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The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Magda Rybczynska, Jean Baudrya, Eyer Klaus
ELSEVIER - 38(33) 5337-5342 - doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Nature Protocols - 15 2920–2955 - https://www.nature.com/articles/s41596-020-0354-0 - 2020
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations
Klaus Eyer, Carlos Castrillon, Guilhem Chenon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths and Jean Baudry
The Journal of Immunology - 205 8 - DOI: https://doi.org/10.4049/jimmunol.2000112 - 2020
One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.
Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers
Marco Molari, Klaus Eyer, Jean Baudry, Simona Cocco, Rémi Monasson
Nature Protocols - 15 2920–2955 - DOI: 10.7554/eLife.55678 - 2020
Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modeling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.
Apollonian packing in polydisperse emulsions
Sylvie Kwok, Robert Botet, Lewis Sharpnack and Bernard Cabane
Nature Protocols - 16 2426-2430 - https://doi.org/10.1039/C9SM01772K - 2020
We have discovered the existence of polydisperse high internal-phase-ratio emulsions (HIPE) in which the internal-phase droplets, present at 95% volume fraction, remain spherical and organise themselves according to Apollonian packing rules. These polydisperse HIPEs are formed by emulsifying oil dropwise in a surfactant-poor aqueous continuous phase. After stirring has ceased, their droplet size distributions begin to evolve spontaneously and continuously through coalescence towards well-defined power laws with the Apollonian exponent. Small-angle X-ray Scattering performed on aged HIPEs demonstrate that the droplet packing structure agrees with that of a numerically simulated random Apollonian packing. We argue that when such concentrated emulsions are allowed to evolve, the coalescing droplets must obey volume and sphericity conservation. This leads to a mechanism that differs from typical coalescence in dilute emulsions.

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay
Millie Heo, Guilhem Chenon, Carlos Castrillon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths, Jean Baudry & Klaus Eyer
Communications Biology - 614 5337-5342 - https://doi.org/10.1038/s42003-020-01296-3 - 2020
Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

Swelling, dewetting and breakup in thin polymer films for cultural heritage
Amélie Castel, Philipp Gutfreund, Bernard Cabaned and Yahya Rharbi
Soft Matter - 16 1485-1497 - https://doi.org/10.1039/C9SM01976F - 2020
The removal of ultrathin amorphous polymer films in contact with nonsolvent/solvent binary mixtures is addressed by means of neutron reflectometry and atomic force microscopy. The high resolution of neutron scattering makes it possible to resolve the distribution profiles of heavy water and benzyl alcohol inside Laropal®A81, often employed as a protective varnish layer for Culture Heritage in restoration of easel paintings. The swelling kinetics and distribution profiles were recorded as a function of time and increasing benzyl alcohol concentration in water. The varnish film swells by penetration of the good solvent. At higher concentrations water-filled cavities appear inside the varnish and grow with time. Contrary to homogeneous dissolution dewetting is observed at late stages of exposure to the liquid which leads to the Breakup of the film. The high resolution measurements are compared to bulk behaviour characterized by the ternary phase diagram and the Flory–Huggins interaction parameters are calculated and used to predict the swelling and solvent partition in the films. Distinct differences of the thin film to bulk behaviour are found. The expectations made previously for the behaviour of solvent/non-solvent mixtures on the removal of thin layers in the restoration of easel paintings should be revised in view of surface interactions.

Using an Untargeted Metabolomics Approach to Identify Salivary Metabolites in Women with Breast Cancer
Daniele Xavier Assad ,Ana Carolina Acevedo, Elisa Cançado Porto Mascarenhas, Ana Gabriela Costa Normando,Valérie Pichon,Helene Chardin,Eliete Neves Silva Guerra and Audrey Combes
Metabolites - 10(12) 1727-1751 - doi.org/10.3390/metabo10120506 - 2020
Metabolic alterations are a hallmark of the malignant transformation in cancer cells, which is characterized by multiple changes in metabolic pathways that are linked to macromolecule synthesis. This study aimed to explore whether salivary metabolites could help discriminate between breast cancer patients and healthy controls. Saliva samples from 23 breast cancer patients and 35 healthy controls were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry and a bioinformatics tool (XCMS Online), which revealed 534 compounds, characterized by their retention time in reverse-phase liquid chromatography and by the m/z ratio detected, that were shared by the two groups. Using the METLIN database, 31 compounds that were upregulated in the breast cancer group (p < 0.05) were identified, including seven oligopeptides and six glycerophospholipids (PG14:2, PA32:1, PS28:0, PS40:6, PI31:1, and PI38:7). In addition, pre-treatment and post-treatment saliva samples were analyzed for 10 patients who experienced at least a partial response to their treatment. In these patients, three peptides and PG14:2 were upregulated before but not after treatment. The area under the curve, sensitivity, and specificity for PG14:2 was 0.7329, 65.22%, and 77.14%, respectively. These results provide new information regarding the salivary metabolite profiles of breast cancer patients, which may be useful biomarkers. View Full-Text

