In this chapter, we describe a fluorescence in vivo hybridization (FIVH) protocol, using nucleic acid probes, for the detection of the bacterium Helicobacter pylori in the gastric mucosa of an ...infected C57BL/6 mouse model. This protocol should be easily extended to other microorganisms not only as a way to identify in vivo important microorganisms and their patterns of distribution within specific or at different anatomic sites, but also to better understand interaction mechanisms involving the microbiome and the human body.
•Helicobacter pylori infection causes mtDNA mutations and a decrease of mtDNA content.•Decreased mtDNA fidelity correlates with decreased electron chain activity.•H. pylori infection targets mtDNA, ...in part, by inhibiting mtDNA repair activity.
Helicobacter pylori infection is an important factor for the development of atrophic gastritis and gastric carcinogenesis. However, the mechanisms explaining the effects of H. pylori infection are not fully elucidated. H. pylori infection is known to induce genetic instability in both nuclear and mitochondrial DNA of gastric epithelial cells. The mutagenic effect of H. pylori infection on nuclear DNA is known to be a consequence, in part, of a down-regulation of expression and activity of major DNA repair pathways. In this study, we demonstrate that H. pylori infection of gastric adenocarcinoma cells causes mtDNA mutations and a decrease of mtDNA content. Consequently, we show a decrease of respiration coupled ATP turnover and respiratory capacity and accordingly a lower level and activity of complex I of the electron transport chain. We wanted to investigate if the increased mutational load in the mitochondrial genome was caused by down-regulation of mitochondrial DNA repair pathways. We lowered the expression of APE-1 and YB-1, which are believed to be involved in mitochondrial base excision repair and mismatch repair. Our results suggest that both APE-1 and YB-1 are involved in mtDNA repair during H. pylori infection, furthermore, the results demonstrate that multiple DNA repair activities are involved in protecting mtDNA during infection.
, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the ...gastric mucosa,
continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that
targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection,
induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the
virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell-cell and cell-matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of
-host interactions and associated diseases. Additionally, it raises awareness for potential interference between
infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.
Background
Polymorphisms in inflammation-related genes have been associated with a risk of gastric carcinoma (GC). However, the biological mechanisms underlying these associations are still elusive. ...Our objective was to determine whether chronic inflammation-associated IL1Β signalling, as seen in the context of
Helicobacter pylori
infection, could be linked to gastric carcinogenesis by modulating the behaviour of gastric epithelial cells.
Methods
The effect of IL1B was assessed by studying the expression and activation status of the IL1Β-activated transcription factors C/EBPβ and CREB in GC cell lines. Interaction between CREB and C/EBPβ was explored through interference RNA, chromatin immunoprecipitation and chemical inhibition. CREB and C/EBPβ expression was analysed in 66 samples of primary GC and in normal gastric mucosa. GC cell growth was analysed in vitro by BrdU incorporation and in vivo employing a chicken embryo chorioallantoic membrane model.
Results
We found that IL1B regulates the expression/activation status of both C/EBPβ and CREB in GC cells through an ERK1/2-dependent mechanism. Our results show that CREB is a direct transactivator of
CEBPB
, acting as an upstream effector in this regulatory mechanism. Furthermore, we found CREB to be overexpressed in 94 % of GC samples and significantly associated with C/EBPβ expression (
P
< 0.05). Finally, we demonstrated both in vitro and in vivo that CREB can mediate IL1B-induced GC cell proliferation.
Conclusions
Our results support the hypothesis that the effect of chronic inflammation on gastric carcinogenesis, as seen in the context of genetically susceptible individuals infected with
Helicobacter pylori
, includes the modulation of signalling pathways that regulate survival mechanisms in epithelial cells.
Summary
IL1B is able to increase the expression/activation status of CREB and its target gene C/EBPβ, which are mandatory for GC cell survival. Our results may help inform new strategies for the prevention and treatment of GC, including the control of chronic inflammation.
The human gastrointestinal tract harbors approximately 100 trillion microorganisms with different microbial compositions across geographic locations. In this work, we used RNASeq data from stomach ...samples of non-disease (164 individuals from European ancestry) and gastric cancer patients (137 from Europe and Asia) from public databases. Although these data were intended to characterize the human expression profiles, they allowed for a reliable inference of the microbiome composition, as confirmed from measures such as the genus coverage, richness and evenness. The microbiome diversity (weighted UniFrac distances) in gastric cancer mimics host diversity across the world, with European gastric microbiome profiles clustering together, distinct from Asian ones. Despite the confirmed loss of microbiome diversity from a healthy status to a cancer status, the structured profile was still recognized in the disease condition. In concordance with the parallel host-bacteria population structure, we found 16 human loci (non-synonymous variants) in the European-descendent cohorts that were significantly associated with specific genera abundance. These microbiome quantitative trait loci display heterogeneity between population groups, being mainly linked to the immune system or cellular features that may play a role in enabling microbe colonization and inflammation.
