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1.
Toxins (Basel) ; 13(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34564597

RESUMO

BACKGROUND: Helicobacter pylori (Hp) colonizes the human stomach and can induce gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. Clinical observations suggest a role for the Hp virulence factor cytotoxin-associated gene A (CagA) in pathogenesis. The pathogenic activity of CagA is partly regulated by tyrosine phosphorylation of C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in host cells. However, CagA differs considerably in EPIYA motifs, whose functions have been well characterized in epithelial cells. Since CagA is fragmented in immune cells, different CagA variants may exhibit undetected functions in B cells. METHODS: B cells were infected with Hp isolates and isogenic mutants expressing different CagA EPIYA variants. CagA translocation and tyrosine phosphorylation were investigated by Western blotting. Apoptosis was analyzed by flow cytometry and metabolic activity was detected by an MTT assay. RESULTS: Isogenic CagA EPIYA variants are equally well translocated into B cells, followed by tyrosine phosphorylation and cleavage. B cell apoptosis was induced in a CagA-independent manner. However, variants containing at least one EPIYA-C motif affected metabolic activity independently of phosphorylation or multiplication of EPIYA-C motifs. CONCLUSIONS: The diverse structure of CagA regulates B cell physiology, whereas B cell survival is independent of CagA.

2.
Life (Basel) ; 11(3)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803490

RESUMO

BACKGROUND: The spatiotemporal profiling of molecular transmission clusters (MTCs) using viral genomic data can effectively identify transmission networks in order to inform public health actions targeting SARS-CoV-2 spread. METHODS: We used whole genome SARS-CoV-2 sequences derived from ten European regions belonging to eight countries to perform phylogenetic and phylodynamic analysis. We developed dedicated bioinformatics pipelines to identify regional MTCs and to assess demographic factors potentially associated with their formation. RESULTS: The total number and the scale of MTCs varied from small household clusters identified in all regions, to a super-spreading event found in Uusimaa-FI. Specific age groups were more likely to belong to MTCs in different regions. The clustered sequences referring to the age groups 50-100 years old (y.o.) were increased in all regions two weeks after the establishment of the lockdown, while those referring to the age group 0-19 y.o. decreased only in those regions where schools' closure was combined with a lockdown. CONCLUSIONS: The spatiotemporal profiling of the SARS-CoV-2 MTCs can be a useful tool to monitor the effectiveness of the interventions and to reveal cryptic transmissions that have not been identified through contact tracing.

3.
Microorganisms ; 8(12)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339161

RESUMO

Helicobacter pylori infection induces a plethora of DNA damages. Gastric epithelial cells, in order to maintain genomic integrity, require an integrous DNA damage repair (DDR) machinery, which, however, is reported to be modulated by the infection. CagA is a major H. pylori virulence factor, associated with increased risk for gastric carcinogenesis. Its pathogenic activity is partly regulated by phosphorylation on EPIYA motifs. Our aim was to identify effects of H. pylori infection and CagA on DDR, investigating the transcriptome of AGS cells, infected with wild-type, ΔCagA and EPIYA-phosphorylation-defective strains. Upon RNA-Seq-based transcriptomic analysis, we observed that a notable number of DDR genes were found deregulated during the infection, potentially resulting to base excision repair and mismatch repair compromise and an intricate deregulation of nucleotide excision repair, homologous recombination and non-homologous end-joining. Transcriptome observations were further investigated on the protein expression level, utilizing infections of AGS and GES-1 cells. We observed that CagA contributed to the downregulation of Nth Like DNA Glycosylase 1 (NTHL1), MutY DNA Glycosylase (MUTYH), Flap Structure-Specific Endonuclease 1 (FEN1), RAD51 Recombinase, DNA Polymerase Delta Catalytic Subunit (POLD1), and DNA Ligase 1 (LIG1) and, contrary to transcriptome results, Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) upregulation. Our study accentuates the role of CagA as a significant contributor of H. pylori infection-mediated DDR modulation, potentially disrupting the balance between DNA damage and repair, thus favoring genomic instability and carcinogenesis.

