Measuring CTL Lytic Granule Secretion and Target Cell Membrane Repair by Fluorescent Lipophilic Dye Uptake at the Lytic Synapse.

CD8+ cytotoxic T lymphocytes (CTL) play a key role in anti-tumor immune response. They are therefore at the heart of current immunotherapy protocols against cancer. Despite current strategies to potentiate CTL responses, cancer cells can resist CTL attack, thus limiting the efficacy of immunotherapies. To optimize immunotherapy, it is urgent to develop rapid assays allowing to assess CTL-cancer cell confrontation at the lytic synapse.In this chapter, we describe a flow cytometry-based method to simultaneously assess the extent of CTL activation and of tumor cell reparative membrane turnover in CTL/target cell conjugates. Such a method can be performed using a limited number of cells. It can therefore be employed in clinical settings when only a few patient-derived cells might be available.

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

The extracellular matrix molecule tenascin-C modulates cell cycle progression and motility of adult neural stem/progenitor cells from the subependymal zone.

Adult neurogenesis has been described in two canonical regions of the adult central nervous system (CNS) of rodents, the subgranular zone (SGZ) of the hippocampus and the subependymal zone (SEZ) of the lateral ventricles. The stem cell niche of the SEZ provides a privileged environment composed of a specialized extracellular matrix (ECM) that comprises the glycoproteins tenascin-C (Tnc) and laminin-1 (LN1). In the present study, we investigated the function of these ECM glycoproteins in the adult stem cell niche. Adult neural stem/progenitor cells (aNSPCs) of the SEZ were prepared from wild type (Tnc+/+) and Tnc knockout (Tnc-/-) mice and analyzed using molecular and cell biological approaches. A delayed maturation of aNSPCs in Tnc-/- tissue was reflected by a reduced capacity to form neurospheres in response to epidermal growth factor (EGF). To examine a potential influence of the ECM on cell proliferation, aNSPCs of both genotypes were studied by cell tracking using digital video microscopy. aNSPCs were cultivated on three different substrates, namely, poly-D-lysine (PDL) and PDL replenished with either LN1 or Tnc for up to 6 days in vitro. On each of the three substrates aNSPCs displayed lineage trees that could be investigated with regard to cell cycle length. The latter appeared reduced in Tnc-/- aNSPCs on PDL and LN1 substrates, less so on Tnc that seemed to compensate the absence of the ECM compound to some extent. Close inspection of the lineage trees revealed a subpopulation of late dividing aNSPCslate that engaged into cycling after a notable delay. aNSPCslate exhibited a clearly different morphology, with a larger cell body and conspicuous processes. aNSPCslate reiterated the reduction in cell cycle length on all substrates tested, which was not rescued on Tnc substrates. When the migratory activity of aNSPC-derived progeny was determined, Tnc-/- neuroblasts displayed significantly longer migration tracks. This was traced to an increased rate of migration episodes compared to the wild-type cells that rested for longer time periods. We conclude that Tnc intervenes in the proliferation of aNSPCs and modulates the motility of neuroblasts in the niche of the SEZ.

Live Imaging Reveals Cerebellar Neural Stem Cell Dynamics and the Role of VNUT in Lineage Progression.

Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored. The fact that neurogenesis occurs in such organized manner in the absence of signals from the local environment, suggests that cerebellar lineage progression is to an important extent governed by cell-intrinsic or pre-programmed events. Finally, we took advantage of the absence of the niche to assay the influence of the vesicular nucleotide transporter inhibition, which dramatically reduced the number of NSCs in vitro by promoting their progression toward neurogenesis.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing.

BACKGROUND: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. METHODS: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose-response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. RESULTS: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. CONCLUSIONS: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment.

Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro.

A comprehensive understanding of the mechanisms controlling the behavior of cell populations with regenerative potential is the first step to design effective therapeutic strategies for many diseases. However, a precise description of the biological events involved, such as proliferation, differentiation, cell fate decisions, migration, or viability, may be hampered by the classical use of experiments based on end-point analysis. By contrast, live imaging and single cell tracking provides researchers with an accurate readout of these features in cells throughout an experiment. Here, we describe a protocol to apply time-lapse video microscopy and post-processing of the data to study critical aspects of the biology and the lineage progression of multiple neural populations.