Synthesis and characterization of molecularly imprinted polymers for the selective extraction of oxazepam from complex environmental and biological samples
Daniele Xavier Assad Elisa Cançado Porto Mascarenhas Ana Gabriela Costa Normando Hélène Chardin Gustavo Barcelos Barra Riccardo Pratesi Yanna Karla De Medeiros Nóbrega Ana Carolina Acevedo Eliete Neves Silva Guerra
Molecular and Clinical Oncology - 155-161 - doi.org/10.3892/mco.2020.2062 - 2020
The early detection of breast cancer enables the use of less aggressive treatment and increases patient survival. The transmembrane glycoprotein mucin 1, which is also known as cancer antigen 15‑3 (CA15‑3), is aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and serves a crucial role in the progression of the disease. CA15‑3 is currently used as a marker of breast cancer. In the present study, CA15‑3 concentrations in saliva and blood of patients with breast cancer were evaluated to test new assays to detect salivary CA15‑3 in addition to ELISA and its diagnostic value. To the best of our knowledge, there are no previous reports of the use of chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLIA) in saliva. Saliva and blood were collected on the same day from patients with breast cancer (n=26) and healthy controls (n=28). For each subject, the level of serum CA15‑3 was measured using ECLIA, and the level of salivary CA15‑3 was measured using ECLIA, CLIA and enzyme‑linked immunosorbent assay (ELISA). ELISA and CLIA were able to detect CA15‑3 in saliva; however, ECLIA could not detect salivary CA15‑3. There was no significant difference between the mean serum and salivary CA15‑3 levels in patients with breast cancer or healthy controls. The levels of CA15‑3 were highest for luminal breast cancer subtypes and stage IV cases. A moderate correlation was observed between salivary and serum CA15‑3 levels as measured by ELISA in breast cancer patients (r=0.56; P=0.0047). The results demonstrated that ECLIA was not a good method to detect salivary CA15‑3, although it is the gold standard for detecting serum CA15‑3. The presence of CA15‑3 in saliva was confirmed, and this will be useful in future research. Further investigations are necessary to confirm the ability to detect salivary CA15‑3 and its correlation with serum CA15‑3.
How high resolution mass spectrometry can help for the accurate quantification of difficult fragrance allergens
Pierre‐Alain Remy, C. Pérès, J. Dugay, E. Corbi, Nathalie David, J. Vial
Flavour and Fragrance Journal - 36(1) - DOI:10.1002/ffj.3639 - 2020
Two high‐resolution mass spectrometers (HRMS) with different analyzer technology, Orbitrap and hybrid quadrupole time‐of‐flight (QTOF), were compared with a low‐resolution mass spectrometer, quadrupole, to analyse a set of 35 difficult allergens. These difficult allergens are commonly coeluted fragrance allergens with matrix compounds, using standard gas chromatography‐mass spectrometer conditions, from the extended list of the Scientific Committee on Consumer Safety (SCCS). Although the fundamental role of chromatographic separation has been demonstrated many times, the aim of this work is to demonstrate the benefits of high‐resolution. The added value of high‐resolution was illustrated in both a qualitative and a quantitative way. For qualitative aspect, the high resolution extracted ion signals of these two detectors were compared with the low‐resolution extracted ion signals. About 50% of the coeluted cases observed with the low‐resolution detector are easily resolved by the two high‐resolution detectors. For the quantitative aspect, an accuracy profile methodology and a performance metric were used to propose an overall evaluation. The Orbitrap mass spectrometer demonstrated a better overall performance, while the QTOF presented similar or even lower quantification performances than the quadrupole on the set of analysed fragrance

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