[Emergency plans in crisis situations]. / Les plans de secours en situation de crise.

In November 2015, in Paris, a wave of terrorist attacks brought horror to France. The medical and nursing teams were severely tested but demonstrated efficiency and courage. The organisation of the emergency response requires fast and essential decision making and actions.

Remote damage control during the attacks on Paris: Lessons learned by the Paris Fire Brigade and evolutions in the rescue system.

On November 13, 2015, in 40 minutes, Paris suffered four suicide bombers attacks; shootings at three different restaurant terraces; and an attack on the Bataclan concert hall, resulting in 130 dead and 495 wounded. How did the Parisian rescue system respond and how did it evolve since?We proved we could deploy quickly wide prehospital and hospital resources and teams' equipment and preparedness is being further developed. To secure a swifter initial response, we need a better integration of the operators of the rescue chain with a simpler and more robust organization as well as improved communications channels. We must continue to anticipate and prepare for possible future attacks.

Single-cell resolution of intracellular T cell Ca2+ dynamics in response to frequency-based H2O2 stimulation.

Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca2+ and H2O2. Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca2+ dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H2O2 and Ca2+, during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

Calcium Dynamics of Ex Vivo Long-Term Cultured CD8+ T Cells Are Regulated by Changes in Redox Metabolism.

T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca2+ signaling does not change with time in culture, older T cells exhibit faster Ca2+ rise and a faster decay. Gene expression analysis of Ca2+ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca2+ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca2+ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca2+ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP3R and STIM1 channels as potential causes for alterations in Ca2+ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging.

Single-cell analysis of embryoid body heterogeneity using microfluidic trapping array.

The differentiation of pluripotent stem cells as embryoid bodies (EBs) remains a common method for inducing differentiation toward many lineages. However, differentiation via EBs typically yields a significant amount of heterogeneity in the cell population, as most cells differentiate simultaneously toward different lineages, while others remain undifferentiated. Moreover, physical parameters, such as the size of EBs, can modulate the heterogeneity of differentiated phenotypes due to the establishment of nutrient and oxygen gradients. One of the challenges in examining the cellular composition of EBs is the lack of analytical methods that are capable of determining the phenotype of all of the individual cells that comprise a single EB. Therefore, the objective of this work was to examine the ability of a microfluidic cell trapping array to analyze the heterogeneity of cells comprising EBs during the course of early differentiation. The heterogeneity of single cell phenotype on the basis of protein expression of the pluripotent transcription factor OCT-4 was examined for populations of EBs and single EBs of different sizes at distinct stages of differentiation. Results from the cell trap device were compared with flow cytometry and whole mount immunostaining. Additionally, single cells from dissociated pooled EBs or individual EBs were examined separately to discern potential differences in the value or variance of expression between the different methods of analysis. Overall, the analytical method described represents a novel approach for evaluating how heterogeneity is manifested in EB cultures and may be used in the future to assess the kinetics and patterns of differentiation in addition to the loss of pluripotency.

Three-dimensional in vitro tri-culture platform to investigate effects of crosstalk between mesenchymal stem cells, osteoblasts, and adipocytes.

The bone marrow niche for mesenchymal stem cells (MSCs) contains different amounts of bone and fat that vary with age and certain pathologies. How this dynamic niche environment may affect their differentiation potential and/or healing properties for clinical applications remains unknown, largely due to the lack of physiologically relevant in vitro models. We developed an enabling platform to isolate and study effects of signaling interactions between tissue-scale, laminated hydrogel modules of multiple cell types in tandem. We applied this platform to co- and tri-culture of primary human MSCs, osteoblasts, and adipocytes over 18 days in vitro. Each cell type was analyzed separately with quantitative polymerase chain reaction (qPCR) and histochemistry for several mesenchymal lineage markers. Distinct expression dynamics for osteogenic, adipogenic, chondrogenic, and myogenic transcriptional regulators resulted within each cell type depending on its culture setting. Incorporating this data into multivariate models produced latent identifiers of each emergent cell type dependent on its co- or tri-culture setting. Histological staining showed sustained triglyceride storage in adipocytes regardless of culture condition, but transient alkaline phosphatase activity in both osteoblasts and MSCs. Taken together, our results suggest novel emergent phenotypes for MSCs, osteoblasts, and adipocytes in bone marrow that are dependent on and result in part from paracrine interactions with their neighboring cell types.

