasis Type="Italic">Proteus vulgaris</Emphasis> TG 251 from <Emphasis Type="Italic">Proteus</Emphasis> serogroup O65

INTRODUCTION: Proteus rods are currently subdivided into five named species, i.e. Proteus mirabilis, P. vulgaris, P. penneri, P. hauseri, and P. myxofaciens, and three unnamed Proteus genomospecies 4 to 6. Based on the serospecificity of the lipopolysaccharide (LPS; O-antigen), strains of P. mirabilis and P. vulgaris were divided into 49 O-serogroups and 11 additional O-serogroups were proposed later. About 15 further O-serogroups have been proposed for the third medically important species, P. penneri. Here the serological classification of P. vulgaris strain TG 251, which does not belong to these serogroups, is reported. Serological investigations also allowed characterization of the epitope specificity of its LPS. MATERIALS AND METHODS: Purified LPSs from five Proteus strains were used as antigens in enzyme immunosorbent assay (EIA), SDS/PAGE, and Western blot and alkali-treated LPSs in the passive immunohemolysis (PIH) test, inhibition of PIH and EIA, and absorption of the rabbit polyclonal O-antisera with the respective LPS. RESULTS: The serological studies of P. vulgaris TG 251 LPS indicated the identity of its O-polysaccharide with that of P. penneri O65. The antibody specificities of P. vulgaris TG 251 and P. penneri O65 O-antisera, were described. CONCLUSIONS: P. vulgaris TG 251 was classified to the Proteus O65 serogroup. Two disaccharide-associated epitopes present in P. vulgaris TG 251 and P. penneri O65 LPSs are suggested to be responsible for cross-reactions with three heterologous Proteus strains.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis>-infection-associated CpG island hypermethylation in the stomach and its possible association with Polycomb repressive marks

Helicobacter pylori infection can induce CpG island (CGI) hypermethylation in gastric mucosa. Recently, genes occupied by Polycomb proteins in embryonic stem cells were shown to be vulnerable to aberrant DNA hypermethylation in cancers. To explore the relationship between H. pylori infection and DNA methylation changes in neoplastic and non-neoplastic stomach, we analyzed 25 CGIs and repetitive DNA elements from 82 chronic gastritis and 69 gastric carcinomas. Twenty-three CGIs showed significantly higher methylation levels in H. pylori-negative gastric carcinoma (n = 28) than in H. pylori-negative chronic gastritis (n = 39; P < 0.05), indicating cancer-associated methylation. Eight CGIs exhibited significantly higher methylation levels in H. pylori-positive chronic gastritis (n = 43) than in H. pylori-negative chronic gastritis (n = 39; P < 0.05). Six CGIs showed both cancer-associated and H. pylori-associated hypermethylation. Six (75%) of the eight H. pylori-associated hypermethylated genes contained at least one of three repressive marks (Suz12 occupancy, Eed occupancy, histone H3 K27 trimethylation), whereas 31% of the remaining cancer-associated hypermethylated genes had at least one mark. The findings suggest that H. pylori infection strongly induces CGI hypermethylation in gastric epithelial cells and that susceptibility to H. pylori-induced DNA hypermethylation may be determined by Polycomb repressive marks in stem or progenitor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Supported by the Korea Research Foundation Grant (MOEHRD; KRF-2005-041-E00081; G.H.K.) and by the second stage Brain Korea 21 Project.     

Multiplex PCR and quality control of <Emphasis Type="Italic">Epinotia aporema</Emphasis> granulovirus production

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1–3 sets of selected 5–6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.     

Interleukin-6 polymorphism and Helicobacter pylori infection in Brazilian adult patients with chronic gastritis

Helicobacter pylori is recognised as the most common cause of chronic active gastritis and this bacterium is also an important pathogenic factor in peptic ulcer disease. The biological factors that influence clinical outcome in H. pylori infection have been extensively studied. In addition to immunological factors in the host, bacterial virulence determinants in H. pylori strains are likely to play a crucial role in gastric cancer development. Singlenucleotide polymorphisms at the 5' flanking region of the interleukin (IL)-6 gene promoter (G or C at -174 base) have been identified and individuals with the G allele at position -174 have been shown to produce higher levels of IL-6 than those with the C/C genotype. The mucosal levels of IL-6 were reported to be increased in H. pylori-associated gastritis. The present study was conducted to examine any relationship between inflammatory cytokine polymorphisms and the inflammatory process in mucosa infected by H. pylori. In our study we did not find any association between the C and G alleles in adult patients with chronic gastritis and inflammatory process in gastric mucosa.     

Bacterial DNA Induces the <Emphasis Type="Italic">Complement</Emphasis> System Activation in Serum and Ascitic Fluid from Patients with Advanced Cirrhosis

Translocation of intestinal bacteria to ascitic fluid is, probably, the first step in the development of spontaneous bacterial peritonitis in patients with cirrhosis. Proteins of the complement system are soluble mediators implicated in the host immune response to bacterial infections and its activation has been traditionally considered to be an endotoxin-induced phenomenon. The aim of this study was to compare the modulation of these proteins in response to the presence of bacterial DNA and/or endotoxin in patients with advanced cirrhosis and ascites in different clinical conditions. Groups I and II consisted of patients without/with bacterial DNA. Group III included patients with spontaneous bacterial peritonitis and Group IV with patients receiving norfloxacin as secondary long-term prophylaxis of spontaneous bacterial peritonitis. Serum and ascitic fluid levels of endotoxin and truncated residues of the complement system were measured by ELISA. The complement system is triggered in response to bacterial DNA, as evidenced by significantly increased levels of C3b, membrane attack complex, and C5a in patients from Groups II and III compared with patients without bacterial DNA (Group I) and those receiving norfloxacin (Group IV). Gram classification did not further differentiate the immune response between patients within groups II and III, even though endotoxin levels were, as expected, significantly higher in patients with bacterial DNA from gram-negative microorganisms. The complement protein activation observed in patients with bacterial DNA in blood and ascitic fluid is indistinguishable from that observed in patients with spontaneous bacterial peritonitis and may occur in an endotoxin-independent manner.     

