Laura Mezzanotte

Cell-based assays: fuelling drug discovery

AbstractIt has been estimated that over a billion dollars in resources can be consumed to obtain clinical approval, and only a few new chemical entities are approved by the US Food and Drug Administration (FDA) each year. Therefore it is of utmost importance to obtain the maximum amount of information about biological activity, toxicological profile, biochemical mechanisms, and off-target interactions of drug-candidate leads in the earliest stages of drug discovery. Cell-based assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of the drug-discovery process. Nevertheless, cellular screening, relying on different strategies ranging from reporter gene technology to protein fragment complementation assays, still presents a variety of challenges. This review focuses on main advantages and limitations of different cell-based approaches, and future directions and trends in this fascinating field. FigureDifferent cell-based strategies can improve the throughput and reliability of the first stages of the drug discovery process

New trends in bioanalytical tools for the detection of genetically modified organisms: an update

Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

Background Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8+ T cell immune response

Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.

Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain

Bioluminescence imaging (BLI) has become the method of choice for optical tracking of cells in small laboratory animals. However, the use of luciferases from different species, depending on different substrates and emitting at distinct wavelengths, has not been optimized for sensitive neuroimaging. In order to identify the most suitable luciferase, this quantitative study compared the luciferases Luc2, CBG99, PpyRE9 and hRluc. Human embryonic kidney (HEK-293) cells and mouse neural stem cells were transduced by lentiviral vector-mediated transfer to express one of the four luciferases, together with copGFP. A T2A peptide linker promoted stoichiometric expression between both imaging reporters and the comparison of cell populations upon flow cytometry. Cell dilution series were used to determine highest BLI sensitivity in vitro for Luc2. However, Coelenterazine h-dependent hRluc signals clearly exceeded d-luciferin-dependent BLI in vitro. For the quantitative in vivo analysis, cells were transplanted into mouse brain and BLI was performed including the recording of emission kinetics and spectral characteristics. Differences in light kinetics were observed for d-luciferin vs Coelenterazine h. The emission spectra of Luc2 and PpyRE9 remained almost unchanged, while the emission spectrum of CBG99 became biphasic. Most importantly, photon emission decreased in the order of Luc2, CBG99, PpyRE9 to hRluc. The feasibility of combining different luciferases for dual color and dual substrate neuroimaging was tested and discussed. This investigation provides the first complete quantitative comparison of different luciferases expressed by neural stem cells. It results in a clear recommendation of Luc2 as the best luciferase selection for in vivo neuroimaging.

A multi-modality platform to image stem cell graft survival in the naïve and stroke-damaged mouse brain.

Neural stem cell implantations have been extensively investigated for treatment of brain diseases such as stroke. In order to follow the localization and functional status of cells after implantation noninvasive imaging is essential. Therefore, we developed a comprehensive multi-modality platform for in vivo imaging of graft localization, density, and survival using 19F magnetic resonance imaging in combination with bioluminescence imaging. We quantitatively analyzed cell graft survival over the first 4 weeks after transplantation in both healthy and stroke-damaged mouse brain and correlated our findings of graft vitality with the host innate immune response. The multi-modality imaging platform will help to improve cell therapy also in context other than stroke and to gain indispensable information for clinical translation.

In Vivo Bioluminescence Imaging of Murine Xenograft Cancer Models with a Red-shifted Thermostable Luciferase

