Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT) studies.

Abstract Inhalation of ambient air particles or engineered nanoparticles (NP) handled as powders, dispersions or sprays in industrial processes and contained in consumer products pose a potential and largely unknown risk for incidental exposure. For efficient, economical and ethically sound evaluation of health hazards by inhaled nanomaterials, animal-free and realistic in vitro test systems are desirable. The new Nano Aerosol Chamber for in-vitro Toxicity studies (NACIVT) has been developed and fully characterized regarding its performance. NACIVT features a computer-controlled temperature and humidity conditioning, preventing cellular stress during exposure and allowing long-term exposures. Airborne NP are deposited out of a continuous air stream simultaneously on up to 24 cell cultures on Transwell® inserts, allowing high-throughput screening. In NACIVT, polystyrene as well as silver particles were deposited uniformly and efficiently on all 24 Transwell® inserts. Particle-cell interaction studies confirmed that deposited particles reach the cell surface and can be taken up by cells. As demonstrated in control experiments, there was no evidence for any adverse effects on human bronchial epithelial cells (BEAS-2B) due to the exposure treatment in NACIVT. The new, fully integrated and transportable deposition chamber NACIVT provides a promising tool for reliable, acute and sub-acute dose-response studies of (nano)particles in air-exposed tissues cultured at the air-liquid interface.

A compact and portable deposition chamber to study nanoparticles in air-exposed tissue

BACKGROUND Epidemiological studies show that elevated levels of particulate matter in ambient air are highly correlated with respiratory and cardiovascular diseases. Atmospheric particles originate from a large number of sources and have a highly complex and variable composition. An assessment of their potential health risks and the identification of the most toxic particle sources would require a large number of investigations. Due to ethical and economic reasons, it is desirable to reduce the number of in vivo studies and to develop suitable in vitro systems for the investigation of cell-particle interactions. METHODS We present the design of a new particle deposition chamber in which aerosol particles are deposited onto cell cultures out of a continuous air flow. The chamber allows for a simultaneous exposure of 12 cell cultures. RESULTS Physiological conditions within the deposition chamber can be sustained constantly at 36-37°C and 90-95% relative humidity. Particle deposition within the chamber and especially on the cell cultures was determined in detail, showing that during a deposition time of 2 hr 8.4% (24% relative standard deviation) of particles with a mean diameter of 50 nm [mass median diameter of 100 nm (geometric standard deviation 1.7)] are deposited on the cell cultures, which is equal to 24-34% of all charged particles. The average well-to-well variability of particles deposited simultaneously in the 12 cell cultures during an experiment is 15.6% (24.7% relative standard deviation). CONCLUSIONS This particle deposition chamber is a new in vitro system to investigate realistic cell-particle interactions at physiological conditions, minimizing stress on the cell cultures other than from deposited particles. A detailed knowledge of particle deposition characteristics on the cell cultures allows evaluating reliable dose-response relationships. The compact and portable design of the deposition chamber allows for measurements at any particle sources of interest.

Responses of lung cells to realistic exposure of primary and aged carbonaceous aerosols

Diesel exhaust and wood burning are important sources of ambient atmospheric particles due to increasing numbers of diesel cars and the importance of wood as a source of renewable energy. Inhalation is the predominant route of entry and uptake for fine and ultrafine particles into the body. Health effects of atmospheric particles are still not completely understood. There is consistent evidence from epidemiology that particle exposure contributes to respiratory and cardiovascular diseases. This study aimed at examining acute responses of airway epithelial cells and luminal macrophages after exposure to freshly emitted and photochemically aged carbonaceous aerosols under realistic atmospheric conditions. In addition to a bronchial epithelial cell line advanced cell cultures namely fully differentiated respiratory epithelia and primary surface macrophages were used. Our results demonstrate that a single exposure of the cells to realistic particle doses of 0.3–3 ng diesel or 3–9 ng wood aerosol per cm2 cell surface induces small, particle-specific responses. The release of interleukin-6 and -8 was found to be decreased in differentiated airway epithelia but not in the other cell models studied. Aerosol exposure decreased macrophage phagocytic activity by 45–90%. Cell and tissue integrity remained unaffected. Overall, primary and aged particles from the same combustion induced similar responses in the cell models tested, whereby particles from diesel exhaust affected the cells more than those from wood combustion.

Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface

BACKGROUND: Due to its antibacterial properties, silver (Ag) has been used in more consumer products than any other nanomaterial so far. Despite the promising advantages posed by using Ag-nanoparticles (NPs), their interaction with mammalian systems is currently not fully understood. An exposure route via inhalation is of primary concern for humans in an occupational setting. Aim of this study was therefore to investigate the potential adverse effects of aerosolised Ag-NPs using a human epithelial airway barrier model composed of A549, monocyte derived macrophage and dendritic cells cultured in vitro at the air-liquid interface. Cell cultures were exposed to 20 nm citrate-coated Ag-NPs with a deposition of 30 and 278 ng/cm2 respectively and incubated for 4 h and 24 h. To elucidate whether any effects of Ag-NPs are due to ionic effects, Ag-Nitrate (AgNO3) solutions were aerosolised at the same molecular mass concentrations. RESULTS: Agglomerates of Ag-NPs were detected at 24 h post exposure in vesicular structures inside cells but the cellular integrity was not impaired upon Ag-NP exposures. Minimal cytotoxicity, by measuring the release of lactate dehydrogenase, could only be detected following a higher concentrated AgNO3-solution. A release of pro-inflammatory markers TNF-alpha and IL-8 was neither observed upon Ag-NP and AgNO3 exposures as well as was not affected when cells were pre-stimulated with lipopolysaccharide (LPS). Also, an induction of mRNA expression of TNF-alpha and IL-8, could only be observed for the highest AgNO3 concentration alone or even significantly increased when pre-stimulated with LPS after 4 h. However, this effect disappeared after 24 h. Furthermore, oxidative stress markers (HMOX-1, SOD-1) were expressed after 4 h in a concentration dependent manner following AgNO3 exposures only. CONCLUSIONS: With an experimental setup reflecting physiological exposure conditions in the human lung more realistic, the present study indicates that Ag-NPs do not cause adverse effects and cells were only sensitive to high Ag-ion concentrations. Chronic exposure scenarios however, are needed to reveal further insight into the fate of Ag-NPs after deposition and cell interactions.

Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

Aggravation of cardiac myofibroblast arrhythmogeneicity by mechanical stress

Aims Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. Methods and results Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. Conclusions The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts.

Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration

Listeria monocytogenes rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that Listeria monocytogenes spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing Listeria monocytogenes inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails. In vitro infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably, in vivo, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.

Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Constitutive IL-2 mRNA expression in lymphocytes, infected with the intracellular parasite Theileria parva.

Theileria parva-infected lymphoblastoid cell lines of T or B cell origin were examined for IL-2 mRNA expression. T. parva-infected T cell lines could be of the CD4-CD8-, CD4+CD8-, CD4-CD8+, or CD4+CD8+ phenotype and express alpha beta or gamma delta TCR. By Northern blot analysis and amplification by the polymerase chain reaction, IL-2 mRNA could be detected in all T. parva-infected cell lines tested. IL-2 mRNA expression was also shown to be dependent on the continuous presence of the parasite in the host cell cytoplasm, because elimination of the parasite by treatment of T. parva-infected cell cultures with the theilericidal drug BW720c resulted in the disappearance of detectable IL-2 mRNA. The effect of anti-IL-2 antibodies on the proliferation of T. parva-infected cells was also tested. Inhibition experiments suggest that although IL-2 mRNA can be detected in all cell lines tested, not all T. parva-infected cell lines are dependent on IL-2 for their proliferation. Our data provide the first example for the constitutive expression of IL-2 mRNA in T and B cells caused by infection with an intracellular parasite.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.