Helicobacter pylori is the major triggering factor for gastric carcinoma, but only a small proportion of infected patients develop this disease. Differences in virulence observed among H. pylori ...strains, namely in the vacuolating cytotoxin vacA gene, may contribute to this discrepancy. Infection with vacA s1, i1 and m1 strains increases the risk for progression of gastric premalignant lesions and for gastric carcinoma. However, in East Asian countries most of the H. pylori strains are vacA s1, regardless of the patients' clinical status, and the significance of the vacA i1 and m1 genotypes for gastric carcinoma in this geographic area remains to be fully elucidated. The aim of the present study was to investigate this relationship in 290 patients from Macau, China. Using very sensitive and accurate genotyping methods, we detected infection with vacA i1 and with vacA m1 strains in, respectively, 85.2% and 52.6% of the patients that were infected with single genotypes. The prevalence of cagA-positive strains was 87.5%. No significant associations were observed between vacA genotypes or cagA and gastric carcinoma. It is worth noting that 37.5% of the infected patients had coexistence of H. pylori strains with different vacA genotypes. Additional studies directed to other H. pylori virulence factors should be performed to identify high risk patients in East Asia.
ABSTRACT Objective: To evaluate nurse safety culture in a teaching hospital, as well as to verify differences in the safety culture dimensions between services. Method: cross-sectional, quantitative ...study, conducted from October to December 2015, in a university hospital. The instrument Hospital Survey on Patient Safety Culture was applied. Results: A total of 195 nurses from four different services participated in the study. Significant difference between services were identified for five dimensions of safety culture: organizational learning (P=0.012); return of information and communication about error (P=0.014); management support for patient safety (P=0.001); general perceptions about patient safety (P=0.005); and frequency of event notification (P=0.003). Conclusion: The medical clinic service had the highest statistical difference between the dimensions. These evaluations allow managers to identify the differences between the same hospital’s services, serving as a warning and assisting in the services’ improvement.
RESUMEN Objetivo: Analizar la cultura de seguridad de los enfermeros de un hospital escuela y examinar las diferencias de los aspectos de esa cultura entre los servicios realizados. Método: Estudio transversal y cuantitativo, realizado de octubre a diciembre de 2015, en un hospital universitario. Se utilizó el instrumento Hospital Survey on Patient Safety Culture. Resultados: Participaron 195 enfermeros de cuatro servicios. Se identificó una diferencia significativa entre los servicios en los cinco aspectos de la cultura de seguridad: el aprendizaje organizacional (p = 0,012); la información de retorno y la comunicación con respecto al error (p = 0,014); el soporte de la gestión para la seguridad del paciente (p = 0,001); las percepciones generales sobre la seguridad del paciente (p = 0,005); y la frecuencia de comunicación de eventos (p = 0,003). Conclusión: El servicio en clínica médica presentó una mayor diferencia estadística entre los aspectos identificados. Estas evaluaciones pueden permitir que los gestores identifiquen las diferencias entre los servicios del mismo hospital, sirviendo como advertencia y perfeccionando los servicios.
RESUMO Objetivo: Avaliar a cultura de segurança dos enfermeiros de um hospital de ensino e verificar as diferenças das dimensões dessa cultura entre os serviços. Método: Estudo transversal e quantitativo, realizado em outubro a dezembro de 2015, em um hospital universitário. Foi aplicado o instrumento Hospital Survey on Patient Safety Culture. Resultados: Participaram 195 enfermeiros de quatro serviços. Identificou-se diferença significativa entre os serviços para cinco dimensões de cultura de segurança: aprendizado organizacional (p = 0.012); retorno da informação e comunicação a respeito de erro (p = 0.014); suporte da gestão para segurança do paciente (p = 0.001); percepções gerais sobre segurança do paciente (p = 0.005); e frequência da notificação de eventos (p = 0.003). Conclusão: O serviço de clínica médica apresentou maior diferença estatística entre as dimensões. Essas avaliações permitem que os gestores identifiquem as diferenças entre os serviços do mesmo hospital, servindo de alerta e aperfeiçoando os serviços.
The continuous characterization of genome-wide diversity in population and case-cohort samples, allied to the development of new algorithms, are shedding light on host ancestry impact and selection ...events on various infectious diseases. Especially interesting are the long-standing associations between humans and certain bacteria, such as the case of
, which could have been strong drivers of adaptation leading to coevolution. Some evidence on admixed gastric cancer cohorts have been suggested as supporting
-
coevolution, but reliable experimental data that control both the bacterium and the host ancestries are lacking. Here, we conducted the first in vitro coinfection assays with dual human- and bacterium-matched and -mismatched ancestries, in African and European backgrounds, to evaluate the genome wide gene expression host response to
. Our results showed that: (1) the host response to
infection was greatly shaped by the human ancestry, with variability on innate immune system and metabolism; (2) African human ancestry showed signs of coevolution with
while European ancestry appeared to be maladapted; and (3) mismatched ancestry did not seem to be an important differentiator of gene expression at the initial stages of infection as assayed here.
H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative ...therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.
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