4.
Cell Host Microbe ; 21(3): 376-389, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28279347

RESUMO

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Concentração de Íons de Hidrogênio
5.
FEBS J ; 283(2): 206-20, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26907789

RESUMO

As a result of Helicobacter pylori adhesion to gastric epithelial cells, the bacterial effector cytotoxin-associated gene A (CagA) is translocated intracellularly, and after hierarchical tyrosine phosphorylation on multiple EPIYA motifs, de-regulates cellular polarity and contributes to induction of an elongation and scattering phenotype that resembles the epithelial to mesenchymal transition (EMT). Stromelysin-1/matrix metalloproteinase-3 (MMP-3) has been reported to induce a sequence of molecular alterations leading to stable EMT transition and carcinogenesis in epithelial cells. To identify the putative role of CagA protein in MMP-3 induction, we exploited an experimental H. pylori infection system in gastric epithelial cell lines. We utilized isogenic mutants expressing CagA protein with variable numbers of EPIYA and phosphorylation-deficient EPIFA motifs, as well as cagA knockout and translocation-deficient cagE knockout strains. Increased levels of MMP-3 transcriptional activation were demonstrated by quantitative real time-PCR for strains with more than two terminal EPIYA phosphorylation motifs in CagA. MMP-3 expression in total cell lysates and the corresponding culture supernatants was associated with CagA expression and translocation and was dependent on CagA phosphorylation. A CagA EPIYA phosphorylation-dependent increase in gelatinase and caseinolytic activity was also detected in culture supernatants by zymography. A significant increase in the transcriptional activity of the mesenchymal markers Vimentin, Snail and ZEB1 and the stem cell marker CD44 was observed in the case of CagA containing phosphorylation-functional EPIYA motifs. Our data suggest that CagA protein induces EMT through EPIYA phosphorylation-dependent up-regulation of MMP-3. Moreover, no significant increase in EMT and stem cell markers was observed following infection with H. pylori strains that cannot effectively translocate CagA protein.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/patologia , Interações Hospedeiro-Patógeno , Humanos , Receptores de Hialuronatos/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Dados de Sequência Molecular , Fosforilação , Vimentina/metabolismo
6.
J Infect Dis ; 213(11): 1767-76, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26802142

RESUMO

Helicobacter pylori colonizes the human stomach and increases the risk for peptic ulcer disease and gastric carcinoma. H. pylori upregulates the expression and activity of several matrix metalloproteinases (MMPs) in cell lines and in the gastric mucosa. The aim of this study was to explore the mechanisms leading to upregulation of MMP10 in gastric epithelial cells induced by H. pylori Infection of gastric cells with H. pylori led to an increase in levels of MMP-10 messenger RNA, protein secretion, and activity. cagA knockout mutants or CagA phosphorylation-defective mutants failed to increase MMP10 expression. These results were confirmed in infection experiments with clinical isolates with known cagA status and in human gastric biopsy specimens. Treatment of cells with chemical inhibitors of the receptor tyrosine kinase EGFR and the kinase Src abrogated H. pylori-induced MMP10 expression. Inhibitors of ERK1/2 and JNK kinases abolished and significantly decreased H. pylori-induced MMP10 expression, respectively, whereas inhibition of the kinase p38 had no effect. Finally, inhibition of MMP10 expression by small interfering RNA led to a decrease in the gastric cell-invasive phenotype mediated by the infection. In conclusion, CagA-positive H. pylori strains stimulate MMP10 expression. MMP-10 modulation occurs via EGFR activation in a process that involves Src, ERK, and JNK pathways. MMP-10 may be implicated in H. pylori-mediated extracellular matrix remodeling.


Assuntos
Receptores ErbB/metabolismo , Mucosa Gástrica/enzimologia , Helicobacter pylori/patogenicidade , Sistema de Sinalização das MAP Quinases , Metaloproteinase 10 da Matriz/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Mucosa Gástrica/microbiologia , Humanos , MAP Quinase Quinase 4/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Virulência/metabolismo
7.
Helicobacter ; 20 Suppl 1: 8-16, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26372819

RESUMO

Three decades have passed since Warren and Marshall described the successful isolation and culture of Helicobacter pylori, the Gram-negative bacterium that colonizes the stomach of half the human population worldwide. Although it is documented that H. pylori infection is implicated in a range of disorders of the upper gastrointestinal tract, as well as associated organs, many aspects relating to host colonization, successful persistence, and the pathophysiological mechanisms of this bacteria still remain controversial and are constantly being explored. Unceasing efforts to decipher the pathophysiology of H. pylori infection have illuminated the crucially important contribution of multifarious bacterial factors for H. pylori pathogenesis, in particular the cag pathogenicity island (PAI), the effector protein CagA, and the vacuolating cytotoxin VacA. In addition, recent studies have provided insight into the importance of the gastrointestinal microbiota on the cumulative pathophysiology associated with H. pylori infection. This review focuses on the key findings of publications related to the pathogenesis of H. pylori infection published during the last year, with an emphasis on factors affecting colonization efficiency, cagPAI, CagA, VacA, and gastrointestinal microbiota.


