Liesbeth Ceelen

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Connexins: sensors and regulators of cell cycling.

It is nowadays well established that gap junctions are critical gatekeepers of cell proliferation, by controlling the intercellular exchange of essential growth regulators. In recent years, however, it has become clear that the picture is not as simple as originally anticipated, as structural precursors of gap junctions can affect cell cycling by performing actions not related to gap junctional intercellular communication. Indeed, connexin hemichannels also foresee a pathway for cell growth communication, albeit between the intracellular compartment and the extracellular environment, while connexin proteins as such can directly or indirectly influence the production of cell cycle regulators independently of their channel activities. Furthermore, a novel set of connexin-like proteins, the pannexins, have lately joined in as regulators of the cell proliferation process, which they can affect as either single units or as channel entities. In the current paper, these multifaceted aspects of connexin-related signalling in cell cycling are reviewed.

Prevalence of Helicobacter pullorum among Patients with Gastrointestinal Disease and Clinically Healthy Persons

ABSTRACT Feces from 531 patients with gastroenteritis and from 100 clinically healthy individuals were tested for Helicobacter pullorum by use of PCR. Samples positive by PCR were qualified for isolation. H. pullorum DNA was demonstrated to be present in feces from 4.3% of patients with gastrointestinal disease but also in feces from 4.0% of clinically healthy persons. One strain was isolated from one patient with gastrointestinal disease.

Helicobacter pullorum in Chickens, Belgium

Helicobacter pullorum is present in approximately one third of the chickens in Belgium.

Inhibition of gap junctional intercellular communication by toxic metals.

As metals are ubiquitous in the environment, humans are continuously exposed to them. Although some metallic ions play key roles in human physiology, most metals are redundant and are actually hazardous to humans. A frequent event in the toxicological cascade triggered by nonessential metals concerns the abrogation of cellular signaling mediated by gap junctions. This paper provides a literature overview of the documented effects of mercury, cadmium, arsenic, aluminum, lead, nickel, vanadium, and indium on gap junctional intercellular communication. Whenever available, particular attention is paid to the mechanistic basis of this deleterious biological outcome, which may involve the gap junction activity level or may arise from modifications in the expression of the gap junction building stones, the connexins.

Modulation of connexin signaling by bacterial pathogens and their toxins

Inherent to their pivotal tasks in the maintenance of cellular homeostasis, gap junctions, connexin hemichannels, and pannexin hemichannels are frequently involved in the dysregulation of this critical balance. The present paper specifically focuses on their roles in bacterial infection and disease. In particular, the reported biological outcome of clinically important bacteria including Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Helicobacter pylori, Bordetella pertussis, Aggregatibacter actinomycetemcomitans, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium species, Streptococcus pneumoniae, and Staphylococcus aureus and their toxic products on connexin- and pannexin-related signaling in host cells is reviewed. Particular attention is paid to the underlying molecular mechanisms of these effects as well as to the actual biological relevance of these findings.

Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

Acute in vivo interactions of Helicobacter equorum with its equine host

REASONS FOR PERFORMING STUDY A novel urease-negative Helicobacter species has been isolated from faecal samples of clinically healthy horses, but no information is available about the main sites of colonisation in the equine gastrointestinal tract nor is the pathogenic potential of this microorganism known. An experimental infection in horses was therefore carried out. METHODS Four horses were infected with H. equorum strain CCUG 52199T and subjected to euthanasia at 10 (n = 2) and 30 days (n = 2) post inoculation. A fifth animal was inoculated with phosphate buffered saline and used as control. Gastrointestinal samples were examined histologically and bacteriologically. These samples, as well as faecal material collected at regular intervals, were also subjected to PCR analysis. RESULTS All horses remained clinically healthy and no specific macroscopic lesions were identified, nor were there any microscopic changes. H. equorum-DNA was detected in the faeces during the whole experiment in all infected animals but not in the negative control. Sites of colonisation were caecum, colon and rectum. CONCLUSIONS H. equorum is able to colonise the equine lower bowel and is excreted in faeces without apparent pathology. No association between the presence of the organism and gastrointestinal disease was demonstrated.

Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model?

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.

Characterization of virulence properties of Listeria monocytogenes serotype 4b strains of different origins.

In this study, the virulence heterogeneity of Listeria monocytogenes serotype 4b strains of different origins was analysed on different levels. On one hand, the survival of L. monocytogenes strains in synthetic gastric fluid was studied. On the other hand, the pathogenic potential of strains with different inlB expression levels was analysed in an A/J mouse model for gastrointestinal listeriosis. Differences in survival capacity in gastric fluid and in in vivo virulence potential were observed between the tested strains. No clear correlation between the origin and the obtained data could be made. However, these results confirm the existence of heterogeneity in virulence potential of L. monocytogenes serotype 4b strains.