COMBImage2: a parallel computational framework for higher-order drug combination analysis that includes automated plate design, matched filter based object counting and temporal data mining.

BACKGROUND: Pharmacological treatment of complex diseases using more than two drugs is commonplace in the clinic due to better efficacy, decreased toxicity and reduced risk for developing resistance. However, many of these higher-order treatments have not undergone any detailed preceding in vitro evaluation that could support their therapeutic potential and reveal disease related insights. Despite the increased medical need for discovery and development of higher-order drug combinations, very few reports from systematic large-scale studies along this direction exist. A major reason is lack of computational tools that enable automated design and analysis of exhaustive drug combination experiments, where all possible subsets among a panel of pre-selected drugs have to be evaluated. RESULTS: Motivated by this, we developed COMBImage2, a parallel computational framework for higher-order drug combination analysis. COMBImage2 goes far beyond its predecessor COMBImage in many different ways. In particular, it offers automated 384-well plate design, as well as quality control that involves resampling statistics and inter-plate analyses. Moreover, it is equipped with a generic matched filter based object counting method that is currently designed for apoptotic-like cells. Furthermore, apart from higher-order synergy analyses, COMBImage2 introduces a novel data mining approach for identifying interesting temporal response patterns and disentangling higher- from lower- and single-drug effects. COMBImage2 was employed in the context of a small pilot study focused on the CUSP9v4 protocol, which is currently used in the clinic for treatment of recurrent glioblastoma. For the first time, all 246 possible combinations of order 4 or lower of the 9 single drugs consisting the CUSP9v4 cocktail, were evaluated on an in vitro clonal culture of glioma initiating cells. CONCLUSIONS: COMBImage2 is able to automatically design and robustly analyze exhaustive and in general higher-order drug combination experiments. Such a versatile video microscopy oriented framework is likely to enable, guide and accelerate systematic large-scale drug combination studies not only for cancer but also other diseases.

COMBImage: a modular parallel processing framework for pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies.

BACKGROUND: Large-scale pairwise drug combination analysis has lately gained momentum in drug discovery and development projects, mainly due to the employment of advanced experimental-computational pipelines. This is fortunate as drug combinations are often required for successful treatment of complex diseases. Furthermore, most new drugs cannot totally replace the current standard-of-care medication, but rather have to enter clinical use as add-on treatment. However, there is a clear deficiency of computational tools for label-free and temporal image-based drug combination analysis that go beyond the conventional but relatively uninformative end point measurements. RESULTS: COMBImage is a fast, modular and instrument independent computational framework for in vitro pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies. Jointly with automated analyses of temporal changes in cell morphology and confluence, it performs and displays conventional cell viability and synergy end point analyses. The image processing algorithms are parallelized using Google's MapReduce programming model and optimized with respect to method-specific tuning parameters. COMBImage is shown to process time-lapse microscopy movies from 384-well plates within minutes on a single quad core personal computer. This framework was employed in the context of an ongoing drug discovery and development project focused on glioblastoma multiforme; the most deadly form of brain cancer. Interesting add-on effects of two investigational cytotoxic compounds when combined with vorinostat were revealed on recently established clonal cultures of glioma-initiating cells from patient tumor samples. Therapeutic synergies, when normal astrocytes were used as a toxicity cell model, reinforced the pharmacological interest regarding their potential clinical use. CONCLUSIONS: COMBImage enables, for the first time, fast and optimized pairwise drug combination analyses of temporal changes in label-free video microscopy movies. Providing this jointly with conventional cell viability based end point analyses, it could help accelerating and guiding any drug discovery and development project, without use of cell labeling and the need to employ a particular live cell imaging instrument.

Cell tracking in vitro reveals that the extracellular matrix glycoprotein Tenascin-C modulates cell cycle length and differentiation in neural stem/progenitor cells of the developing mouse spinal cord.