Assuntos
Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Fatores de Virulência/metabolismo , Humanos
8.
Influenza Other Respir Viruses ; 8(1): 107-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24373295

RESUMO

OBJECTIVES: The genotypic analysis of human metapneumo-(HMPV) and boca-(HBoV) viruses circulating in Greece and their comparison to reference and other clinical strains. DESIGN: Genetic analysis of representative strains over three consecutive winter seasons of the years 2005-2008. SETTING: Representative positive specimens for HMPV and HBoV from paediatric patients of healthcare units and hospitals in Southern Greece with influenza-like illness or other respiratory tract infections. SAMPLE: Seven to ten positive specimens for either HMPV or HBoV from each winter period. In total, 24 specimens positive for HMPV and 26 for HBoV, respectively. MAIN OUTCOME MEASURES: Sequence diversity of HMPV and HBoV strains by sequencing the complete G and VP1/VP2 genes, respectively. RESULTS: In total, 24 HMPV strains were found to have a 92-100% nucleotide and a 85.9-100% amino acid identity. Phylogenetic analysis based on the number of amino acid differences, revealed circulation of 4 different subclusters belonging to genetic lineage B2. Similarly, analysis of 26 HBoV strains indicated that 22 clustered within genotype St2, 2 into genotype St1 and the remaining 2 formed a third cluster derived from potential recombination between different St1 genotype strains. St2 HBoV genotype was observed throughout the whole observation period whereas St1 only during the second and the third winter period. Higher levels of heterogeneity were observed between HMPV compared to HBoV strains. CONCLUSIONS: Phylogenetic analysis revealed circulation of one single lineage (B2) for HMPV viruses and predominance of St2 genotype for HBoV viruses. A possible recombination between St1 genotype strains of HBoV was observed.


Assuntos
Variação Genética , Bocavirus Humano/classificação , Metapneumovirus/classificação , Infecções por Paramyxoviridae/virologia , Infecções por Parvoviridae/virologia , Infecções Respiratórias/virologia , Adolescente , Criança , Pré-Escolar , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Feminino , Genótipo , Grécia/epidemiologia , Bocavirus Humano/genética , Bocavirus Humano/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Masculino , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Epidemiologia Molecular , Dados de Sequência Molecular , Infecções por Paramyxoviridae/epidemiologia , Infecções por Parvoviridae/epidemiologia , Filogenia , RNA Viral/genética , Infecções Respiratórias/epidemiologia , Análise de Sequência de DNA , Homologia de Sequência , Proteínas Estruturais Virais/genética
9.
Helicobacter ; 18(6): 459-67, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23714140

RESUMO

BACKGROUND: Increasing clarithromycin resistance reduces Helicobacter pylori eradication rates with conventional triple regimens. We evaluated effectiveness and safety of a 10-day-quadruple nonbismuth containing regimen, as first-line treatment or second-line treatment (after conventional triple) for H. pylori, and assessed impact of antibiotic resistance on treatment success. MATERIALS AND METHODS: Eligible patients had upper GI endoscopy and positive CLO-test, also confirmed by histology and/or culture. The eradication scheme comprised: Esomeprazole 40 mg, Metronidazole 500 mg, Amoxicillin 1000 mg, and Clarithromycin 500 mg, twice daily, for 10 days. Treatment adherence and adverse effects were recorded. Eradication was tested by (13) C-urea breath test or histology. RESULTS: One hundred and ninety out of 198 patients (115M/83F, aged 18-81, mean 52 years, 37% smokers, 27% ulcer disease) who completed the study protocol were evaluated for eradication. Adherence to treatment was 97.7% (95% CI 95.9-99.6). Six (3.2%) patients experienced severe side effects and discontinued treatment. Intention to treat and per protocol analysis in first line was 91.5% (95% CI 86.2-94.8) and 95% (95% CI 90.4-97.4) and in second line was 60.6% (95% CI 43.6-75.3) and 64.5% (95% CI 46.9-78.8), respectively. Antibiotic susceptibility tests were performed in 106 of 124 (85%) patients who gave consent. Among them 42 (40%) harbored clarithromycin resistant strains. Eradication rates were significantly higher in sensitive and single clarithromycin or metronidazole resistant (37/37, 100% and 43/47, 91%) than in dual resistant strains (12/22, 55%) (p < .0001). Specifically, concomitant regimen eradicated 7/10, 70% of dual resistant strains as first-line treatment and 5/12, 42% as second-line treatment. Multivariate analysis showed that dual resistance was the only independent significant predictor of treatment failure. CONCLUSIONS: The 10-days "concomitant" regimen is effective and safe first-line H. pylori treatment, in a high clarithromycin resistance area, although dual antibiotic resistance may compromise its effectiveness.


