Fabrice Malergue

Novel Approach in Monocyte Intracellular TNF Measurement: Application to Sepsis-Induced Immune Alterations.

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 results were expressed as medians of fluorescence intensity (median: 16.1 [IQR: 14.5–19.1] vs. 5 [4.0–8.0], P = 0.0001) or as percentages of positive cells (99.7 [99.6–99.8] vs. 85 [74–97], P = 0.0001). 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 widely assessed 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.

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.

Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation.

Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers’ platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers’ platelets, presumably due to a biogenesis defect. Additionally, 25–29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7–9%) and autophagosome-like structures (0–3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.