New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

Supported by Grants from the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal), No. PPCDT/SAL-IMI/57297/2004 and No. PTDC/BIM-MEC/1051/2012; The Swedish Cancer foundation; The Swedish Research Council, No. K2010-79X-21372-01-3; Forska utan djurforsok, Animal Free Research; and by Research fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia (Portugal).AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Le(x)), Le(y) and Le(a) and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Le(x) and Le(a) glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions. CONCLUSION: These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.publishersversionpublishe

Ulcerogenic Helicobacter pylori Strains Isolated from Children: A Contribution to Get Insight into the Virulence of the Bacteria

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA “on” status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors

Microbiologic quality of an untreated water sample analyzed by a novel DNA chip for simultaneous detection of microorganisms

Consumption of contaminated drinking water heavily contributes to the burden of gastrointestinal waterborne diseases. Conventional detection methodologies present several shortcomings, such as indirect measure of indicator species, low throughput and time consume. DNA chips have the potential to serve as surveillance systems for the simultaneous detection of pathogens overcoming these limitations. We have developed a DNA chip for simultaneous detection of multiple waterborne pathogens. Species specific DNA probes were implemented on a microarray, for microorganism detection. Present study reports the results of one untreated water sample analyzed by conventional methods and by the DNA chip. The results were concordant for the mandatory organisms (total coliforms, <em>Escherichia coli</em>, fecal enterococci) using both methods, reinforcing the utility and proof-ofconcept of the DNA chip. However, it is necessary a prior enrichment in a culture medium in order to obtain a positive signal using the DNA chip. The DNA chip may be a valuable distinctive tool for waterborne pathogens detectio

Identification of proteins found to be differentially expressed in <i>H. pylori</i> strains from PUD patients (n = 5) compared to NUD patients (n = 5).

<p>“Comparison to 2DE databases” indicates spots for which identification was done by finding the corresponding spot on the standard <i>H. pylori</i> 2DE gel from the database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026265#pone.0026265-Max1" target="_blank">[14]</a>. All of the others were identified by PMF analysis. For each spot the average of the % Vol ± S.D. is indicated. In the Variation (DU/NUD) column: ↑, proteins for which the average of the % Vol of spots on DU 2DE maps was higher than that on NUD 2DE maps; ↓, proteins for which the average of the % Vol of spots on DU 2DE maps was lower than that on NUD 2DE maps; ↔, proteins that were shifted right in the DU 2DE-maps regarding their position on the NUD 2DE-maps. In the Variation (GU/NUD) column: ↑, proteins for which the average of the % Vol of spots on GU 2-DE maps was higher than that on the DU 2DE maps; ↓, proteins for which the average of the % Vol of spots on GU 2DE maps was lower than that on DU 2DE maps;  = , proteins showing the same pattern of expression observed for the <i>H. pylori</i> strains associated with DU. Asterisks indicate statistical significant results (<i>p</i><0.05). ¹Indicates the identified ORF in the 26695 reference strain.</p

Profile of the soluble proteome of the 10 studied pediatric <i>H. pylori</i> strains.

<p>These include five strains associated with NUD (173/00, 207/99, 228/99, 655/99 and 1786/05), four from patients affected by DU (1089/03, 1152/04, 1198/04 and 1846/05) and a strain associated with GU (499/02). Proteins from the total biomass recovered from a 24 h grown plate were separated by 2DE, <i>i.e.</i>, first in a non-linear pH 3–11 and than in a 7–16% (w/v) SDS-PAGE. After CBB-staining, gels were analyzed using ImageMaster™ 2-D Platinum software. Black arrow indicates spots that were more abundant in PUD (DU+GU) strains when compared to NUD strains; Dotted arrow indicates spots that were less abundant in PUD (DU+GU) strains when compared to NUD strains; Dashed arrow indicates the spot that was shifted right in DU strains when compared to NUD strains. Spot numbers indicated in the 2DE maps are the same as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026265#pone-0026265-t002" target="_blank">Table 2</a>.</p

Impact of the two isogenic groups of pediatric <i>H. pylori</i> strains on the NCI-N87 cells morphology.

<p>NCI-N87 cells grown to 80–90% confluence were co-cultured for 24 h with the pool of five isogenic NUD-associated <i>H. pylori</i> strains (NUD) and in parallel with the pool of five isogenic PUD strains (PUD). The control corresponds to non-infected NCI-N87 cells. A), B), and C) light microscopy observations. D), E), and F) immunodetection of the microtubule network (green) (1∶1000 α-tubulin antibody plus a FITC conjugated secondary antibody). G), H), and I) PAS staining alone, and in J), L), and M) in conjugation with hematoxylin. Dark arrow indicates extracellular vesicles that reacted with PAS.</p

Impact of the two isogenic groups of pediatric <i>H. pylori</i> strains on NCI-N87 cells viability.

<p>The viability of NCI-N87 cells was assessed by the trypan blue exclusion assay after 1, 12 and 24 h of co-culture with a pool of five pediatric PUD-associated strains (▪ in full line) and a pool of five pediatric NUD-associated strains (• in dashed line). For normalization, the value of 100% corresponds to the viability of non-infected NCI-N87 cells. * indicates values that were determined as significantly different (<i>p</i><0.05) between the viability of cells infected with PUD strains and that of cells infected with NUD-strains at the same time-point of co-culture. Symbols and error bars are means ± SD of the values at each point for 3 observations.</p

Schematic diagram of the DU development in children upon <i>H. pylori</i> infection.

<p>The virulence of the pediatric PUD-associated <i>H. pylori</i> strains results from a synergy between their natural ability to better adapt to the hostile human stomach and their virulence factors. Adaptation is ensured by: higher motility (↑ FlaA, ↑ FlgE, ↓ HELP_0944, ↑ HPAG1_1081, and ↑ HPG27_1480), higher ability to survive under low pH conditions (↑ UreA and B and ↑ putative aldo-keto reductase), better antioxidant defenses against inflammation (↑ Pfr, ↑ NapA and ↓ KatA), modified metabolism (↓ HyuA, ↓ AspA, ↑ Pgk, ↓ ScoA and B, ↑AroQ, ↓ Porγ, ↑ FldA, ↓ RpsA and ↓ CysS), adhesion (indicated by a anchor) mediated by “on” OipA and HomB. Virulence factors (indicated by a bomb): cagA and VacAs1. 1 – Infection; 2 – Host response (acid hypersecretion and inflammation); 3 – duodenal acid injury (Duodenal Ulcer, indicated by an explosion symbol). Dotted arrow - Time line.</p

Motility of the two isogenic groups of pediatric <i>H. pylori</i> strains in study.

<p>The five strains of each class, recovered from 24 h grown plates, were pooled together, inoculated on semi-solid BHI broth −5% FBS medium plates and incubated for 11 days at 37°C under microaerophilic conditions. A) The growth halo observed at day 5 for the pool of PUD and of NUD strains. B) Variation of the diameter of the growth halo along the time (▪ in full line refers to PUD strains and • in dashed line refers to NUD strains). Symbols are means of the values at each point for 2 observations.</p