Assuntos
Amoxicilina/uso terapêutico , Antibacterianos/uso terapêutico , Claritromicina/uso terapêutico , Farmacorresistência Bacteriana , Esomeprazol/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Metronidazol/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Combinada , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
10.
PLoS One ; 8(2): e56291, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23409168

RESUMO

CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1(-/-) mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Interleucina-8/metabolismo , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Helicobacter pylori/fisiologia , Humanos , Interleucina-8/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Transcricional/efeitos dos fármacos
11.
Helicobacter ; 18(3): 229-41, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23190444

RESUMO

BACKGROUND: CagA protein of Western origin Helicobacter pylori isolates contains at its carboxyl-terminal end repeating types of EPIYA motifs, depending on the surrounding sequence, which dictate hierarchic tyrosine phosphorylation. To produce, in an isogenic background, mutant strains expressing CagA protein with variable numbers of EPIYA-C terminal motifs, we have adopted a mutagenesis assay using a megaprimer approach. MATERIALS AND METHODS: The H. pylori P12 reference strain containing two terminal EPIYA-C motifs was utilized. Initially, we cloned, full-length cagA gene, next to the Campylobacter jejuni kanamycin-resistance cassette, followed by the 1200-bp region located immediately after cagA gene (metacagA region). Then, we generated a megaprimer consisting of three consecutive copies of the EPIYA-C coding sequence of cagA gene, followed by the 140-bp region of the cagA genomic sequence present immediately after the second EPIYA-C repeat. We utilized these two products to perform a QuikChange mutagenesis assay and were able to obtain all desired combinations of EPIYA-C motifs, followed by Kan(r) cassette and metacagA region. These constructions were used to perform natural transformation of the P12 parental strain, by directional homologous recombination. RESULTS: We produced isogenic H. pylori strains that express CagA with variable number of EPIYA-C motifs (AB, ABC, ABCCC) and their phosphorylation-deficient counterparts. They exhibited similar growth characteristics to the parental strain, adhered equally well to gastric cells and successfully translocated CagA, following pilus induction. CONCLUSIONS: Our method can be used in other cases where highly repetitive sequences need to be reproduced.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Helicobacter pylori/genética , Mutagênese , Recombinação Genética , Motivos de Aminoácidos , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Linhagem Celular , Meios de Cultura , Células Epiteliais/microbiologia , Mucosa Gástrica/citologia , Mucosa Gástrica/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Sequências Repetitivas de Aminoácidos , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência
12.
J Med Microbiol ; 61(Pt 4): 559-566, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22160312

RESUMO

Helicobacter pylori infection is often acquired in childhood and can persist for life. Previous studies in adult patients have shown that H. pylori isolates from North American and European hosts express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes, while Asian strains have the capacity to express type 1 Le(a) and Le(b) structures. In order to understand the influence of environmental and host factors on the expression of Le antigens, we analysed 50 Greek H. pylori isolates from symptomatic children. Both CagA-positive and -negative strains were evaluated. The expression of Le antigens was determined by whole-cell indirect ELISA (WCE), and LPS profiles were assessed by gel electrophoresis and immunoblotting. Occurrence of Le(x) and/or Le(y) antigens was confirmed in 35 of the isolates (70 %) while 15 of the isolates were non-typable. It was found that 11 of the paediatric isolates had the propensity to express type 1 Le(b) blood-group antigen (22 %), a feature relatively uncommon in H. pylori isolates from adults. One strain expressed both Le(b) and Le(a) antigens. The majority of the isolates (49/50, 98 %) expressed α1,6-glucan, an antigenic non-Le determinant present in the outer core region of H. pylori LPS. All Le(x)- and Le(y)-expressing strains also carried a functional cag pathogenicity island-encoding a type IV secretion system, capable of translocating CagA protein, as well as the vacAs1 allele, suggesting that Le(x) and Le(y) epitopes may aid the persistence of more aggressive strains. No association between bacterial virulence characteristics and the histopathological observations was evident.


