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.

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), ß-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.

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.

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.

Postmortem redistribution of fentanyl in the rabbit blood.

Postmortem redistribution of fentanyl in the rabbit was investigated after application of the 50-µg/h Durogesic pain patch. Patches were applied for 48 hours. Two cycles of patch administration were used before characterization of the postmortem redistribution. Fentanyl showed marked redistribution into the femoral and pulmonary veins of the rabbit through 48 hours after the animals were humanely killed and the pain patches removed. The plasma concentration of 2.34 ng/mL in the femoral blood before killing the animals increased 5.6-fold by 48 hours after patch removal to 13.2 ng/mL. This postmortem concentration is approximately 3-fold the C(max) determined during antemortem pharmacokinetic analysis, 4 ng/mL, which was achieved 24 hours after the application of the second 50-µg/h Durogesic pain patch. After blood sampling for 48 hours after animal termination with patch removal compared with sampling for 48 hours from animals not terminated and with patch removal, the exposure ratios in the terminated animals were approximately 30-fold, indicating that between the postmortem redistribution of fentanyl and the cessation of hepatic clearance of fentanyl in the rabbit, the postmortem redistribution of fentanyl leads to an elevated measures of postmortem blood concentrations relative to antemortem blood concentrations.

Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool?

Proof of principle of organ reengineering through the development of a transplantable recellularized liver graft was published recently. As the decellularization time of the rat liver took 72 h, loss of some key matrix proteins seemed inevitable. Here, we describe the development of a three-dimensional naturally derived liver scaffold with an intact microvascular system that is capable of withstanding fluid flows in the three hepatic circular systems and that is obtained within 60 min. For this purpose, whole rat livers were sequentially perfused with a selection of mild tensioactive substances to remove the cellular components while preserving the major extracellular matrix proteins, including laminin, collagen I, collagen IV, and fibronectin. In addition, we could show the presence of extracellular matrix--bound growth factor islets, important for cell engraftment, migration, proliferation, and differentiation. This easy to prepare scaffold could represent a remarkable tool in the bioengineering of complex three-dimensional in vitro systems for advanced preclinical drug development.

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.

Virulence properties of Campylobacter jejuni isolates of poultry and human origin.

Campylobacter jejuni is one of the leading causes of food-borne gastroenteritis. Because of the high prevalence of C. jejuni in poultry, poultry meat is considered a major source of C. jejuni infections for humans. However, it is not known whether all poultry-associated C. jejuni strains are capable of causing disease in humans. Four different virulence properties of C. jejuni strains were compared between 20 poultry isolates and 24 human isolates. Strains were chosen based on their PFGE pattern to represent a heterogeneous population. The isolates were compared for their ability to invade and induce interleukin-8 (IL-8) production in T84 cells, their production of functional cytolethal distending toxin (CDT) using HEp-2 cells, and their sodium deoxycholate resistance. All four virulence factors were present among strains of human and poultry origin, with strong differences observed among strains. For invasion and IL-8 induction, no difference was observed between the two populations. However, on average, human isolates arrested more HEp-2 cells in their cell cycle than did the poultry isolates (P=0.041), suggesting higher CDT production by the former. The ability to survive 16 000 mug sodium deoxycholate ml(-1) was significantly more pronounced (P=0.006) among human isolates than poultry isolates, although all strains possessed the cmeABC operon. These data suggest that all four virulence properties are widespread among C. jejuni isolates, but that a higher degree of bile-salt resistance and more pronounced CDT production are associated with strains causing enteritis in humans.

Pathogenesis of Helicobacter pullorum infections in broilers.

Four groups of 23 one-day-old broiler chickens were each inoculated by gavage with a different Helicobacter pullorum strain isolated from humans or poultry. As a control, a fifth group of eight animals was inoculated with phosphate-buffered saline. Faecal samples were collected weekly and tested for the presence of H. pullorum DNA using PCR. At 1, 8, 15, 22 and 42 days postinoculation, birds were euthanized and samples from the liver and intestinal tract were histologically, immunohistochemically and bacteriologically examined. The samples were also tested for the presence of H. pullorum DNA by PCR. All animals remained clinically healthy throughout the experiment although mild lesions in the caeca were present in animals inoculated with H. pullorum. In all H. pullorum-inoculated groups, DNA of this bacterium was detected in faecal samples until 42 days postinoculation. The main site of colonization was the caecum. Immunohistochemical examination revealed that the bacterium was closely associated with the caecal epithelial cells. It was concluded that H. pullorum may colonize the caecum of broilers and is excreted in their faeces until slaughter age. This implies that chicken meat might constitute a source of infection for human beings.

The cytolethal distending toxin among Helicobacter pullorum strains from human and poultry origin.

Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro.

Cytolethal distending toxin generates cell death by inducing a bottleneck in the cell cycle.