PurposeConventional in vivo bioluminescence imaging using wild-type green-emitting luciferase is limited by absorption and scattering of the bioluminescent signal through tissues. Imaging methods using a red-shifted thermostable luciferase from Photinus pyralis were optimized to improve the sensitivity and image resolution. In vivo bioluminescence imaging performance of red- and green-emitting luciferases were compared in two different xenograft mouse models for cancer.MethodsHuman hepatoblastoma cell line (HepG2) and human acute monocytic leukemia cell line (Thp1) cells were genetically engineered using retroviral vector technology to stably express the red-shifted or the wild-type green luciferase. A xenograft model of liver cancer was established by subcutaneous injection of the HepG2-engineered cells in the flank regions of mice, and a leukemia model was generated by intravenous injection of the engineered Thp1 cells. The cancer progression was monitored with an ultrasensitive charge-coupled device camera. The relative intensities of the green- and red-emitting luciferases were measured, and the resulting spatial resolutions of the images were compared. Imaging was performed with both intact and scarified live animals to quantify the absorption effects of the skin and deep tissue.ResultsThe red-emitting luciferase was found to emit a bioluminescence signal with improved transmission properties compared to the green-emitting luciferase. By imaging the HepG2 models, which contained tumors just beneath the skin, before and after scarification, the percentage of light absorbed by the skin was calculated. The green bioluminescent signal was 75 ± 8% absorbed by the skin, whereas the red signal was only 20 ± 6% absorbed. The Thp1 model, which contains cancer cells within the bones, was likewise imaged before and after scarification to calculate the percentage of light absorbed by all tissue under the skin. This tissue was responsible for 90 ± 5% absorption of the green signal, but only 65 ± 6% absorption of the red signal.ConclusionTwo different bioluminescent mouse cancer models demonstrate the utility of a new red-shifted thermostable luciferase, Ppy RE-TS, that improved the in vivo imaging performance when compared with wild-type P. Pyralis luciferase. While wild-type luciferase is currently a popular reporter for in vivo imaging methods, this study demonstrates the potential of red-emitting firefly luciferase mutants to enhance the performance of bioluminescence imaging experiments.

New proteomic approaches for biomarker discovery in inflammatory bowel disease

&NA; There is an increasing interest in the discovery of new inflammatory bowel disease (IBD) biomarkers able to predict the future patterns of disease and to help in diagnosis, treatment, and prognosis. A biomarker is a substance that can be measured biologically and is associated with an increased risk of the disease. Biomarkers can be a genetic testing factor or proteins in biological samples such as serum, plasma, and cellular subpopulations. All of them should be studied to find out their utility in the management of IBD. Ulcerative colitis and Crohns disease are relapsing and remitting chronic IBDs characterized by a global immune defect. The gold standard of their diagnosis is histological evaluation performed during endoscopic procedures. Several studies have focused on the identification and combination of less invasive diagnostic serum biomarkers. Nowadays, diagnostic serum tests are not able either to determine whether and when the relapse will occur once the disease is in remission state or to select a patient phenotype more responsive to a specific therapy and more susceptible to different types of complication. In this review we analyze and report the current understanding in IBD biomarkers and discuss potential future biomarkers and new developments of proteomics, such as subproteomics, as an innovative approach for the classification of patients according to their pattern of protein expression. (Inflamm Bowel Dis 2010)

A new gastric-emptying mouse model based on in vivo non-invasive bioluminescence imaging

Background  Different techniques were used to assess gastric emptying (GE) in small animals; most of them require sophisticated equipment, animal sacrifice and are expensive. In the present investigation a simple, non‐invasive method based on bioluminescence imaging (BLI) is reported to study GE, using light‐emitting Escherichia coli cells as a marker of the gastric content.

Luminescent Probes and Visualization of Bioluminescence

Bioluminescence (BL) has revealed an extremely useful analytical tool enabling ultrasensitive detection in biotechnological applications. Following the discovery of luciferin and luciferases, molecular biology techniques allowed the cloning of several luciferases and photoproteins. Among most used BL reporters, we find firefly and click-beetle luciferases, bacterial luciferase, Renilla, Gaussia, and Cypridina luciferases, and calcium-activated photoproteins. According to the specific bioluminescent protein, different substrates and protocols must be applied in the experimental procedure for BL measurement. By conjugating (either chemically or by molecular biology techniques) bioluminescent probes to specific targets, it is in fact possible to track a wide range of events and analytes. To aid investigators in the choice and applications of reporter genes, the materials and methods required for BL measurements and experimental protocols are described.