Assuntos
Antígenos de Bactérias/metabolismo , Glucanos/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Adolescente , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Criança , Pré-Escolar , Mucosa Gástrica/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Glucanos/genética , Grécia/epidemiologia , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética
13.
Infect Genet Evol ; 11(8): 1899-905, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21871583

RESUMO

Helicobacter pylori infection is associated with gastric cancer (GC). The highest incidence rates have been described in Asia, but regional variations exist that do not match the distribution of infection prevalence rates. The aim of the study was to examine the possible contribution of H. pylori virulence factors to geographic differences in the incidence of GC across East and Southeast Asia. We studied 66 isolates from Cambodian patients that had previously been assigned to two genetic populations based on sequences of seven housekeeping genes, namely hpEurope (n = 34, 51.5%) and hpEastAsia, subpopulation hspEAsia (n = 32, 48.5%). These strains were characterized with respect to vacA polymorphism and cagA status by PCR, and the CagA C-terminal region was sequenced. We also sequenced the complete cagA gene from 10 hpEurope and 10 hspEAsia strains chosen at random. The cagA gene was present in 92.4% of the 66 isolates and was mainly of Western type (n = 36, 59.0%). hspEAsia strains carrying East-Asian CagA and the m1-type vacA allele (15.2%) were less frequent among the 66 Cambodian isolates than reported in East Asian countries, a finding that might partly explain the intermediate incidence of GC in Cambodia, and by extension, in Southeast Asia (except for Vietnam). The observed high prevalence of s1a alleles (34.4%) and Western CagA (28.1%) among hspEAsia strains indicates frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains. This expansion might have severe consequences for individual disease outcome.


Assuntos
Alelos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Helicobacter pylori/genética , Adolescente , Adulto , Idoso , Camboja/epidemiologia , Emigração e Imigração , Europa (Continente)/epidemiologia , Extremo Oriente/epidemiologia , Feminino , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Variação Genética , Infecções por Helicobacter/complicações , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , Análise de Sequência de DNA , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/microbiologia , Adulto Jovem
14.
Helicobacter ; 15(3): 227-32, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20557365

RESUMO

BACKGROUND: Rapid urease test (CLO-test) is an inexpensive and quick method for diagnosis of Helicobacter pylori infection with controversial results in children. We evaluated the performance of CLO-test in relation to endoscopic and histological findings in children with H. pylori infection. MATERIALS AND METHODS: We studied the medical records of children with H. pylori infection who were diagnosed between 1989 and 2009. Noninfected children were used as controls. H. pylori infection was defined by positive culture or by two other positive tests (histology and CLO-test, or urea breath test when a single test was positive). All children had histology together with CLO-test. Tissue culture was performed whenever possible. RESULTS: Five hundred thirty infected children (10.4 +/- 3.0 years) and 1060 controls (7.3 +/- 4.4 years) were studied. Sensitivity of CLO-test was 83.4% (95% CI, 79.9-86.3%), of culture 84.6% (95% CI, 78.7-89.1%), of histology 93.2% (95% CI, 90.7-95.1%), and specificity 99% (95% CI, 98.2-99.4%), 100%, and 100% respectively. CLO-test positivity was correlated with higher bacterial density (p < .001), activity (p < .001) and severity of gastritis (p < .01), older age (p < .01), and the presence of antral nodularity (p < .001). When CLO-test was positive, the concordance with histology and culture was high (95.5 and 89.2% respectively), whereas low concordance was observed when CLO-test was negative (17.05 and 45.83% respectively). CONCLUSIONS: CLO-test had lower sensitivity and comparable specificity with histology. Both tests should be performed concurrently to accurately diagnose H. pylori infection in children.


