Intracellular Flow Cytometry ("Phosphoflow") to Assess Signal Transduction in Rare Populations Such As Memory B Cell Subsets and Plasma Cells.

Intracellular flow cytometry is a powerful technique that can be used to interrogate signalling in rare cellular populations. The strengths of the technique are that massively parallel readouts can be gained from thousands of single cells simultaneously, and the assay is fast and relatively straightforward. This plate-based protocol enables different doses and different timepoints of stimulation to be assessed and has been optimized for rare B cell populations. Combining this technique with high-dimensional flow cytometry enables multiple signalling proteins to be measured with high confidence.

Design, synthesis, antiviral evaluation, and In silico studies of acrylamides targeting nsP2 from Chikungunya virus.

The Togaviridae family comprises several New- and Old-World Alphaviruses that have been responsible for thousands of human illnesses, including the RNA arbovirus Chikungunya virus (CHIKV). Firstly, it was reported in Tanzania in 1952 but rapidly it spread to several countries from Europe, Asia, and the Americas. Since then, CHIKV has been circulating in diverse countries around the world, leading to increased morbidity rates. Currently, there are no FDA-approved drugs or licensed vaccines to specifically treat CHIKV infections. Thus, there is a lack of alternatives to fight against this viral disease, making it an unmet need. Structurally, CHIKV is composed of five structural proteins (E3, E2, E1, C, and 6k) and four non-structural proteins (nsP1-4), in which nsP2 represents an attractive antiviral target for designing novel inhibitors since it has an essential role in the virus replication and transcription. Herein, we used a rational drug design strategy to select some acrylamide derivatives to be synthesized and evaluated against CHIKV nsP2 and also screened on CHIKV-infected cells. Thus, two regions of modifications were considered for these types of inhibitors, based on a previous study of our group, generating 1560 possible inhibitors. Then, the 24 most promising ones were synthesized and screened by using a FRET-based enzymatic assay protocol targeting CHIKV nsP2, identifying LQM330, 333, 336, and 338 as the most potent inhibitors, with Ki values of 48.6 ± 2.8, 92.3 ± 1.4, 2.3 ± 1.5, and 181.8 ± 2.5 µM, respectively. Still, their Km and Vmax kinetic parameters were also determined, along with their competitive binding modes of CHIKV nsP2 inhibition. Then, ITC analyses revealed KD values of 127, 159, 198, and 218 µM for LQM330, 333, 336, and 338, respectively. Also, their ΔH, ΔS, and ΔG physicochemical parameters were determined. MD simulations demonstrated that these inhibitors present a stable binding mode with nsP2, interacting with important residues of this protease, according to docking analyzes. Moreover, MM/PBSA calculations displayed that van der Waals interactions are mainly responsible for stabilizing the inhibitor-nsP2 complex, and their binding energies corroborated with their Ki values, having -198.7 ± 15.68, -124.8 ± 17.27, -247.4 ± 23.78, and -100.6 ± 19.21 kcal/mol for LQM330, 333, 336, and 338, respectively. Since Sindbis (SINV) nsP2 is similar to CHIKV nsP2, these best inhibitors were screened against SINV-infected cells, and it was verified that LQM330 presented the best result, with an EC50 value of 0.95 ± 0.09 µM. Even at 50 µM concentration, LQM338 was found to be cytotoxic on Vero cells after 48 h. Then, LQM330, 333, and 336 were evaluated against CHIKV-infected cells in antiviral assays, in which LQM330 was found to be the most promising antiviral candidate in this study, exhibiting an EC50 value of 5.2 ± 0.52 µM and SI of 31.78. The intracellular flow cytometry demonstrated that LQM330 is able to reduce the CHIKV cytopathogenic effect on cells, and also reduce the percentage of CHIKV-positive cells from 66.1% ± 7.05 to 35.8% ± 5.78 at 50 µM concentration. Finally, qPCR studies demonstrated that LQM330 was capable of reducing the number of viral RNA copies/µL, suggesting that CHIKV nsP2 is targeted by this inhibitor as its mechanism of action.

Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions.

Background: Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that ASCs regulate multiple aspects of biology through the secretion of various cytokines. In this regard, ICFC is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Methods: Paraformaldehyde plus saponin or the eBioscience Foxp3/Transcription Factor Staining Buffer Set were used to evaluate the non-specific intracellular retention of phycoerythrin-containing antibody conjugates by ASCs. Results: We showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of ASC intracellular protein expression compared with other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within ASCs. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between ASCs and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen ASCs expressed toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1ß. Conclusion: Increasing the number of centrifugation steps performed on ASCs post-fixation leads to inappropriate retention of phycoerythrin-containing antibody conjugates during ICFC.

Intracellular Flow Cytometry Improvements in Clinical Studies.

Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.

T cell cytokine responses to stimulation with Ureaplasma parvum in pregnancy.

UNLABELLED: Ureaplasma spp. are a common vaginal microorganism causally linked to inflammation-driven preterm birth (PTB). The nature of the immune response to Ureaplasma spp. may influence PTB risk. This study sought to define maternal T cell cytokine responses to in vitro stimulation with Ureaplasma parvum serovar 3 (UpSV3) in vaginally colonised (UP+) and non-colonised (UP-) pregnant women. Whole blood flow cytometry demonstrated an increase (p=0.027) in the baseline frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells in UP+ women. UpSV3 stimulation resulted in a significant and specific increase (p=0.001) in the frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells, regardless of vaginal colonisation status. UpSV3 stimulation also increased the frequency of IFNγ-positive CD3(+)CD4(+) T cells, particularly in the UP+ group (p=0.003). This is the first published study to examine T cell responses to Ureaplasma spp. EXPOSURE: Future appropriately-powered studies are needed to assess whether insufficient priming or a loss of tolerance to Ureaplasma spp. is occurring in UP+ women at risk of PTB.

Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.