The Loss of PTEN Allows TCR αβ Lineage Thymocytes to Bypass IL-7 and Pre-TCR–mediated Signaling

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates cell survival and proliferation mediated by phosphoinositol 3 kinases. We have explored the role of the phosphoinositol(3,4,5)P3-phosphatase PTEN in T cell development by analyzing mice with a T cell–specific deletion of PTEN. Ptenflox/floxLck-Cre mice developed thymic lymphomas, but before the onset of tumors, they showed normal thymic cellularity. To reveal a regulatory role of PTEN in proliferation of developing T cells we have crossed PTEN-deficient mice with mice deficient for interleukin (IL)-7 receptor and pre–T cell receptor (TCR) signaling. Analysis of mice deficient for Pten and CD3γ; Pten and γc; or Pten, γc, and Rag2 revealed that deletion of PTEN can substitute for both IL-7 and pre-TCR signals. These double- and triple-deficient mice all develop normal levels of CD4CD8 double negative and double positive thymocytes. These data indicate that PTEN is an important regulator of proliferation of developing T cells in the thymus

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule

Role of the interferons in CD64 and CD169 expressions in whole blood: Relevance in the balance between viral- or bacterial-oriented immune responses

International audienceIntroduction CD64 expression increases on neutrophils during bacterial infections. Recently an increase in CD169 expression has been discovered on monocytes during viral infections. Generally, interferons alpha (IFNs alpha) and IFNs gamma are key drivers of the infectious host immune response. The purpose of this study was to explore if a link exists between these IFNs and both biomarkers. Methods Whole blood samples from healthy volunteers were stimulated with either IFNs, interleukins, or infectious extracts, to mimic an infectious state. Expressions of CD64 and CD169 were assessed in these samples by multiple flow cytometry methods, over precise kinetics. Results The expression of CD64 was statistically higher in samples stimulated with IFN gamma, and CD169 in those stimulated with IFN alpha (and all other type I IFNs). Surface expressions are directly induced by their respective IFNs via Janus kinase/signal transducer and activator of transduction pathways within 6 to 8 hours of incubation. Mixing both types of IFNs seemed to indicate that they partially inhibit each other. Conclusions The induction of CD169 on monocytes and CD164 on neutrophils by type I and type II IFNs confirms the relevance of these markers for assessing between a viral- vs bacterial-oriented immune response

One-step White Blood Cell Extracellular Staining Method for Flow Cytometry

International audienceFlow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one- step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format.The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields

Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery

Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions

Direct freezing of whole blood enables analysis of leucocyte markers by flow cytometry: a proof-of-concept study

International audienceAim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry

Novel Approach in Monocyte Intracellular TNF Measurement

Objective: The monitoring of septic shock induced immunosuppression has been proposed to identify patients who could benefit from specific immunoadjuvant therapies. Among potential biomarkers to monitor immunological status, functional testing remains the gold standard because it directly measures the capacity of a cell population to respond to an immune challenge. We investigated a new approach in intracellular staining for flow cytometry to measure tumor necrosis factor (iTNF) produced in vitro by monocytes in response to lipopolysaccharide. Design, Setting, Subjects, and Interventions: Observational study in intensive care unit and immunology laboratory of a university medical center. Sixteen septic shock patients and eight control subjects were included. Main Results: Monocyte iTNF was determined by flow cytometry in whole blood and completed in 2.5 h according to a no-wash, no centrifuge procedure. Lipopolysaccharide challenge induced a tremendous expression of iTNF that was statistically more pronounced in controls than in patients. This was observed when resultswere expressed as medians of fluorescence intensity (median: 16.1 [IQR: 14.5–19.1] vs. 5 [4.0– 8.0], P¼0.0001) or as percentages of positive cells (99.7 [99.6–99.8] vs. 85 [74–97], P¼0.0001). iTNF expression was correlated to monocyte HLA-DR expression in patients and controls. Conclusions: These preliminary results illustrate the feasibility of immune functional testing on a routine manner in septic shock patients. They now deserve to be widelyassessed and validated in various intensive care unit conditions. This could be a major step to characterize the rapidly changing immune response overtime and thus permit personalized medicine

Flow cytometry evaluation of infection-related biomarkers in febrile subjects in the emergency department

International audienceAim: In an Emergency Department (ED), the etiological identification of infected subjects is essential. 13 infection-related biomarkers were assessed using a new flow cytometry procedure. Materials & methods: If subjects presented with febrile symptoms at the ED, 13 biomarkers' levels, including CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), were tested and compared with clinical records. Results: Among 50 subjects, 78% had bacterial infections and 8% had viral infections. nCD64 showed 82% sensitivity and 91% specificity for identifying subjects with bacterial infections. mCD169, HLA-ABC ratio and HLA-DR on monocytes had high values in subjects with viral infections. Conclusion: Biomarkers showed promising performances to improve the ED's infectious stratification

A Novel Rapid Method of Red Blood Cell and Platelet Permeabilization and Staining for Flow Cytometry AnalysisKey Terms

International audienceBACKGROUND Flow cytometry essentially focuses on surface-expressed proteins, with few protocols being devoted to intracellular components. We evaluated a two-step procedure using new formaldehyde-free permeabilization and staining reagents that allow the staining of platelets and red blood cells (RBCs) from whole blood. METHODS Citrated blood was treated with the new staining protocol (NSP) or control reagent (phosphate-buffered solution bovine serum albumin) and stained with antibodies against surface or intracellular markers. The effects of the NSP on cell integrity, morphology, and content were evaluated. RESULTS The NSP slightly reduced the cell count (similar to 20%) and changed the RBC morphology with a 42% mean diameter reduction. Conversely, the NSP did not affect platelet discoid morphology and led to a minor size decrease (11%). These morphological changes neither impelled a gating strategy modification nor interfered with the discrimination among populations based on surface markers. The NSP provided intracellular access to all the tested antigens: CD62P, FXIII, and CD63 in platelets and glycated and fetal hemoglobin (HbA1c and HbF) and nucleic acid in RBCs. The NSP gave excellent intra-assay precision with minimal impact on cell morphology and fluorescence labelling over time (up to 24 h). CONCLUSIONS With the ability to detect surface and intracellular antigens through a rapid preparation protocol without washing steps or toxic formaldehyde treatment, this NSP designed for research offers a marked improvement in the analysis of platelets and RBCs isolated directly from whole blood. Consequently, the NSP opens new avenues to investigate platelet degranulation and erythrocyte subpopulations