Assuntos
Endoscopia Gastrointestinal , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/enzimologia , Urease/análise , Adolescente , Testes Respiratórios , Criança , Pré-Escolar , Feminino , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/metabolismo , Histocitoquímica , Humanos , Masculino , Sensibilidade e Especificidade
15.
J Clin Microbiol ; 48(6): 2237-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20392907

RESUMO

Polymorphic variability in Helicobacter pylori factors CagA and VacA contributes to bacterial virulence. The presence of one CagA EPIYA-C site is an independent risk factor for gastroduodenal ulceration (odds ratio [OR], 4.647; 95% confidence interval [CI], 2.037 to 10.602), while the presence of the vacA i1 allele is a risk factor for increased activity (OR, 5.310; 95% CI, 2.295 to 12.287) and severity of gastritis (OR, 3.862; 95% CI, 1.728 to 8.632).


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Polimorfismo Genético , Fatores de Virulência/genética , Adulto , Idoso , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Úlcera Péptica/microbiologia , Análise de Sequência de DNA , Índice de Gravidade de Doença
16.
J Clin Microbiol ; 47(8): 2426-34, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19535517

RESUMO

The presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pylori isolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacA isotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pylori isolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3' variable region of the cagA gene as well as vacA isotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pylori colonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacA genotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Polimorfismo Genético , Adolescente , Adulto , Criança , Pré-Escolar , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Gastrite/patologia , Grécia , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Estudos Prospectivos , Análise de Sequência de DNA , Índice de Gravidade de Doença , Estatística como Assunto , Virulência , Fatores de Virulência/genética
17.
Int J Food Microbiol ; 133(1-2): 141-7, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19515446

RESUMO

Streptococcus macedonicus ACA-DC 198, a bacteriocin producer isolated from Greek Kasseri cheese, was used in a series of in vitro and in vivo experiments in order to evaluate its pathogenic potential. The strain was examined in vitro for haemolytic activity, antibiotic resistance and presence of pathogenicity genes encountered in Streptococcus pyogenes. Subsequently, the strain was orally administered to mice (8.9 log cfu daily), continuously over a period of 12 weeks, in order to ascertain the effects of its long term consumption on animal health and gastric inflammation. S. macedonicus ACA-DC 198 was found to be non-haemolytic and sensitive to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin, tetracycline, and vancomycin, with the only resistance observed against kanamycin. PCR amplification and DNA-DNA hybridization did not reveal the presence of any of the S.pyogenes pathogenicity genes examined, namely emm, scpA, hasA, speB, smez2, speJ, sagAB, hylA, ska, speF, speG, slo, hylP2 and mga. In the mouse study, no detrimental effects were observed in the behaviour, general well being, weight gain and water consumption of the animals receiving S. macedonicus ACA-DC 198. Histologic analysis showed no evidence of inflammation in the stomach of the animals receiving S. macedonicus ACA-DC 198, while faecal microbiological analysis revealed that the strain retained its viability passing through the mouse gastrointestinal tract. Finally, no evidence of translocation to the liver, spleen and mesenteric lymph nodes was observed. In conclusion, none of the examined virulence determinants were detected in S. macedonicus ACA-DC 198 and its long term, high dosage oral administration did not appear to induce any pathogenic effect in mice.


Assuntos
Genes Bacterianos , Streptococcus/patogenicidade , Animais , Antibacterianos/farmacologia , Translocação Bacteriana , Bacteriocinas/biossíntese , Queijo/microbiologia , Resistência Microbiana a Medicamentos , Microbiologia de Alimentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Fenótipo , Streptococcus/efeitos dos fármacos , Streptococcus/genética , Streptococcus/fisiologia , Virulência/genética
18.
J Clin Microbiol ; 45(2): 488-95, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17151214

RESUMO

Cytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pylori pathogenesis, and therefore determination of these motifs in H. pylori clinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pylori isolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagA negative by cagA-specific PCR, empty-site cagA PCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylori subclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagA screening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Criança , Feminino , Grécia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
19.
Clin Diagn Lab Immunol ; 12(12): 1378-86, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16339060

RESUMO

In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P=0.038) and neutrophilic (P=0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P=0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P=0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.


Assuntos
Mucosa Gástrica/imunologia , Gastrite/terapia , Infecções por Helicobacter/terapia , Helicobacter pylori , Lactobacillus , Animais , Anticorpos Antibacterianos/fisiologia , Contagem de Colônia Microbiana , Seguimentos , Mucosa Gástrica/microbiologia , Gastrite/imunologia , Gastrite/microbiologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Imunoglobulina G/fisiologia , Interleucina-8/metabolismo , Linfócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos
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