Generation of astrocytes during the development of the mammalian spinal cord is poorly understood. Previously, we have shown that the glycoprotein of the extracellular matrix (ECM) tenascin-C (Tnc) modulates the expression territories of the patterning genes Nkx6.1 and Nkx2.2 in the developing ventral spinal cord, tunes the responsiveness of neural stem/progenitor cells towards the cytokines FGF2 and EGF and thereby promotes astrocyte maturation. In order to obtain further mechanistic insight into these processes, we have compared embryonic day-15 spinal cord neural progenitor cells (NPCs) from wild-type and Tnc knockout mice using continuous single-cell live imaging and cell lineage analysis in vitroTnc knockout cells displayed a significantly reduced rate of cell division both in response to FGF2 and EGF. When individual clones of dividing cells were investigated with regard to their cell lineage trees using the tTt tracking software, it appeared that the cell cycle length in response to growth factors was reduced in the knockout. Furthermore, when Tnc knockout NPCs were induced to differentiate by the removal of FGF2 and EGF glial differentiation was enhanced. We conclude that the constituent of the stem cell niche Tnc contributes to preserve stemness of NPCs.

Novel aspects of live intestinal epithelial cell function revealed using a custom time-lapse video microscopy apparatus.

Many aspects of cell physiology, including migration, membrane function, and cell division, are best understood by observing live cell dynamics over time using video microscopy. To probe these phenomena in colon epithelial cells using simple components with a limited budget, we have constructed an inexpensive (<$410) self-contained apparatus, consisting of a closed-loop, feedback-controlled system regulated by a PID (proportional-integrative-derivative) controller contained within a 0.077 m3 insulated acrylic box. Temperature, humidity, pH, and proliferative capacity of colon epithelial cells in this system mimic those in a standard tissue culture incubator for over four days. Our system offers significant advantages over existing cost-prohibitive commercially available and custom-made devices because of its very low cost, use of PID temperature control, lack of reliance on constant infusion of external humidified, heated air or carbon dioxide, ability to directly measure cell culture medium temperature, and combination of exquisite cellular detail with minimal focus drift under physiological conditions for extended periods of time. Using this apparatus, coupled with an inverted microscope equipped with phase contrast optics and a programmable digital camera, we have observed many events in colon epithelial cells not visible by static imaging, including kinetics of normal and abnormal mitoses, dynamic membrane structures, intracellular vesicle movements, and cell migration. © 2018 International Society for Advancement of Cytometry.

Isolation of Neural Stem and Progenitor Cells from the Adult Brain and Live Imaging of Their Cell Cycle with the FUCCI System.

Neural stem cells (NSCs) enter quiescence in early embryonic stages to create a reservoir of dormant NSCs able to enter proliferation and produce neuronal precursors in the adult mammalian brain. Various approaches of fluorescent-activated cell sorting (FACS) have emerged to allow the distinction between quiescent NSCs (qNSCs), their activated counterpart (aNSCs), and the resulting progeny. In this article, we review two FACS techniques that can be used alternatively. We also show that their association with transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice allows an unprecedented overlook on the cell cycle dynamics of adult NSCs.

Contractility Assay for Established Myoepithelial Cell Lines.

The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.

Effects of hemin, CO2, and pH on the branching of Candida albicans filamentous forms.

Morphological transitions of wild-type and oxidative stress-tolerant Candida albicans strains were followed in the RPMI-FBS culture medium at pH values and CO2 levels characteristic for the anatomical niches inhabited by this opportunistic human pathogen fungus, including the oral cavity as well as the intestinal and vaginal lumens. Selected cultures were also supplemented with hemin modeling bleedings. Germination as well as elongation and branching of hyphae were monitored in the cultures using time-lapse video microscopy. Unexpectedly, branching time, which is defined as the time taken until the first branch of hypha emerges for the first time after germination, correlated well with alterations in the environmental conditions meanwhile no such correlations were found for germination time (time lasted until the appearance of the germination tube). Based on these observations, hypotheses were set up to estimate the significance of branching time in the pathogenesis of both superficial and systemic candidiases.