Cytolethal distending toxin (CDT) is a bacterial protein that is widely distributed among gram-negative bacteria including Escherichia coli, Campylobacter spp., enterohepatic Helicobacter spp., Actinobacillus actinomycetemcomitans and Haemophilus ducreyi. In vitro studies demonstrated that it is able to stop proliferation of various cell lines. The toxin is composed of three subunits designated CDTs A, B and C. The B subunit targets the eukaryotic DNA and triggers a signalling pathway involving different protein kinases which results in a cell block before entering into mitosis. To date, the individual role of the A and C subunits has not been totally elucidated. There are indications that the CDT is also produced in vivo. Its exact role in pathogenesis is not yet clear, but possible actions include inhibition of epithelial cell proliferation, apoptosis of immune cells and inhibition of a fibrotic response.

Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates.

Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage.

In vitro susceptibility of Helicobacter pullorum strains to different antimicrobial agents.

The in vitro activity of 13 antimicrobial agents against 23 Helicobacter pullorum strains from poultry (21) and human (two) origin, and one human H. canadensis strain was tested by the agar dilution method. With the H. pullorum strains, monomodal distributions of Minimum Inhibitory Concentrations (MICs) were seen with lincomycin, doxycycline, gentamicin, tobramycin, erythromycin, tylosin, metronidazole, and enrofloxacin in concentration ranges considered as indicating susceptibility in other bacteria. The normal susceptibility level for nalidixic acid was situated at or slightly above the MIC breakpoints proposed for Campylobacteriaceae. Ampicillin, ceftriaxone, and sulphamethoxazole-trimethoprim showed poor activity against H. pullorum. For the H. canadensis strain, a similar susceptibility pattern was seen, except for nalidixic acid and enrofloxacin, whose MIC of >512 and 8 microg/ml, respectively, indicated resistance of this agent. With spectinomycin, a bimodal distribution of the MICs was noted for the tested strains; eight H. pullorum isolates originating from one flock showed acquired resistance (MIC>512 microg/ml).

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.

The HepaRG cell line: a valuable in vitro tool for hepatitis virus infection studies.

Hepatitis virus infections, mainly hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, constitute a major problem for public health since they have a worldwide distribution and because they are associated with hepatocellular carcinoma and death. Current anti-HBV vaccines seem to be effective in the majority of people. However, an important issue waiting to be tackled nowadays is how to cure patients with chronic hepatitis B. Moreover, no vaccine is available today for the prevention of HCV infection. Therefore, the use of adequate in vitro infection systems is a prerequisite for the molecular understanding of the infection events of these viruses, which could result in the development of novel powerful therapeutics. In this respect, the HepaRG cell line exhibits a hepatocyte-like morphology and displays drug metabolism capacity similar to that of primary hepatocytes. HepaRG cells have yet been proven to be a useful tool in the study of viral infections, particularly for deciphering the mechanism of HBV entry into hepatocytes.

Critical selection of reliable reference genes for gene expression study in the HepaRG cell line.

The human HepaRG cell line has shown to be a valuable in vitro tool for repeated exposure to chemical compounds and to evaluate their potential toxic outcome. Seen the importance given by the actual EU legislation of cosmetics and chemical substances to the use of in vitro methods in human safety evaluation, one can expect that HepaRG cells will gain importance as human-relevant cell source. At the transcriptional level, RT-qPCR assays are often used to obtain quantitative results. The choice of internal control is important since it may affect the study outcome. Indeed, it is well-known that expression levels of traditional reference genes can vary across tissue types and across experimental settings within one specific tissue type. From a review of the scientific literature, it appears that, for HepaRG cells, S18 often is used as internal control, but without any evidence of its expression stability in this cell line. Therefore, we aimed to select the most optimal reference genes for gene expression studies in HepaRG cells and to check whether S18 is a suitable reference gene. Twelve candidate genes' expression stability level was analyzed by three algorithms (geNorm, BestKeeper, Normfinder), which identified the optimal single reference gene (TBP) and the most suitable set of reference genes (TBP, UBC, SDHA, RLP13, YHWAZ, HMBS, B2M and HPRT1) for HepaRG transcriptional profiling. This study provides a new set of reference genes that is suitable for testing whenever RT-qPCR data for HepaRG cells are generated. The most stable ones can then be selected for further normalization.

Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates.

Helicobacter pullorum infections have been associated with several enterohepatic diseases, but the mechanism of action is currently undefined. The present study was therefore set up to investigate possible cytotoxic effects of this pathogen on liver cells. A mouse hepatic cell line was exposed to H. pullorum sonicate and cytotoxicity was observed for all isolates after incubation for 72 h. Features characteristic for mitotic catastrophe characterized by chromatin condensation, formation of multinuclear distended cells and micronucleation, were recorded. In addition, intranuclear pseudoinclusions were seen in sonicate-treated cells. Finally, cells exposed to sonicate eventually underwent cell death with the morphological features of necrosis, which occurred without activation of caspase-3. The toxic factor involved in the cytotoxic activity proved to be soluble, trypsin-sensitive and stable at 56 degrees C and at -70 degrees C with a molecular weight to be over 50 kDa. The current study shows for the first time that H. pullorum causes mitotic catastrophe resulting in primary necrosis in mouse hepatocytes.

Helicobacter pullorum in chickens, Belgium.

A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.

Prevalence of Helicobacter pullorum among patients with gastrointestinal disease and clinically healthy persons.

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.