Production of proinflammatory and regulatory monokines in hemodialysis patients shown at a single-cell level.

Immunologic complications of chronic renal failure are associated with the overproduction of proinflammatory cytokines by monocytes. This is partly due to renal failure itself but is further enhanced by hemodialysis treatment with frequent contact between blood and dialyzer membranes. Previous studies have shown an imbalance of proinflammatory and regulatory monokines in these patients. This study examines monokine production in hemodialysis patients using for the first time a very sensitive method of cytokine detection at a single-cell level by flow cytometry ("cytoflow technique"). Monocytes were stained intracellularly for the production of interleukin-6 (IL-6) and IL-10 after 20 h of culture with lipopolysaccharide. It was shown that high levels of proinflammatory IL-6 in hemodialysis patients are due to an increased number of monocytes producing this cytokine, while IL-6 synthesis per cell remains unchanged. In contrast, elevated levels of regulatory IL-10 are due to an increased synthesis per cell. This study demonstrates that in healthy subjects there is a population of monocytes producing exclusively IL-10 after 20 h of stimulation by lipopolysaccharide. This distinct population of regulatory monocytes is infrequent in dialysis patients, in whom most of the IL-10-positive monocytes also produce IL-6. These findings indicate that overproduction of proinflammatory factors in dialysis patients is at least in part due to a loss of cytokine-specific differentiation in monocytes.

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Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

Journal of Virology ◽

10.1128/jvi.01448-16 ◽

2016 ◽

Vol 90 (20) ◽

pp. 9018-9028 ◽

Cited By ~ 33

Author(s):

G. Martrus ◽

A. Niehrs ◽

R. Cornelis ◽

A. Rechtien ◽

W. García-Beltran ◽

...

Keyword(s):

Single Cell ◽

Single Cell Level ◽

Cell Level ◽

Gag Protein ◽

Novel Approach ◽

Latently Infected ◽

Infected Cells ◽

First Time ◽

Kinetics Of ◽

Hiv 1

ABSTRACTHIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection ofgagpolmRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4+T cells, four cell populations were detected according to their expression levels of viral mRNA and protein.gagpolmRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4+T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle.IMPORTANCEEarly after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation orde novoinfection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected andde novo-infected cells dependent on the presence of viral RNA or protein.

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