The effect of pulsed electric fields on the electrotactic migration of human neural progenitor cells through the involvement of intracellular calcium signaling.

Endogenous electric fields (EFs) are required for the physiological control of the central nervous system development. Application of the direct current EFs to neural stem cells has been studied for the possibility of stem cell transplantation as one of the therapies for brain injury. EFs generated within the nervous system are often associated with action potentials and synaptic activity, apparently resulting in a pulsed current in nature. The aim of this study is to investigate the effect of pulsed EF, which can reduce the cytotoxicity, on the migration of human neural progenitor cells (hNPCs). We applied the mono-directional pulsed EF with a strength of 250mV/mm to hNPCs for 6h. The migration distance of the hNPCs exposed to pulsed EF was significantly greater compared with the control not exposed to the EF. Pulsed EFs, however, had less of an effect on the migration of the differentiated hNPCs. There was no significant change in the survival of hNPCs after exposure to the pulsed EF. To investigate the role of Ca2+ signaling in electrotactic migration of hNPCs, pharmacological inhibition of Ca2+ channels in the EF-exposed cells revealed that the electrotactic migration of hNPCs exposed to Ca2+ channel blockers was significantly lower compared to the control group. The findings suggest that the pulsed EF induced migration of hNPCs is partly influenced by intracellular Ca2+ signaling.

Nanoparticles for the delivery of zoledronic acid to prostate cancer cells: a comparative analysis through time lapse video-microscopy technique.

Time-lapse live cell imaging is a powerful tool for studying the responses of cells to drugs. Zoledronic acid (ZOL) is the most potent aminobiphosphonate able to induce cell growth inhibition at very low concentrations. The lack of clear evidence of ZOL-induced anti-cancer effects is likely due to its unfavorable pharmacokinetic profile. The use of nanotechnology-based formulations allows overcoming these limitations in ZOL pharmaco-distribution. Recently, stealth liposomes (LIPOs) and new self-assembly PEGylated nanoparticles (NPs) encapsulating ZOL were developed. Both the delivery systems showed promising anticancer activity in vitro and in vivo. In this work, we investigated the cytostatic effect of these novel formulations (LIPOs and NPs) compared with free ZOL on 2 different prostate cancer cell lines, PC 3 and DU 145 and on prostate epithelial primary cells EPN using time lapse video-microscopy (TLVM). In PC3 cells, free ZOL showed a significant anti-proliferative effect but this effect was lower than that induced by LIPOs and NPs encapsulating ZOL; moreover, LIPO-ZOL was more potent in inducing growth inhibition than NP-ZOL. On the other hand, LIPO-ZOL slightly enhanced the free ZOL activity on growth inhibition of DU 145, while the anti-proliferative effect of NP-ZOL was not statistically relevant. These novel formulations did not induce anti-proliferative effects on EPN cells. Finally, we evaluated cytotoxic effects on DU145 where, LIPO-ZOL induced the highest cytotoxicity compared with NP-ZOL and free ZOL. In conclusion, ZOL can be transformed in a powerful anticancer agent, if administered with nanotechnology-based formulations without damaging the healthy tissues.

Detailed process of shell construction in the photosynthetic testate amoeba Paulinella chromatophora (euglyphid, Rhizaria).

Most euglyphids, a group of testate amoebae, have a shell that is constructed from numerous siliceous scales. The euglyphid Paulinella chromatophora has photosynthetic organelles (termed cyanelles or chromatophores), allowing it to be cultivated more easily than other euglyphids. Like other euglyphids, P. chromatophora has a siliceous shell made of brick-like scales. These scales are varied in size and shape. How a P. chromatophora cell makes this shell is still a mystery. We examined shell construction process in P. chromatophora in detail using time-lapse video microscopy. The new shell was constructed by a specialized pseudopodium that laid out each scale into correct position, one scale at a time. The present study inferred that the sequence of scale production and secretion was well controlled.