MUC 1 mucin content in gastric juice of duodenal ulcer patients: effect of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> eradication therapy

It has been suggested that Helicobacter pylori can interact via carbohydrate structures with gastric mucins. Particularly, the Lewis b structures of the secretory MUC 5AC mucin are considered to be putative receptors for bacterial adhesins. Also the epithelial MUC 1 mucin is implicated by some authors to have a major role in the mechanism of infection. The main objective of our study was to evaluate MUC 1 mucin levels in human gastric juice before and at the end of eradication therapy. Any possible changes could suggest the participation of MUC 1 in H. pylori infection. We assume that the amount of the soluble form of MUC 1 exfoliated to the juice correlates with MUC 1 expressed on epithelial cells. Gastric juice samples of 14 duodenal ulcer patients infected with H. pylori were assayed before and at the end of eradication. In all samples, DNA content was determined. Mucin fractions were isolated by gel exclusion chromatography. High molecular mass material containing MUC 1 was subjected to 4%–12% polyacrylamide gradient gels, electrotransfer to Immobilon P and immunodetection. In 12 infected patients (86%), the initial low level of MUC 1 mucin in gastric juice increased at the end of eradication. In comparison to the infected patients, neutral carbohydrate and DNA content in gastric juice diminished after treatment. Our results indicate that the bacterium affects the soluble form of MUC 1 mucin, thus suggesting a likely role of this mucin in the course of H. pylori infection.     

Mutation and haplotype analyses of the <Emphasis Type="Italic">MUT</Emphasis> gene in Japanese patients with methylmalonic acidemia

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of l-methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding – that a limited number of mutations account for most of the mutations in Japanese mut MMA patients – is in contrast with results of a previous study in Caucasian patients.     

Serological and structural characterization of the O-antigens of the unclassified <Emphasis Type="Italic">Proteus mirabilis</Emphasis> strains TG 83, TG 319, and CCUG 10700 (OA)

INTRODUCTION: Lipopolysaccharide (endotoxin, LPS) is an important potential virulence factor of Proteus rods. The serological specificity of the bacteria is defined by the structure of the O-polysaccharide chain (O-antigen) of the LPS. Until now, 76 O-serogroups have been differentiated among Proteus strains. MATERIALS AND METHODS: LPSs were isolated from Proteus mirabilis TG 83, TG 319, and CCUG 10700 (OA) strains by phenol/water extraction. Antisera were raised by immunization of rabbits with heat-killed bacteria. Serological investigations were performed using enzyme immunosorbent assay, passive immunohemolysis, inhibition of both assays, absorption of antisera, and Western blot. RESULTS: The cross-reactive epitope shared by these strains and P. penner O72a,O72b is located on the O-polysaccharide and is most likely associated with an alpha-D-Glcp-(1-->6)-beta-D-GalpNAc disaccharide fragment. The serological data indicated the occurrence of two core types in the LPSs studied, one characteristic for P. mirabilis TG 319 and CCUG 10700 (OA) and the other for P. mirabilis TG 83 and O57. CONCLUSIONS: The serological and structural data showed that P. mirabilis TG 83, TG 319, CCUG 10700 (OA), and O57 have the same O-antigen structure and could be qualified to the Proteus O57 serogroup.     

<Emphasis Type="Italic">SCN1A,</Emphasis><Emphasis Type="Italic">SCN1B</Emphasis>, and <Emphasis Type="Italic">GABRG2</Emphasis> gene mutation analysis in Chinese families with generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+; MIM#604233) is a familial epilepsy syndrome characterized by phenotypic and genetic heterogeneity. It was associated with mutations in the neuronal voltage-gated sodium channel subunit gene (SCN1A, SCN2A, SCN1B) and ligand-gated gamma aminobutyric acid receptors genes (GABRG2, GABRD). We investigated the roles of SCN1A, SCN1B, and GABRG2 mutations in the etiology of Chinese GEFS+ families. Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood lymphocytes of 23 probands and their family members. The sequences of SCN1A, SCN1B, and GABRG2 genes were analyzed by polymerase chain reaction (PCR) and direct sequencing. The major phenotypes of affected members in the 23 GEFS+ families exhibited FS and FS+, whereas rare phenotypes afebrile generalized tonic-clonic seizures (AGTCS), myoclonic-astatic epilepsy (MAE), and partial seizures were also observed. A novel SCN1A mutation, p.N935H, was identified in one family and another novel mutation in GABRG2, p.W390X, in another family. However, no SCN1B mutation was identified. The combined frequency of SCN1A, SCN1B, and GABRG2 mutations was 8.7% (2/23), extending the distribution of SCN1A and GABRG2 mutations to Chinese GEFS+ families. There were still unidentified genes contributing to the pathogenesis of GEFS+.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

<Emphasis Type="Italic">MECP2 </Emphasis>and <Emphasis Type="Italic">CDKL5</Emphasis> gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.