Imaging single-cell signaling dynamics with a deterministic high-density single-cell trap array.

Stochasticity in gene expression, protein or metabolite levels contributes to cell-cell variations, the analysis of which could lead to a better understanding of cellular processes and drug responses. Current technologies are limited in their throughput, resolution (in space, time, and tracking individual cells instead of population average) and the ability to control cellular environment. A few microfluidic tools have been developed to trap and image cells; however, in most designs available to date, there is a compromise among loading efficiency, speed, the ability to trap single cells, and density or number of trapped cells. To meet the needs of single-cell imaging studies, we developed a microfluidic platform for high-throughput capture and imaging of thousands of single cells. The optimized trapping mechanism enables 95% of the traps to be occupied with single cells, with a trap density of 860 traps/mm(2). The dense array allows up to 800 cells to be imaged simultaneously with a 4x objective and a typical camera setup. Capture occurs with low shear and 94% viability after 24 h. This platform is compatible with other upstream microfluidic components for complex cell stimulation patterns, and we show here the ability to measure heterogeneity in calcium oscillatory behavior in genetically identical cells and monitor kinetic cellular response to chemical stimuli.

Predicting cytotoxic T-cell age from multivariate analysis of static and dynamic biomarkers.

Adoptive T-cell transfer therapy relies upon in vitro expansion of autologous cytotoxic T cells that are capable of tumor recognition. The success of this cell-based therapy depends on the specificity and responsiveness of the T cell clones before transfer. During ex vivo expansion, CD8+ T cells present signs of replicative senescence and loss of function. The transfer of nonresponsive senescent T cells is a major bottleneck for the success of adoptive T-cell transfer therapy. Quantitative methods for assessing cellular age and responsiveness will facilitate the development of appropriate cell expansion and selection protocols. Although several biomarkers of lymphocyte senescence have been identified, these proteins in isolation are not sufficient to determine the age-dependent responsiveness of T cells. We have developed a multivariate model capable of extracting combinations of markers that are the most informative to predict cellular age. To acquire signaling information with high temporal resolution, we designed a microfluidic chip enabling parallel lysis and fixation of stimulated cell samples on-chip. The acquisition of 25 static biomarkers and 48 dynamic signaling measurements at different days in culture, integrating single-cell and population based information, allowed the multivariate regression model to accurately predict CD8+ T-cell age. From surface marker expression and early phosphorylation events following T-cell receptor stimulation, the model successfully predicts days in culture and number of population doublings with R2=0.91 and 0.98, respectively. Furthermore, we found that impairment of early signaling events following T cell receptor stimulation because of long term culture allows prediction of costimulatory molecules CD28 and CD27 expression levels and the number of population divisions in culture from a limited subset of signaling proteins. The multivariate analysis highlights the information content of both averaged biomarker values and heterogeneity metrics for prediction of cellular age within a T cell population.

Parallel multi-time point cell stimulation and lysis on-chip for studying early signaling events in T cell activation.

Dynamics of complex signaling networks are important to many biological problems. Quantitative data at early time points after cellular stimulation are necessary for accurate model generation. However, the large amount of data needed is often extremely time-consuming and expensive to acquire with conventional methods. We present a two-module microfluidic platform for simultaneous multi-time point stimulation and lysis of T cells for early time point signaling activation with a resolution down to 20 s using only small amounts of cells and reagents. The key design features are rapid mixing of reagents and uniform splitting into eight channels for simultaneous collection of multi-time point data. Chaotic mixing was investigated via computational fluid dynamic modeling, and was used to achieve rapid and complete mixing. This modular device is flexible-with easy adjustment of the setup, a wide range of time points can be achieved. We show that treatment in the device does not elicit adverse cellular stress in Jurkat cells. The activation of six important proteins in the signaling cascade was quantified upon stimulation with a soluble form of alpha-CD3. The dynamics from device and conventional methods are similar, but the microdevice exhibits significantly less error between experiments. We envision this high-throughput format to enable simple and fast generation of large sets of quantitative data, with consistent sample handling, for many complex biological systems.