Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
J Immunol Methods ; 470: 59-63, 2019 07.
Article in English | MEDLINE | ID: mdl-31082373

ABSTRACT

BACKGROUND: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression. METHODS: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR. RESULTS: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 µL to 5 µL, lysis from 1 mL to 500 µL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed. CONCLUSIONS: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications.


Subject(s)
Flow Cytometry/methods , HLA-DR Antigens/blood , Receptors, IgG/blood , Shock, Septic/diagnosis , Staining and Labeling/methods , Aged , Biomarkers/blood , Case-Control Studies , Female , Gene Expression , HLA-DR Antigens/genetics , Humans , Male , Monocytes/metabolism , Monocytes/pathology , Neutrophils/metabolism , Neutrophils/pathology , Receptors, IgG/genetics , Shock, Septic/blood , Shock, Septic/genetics , Shock, Septic/mortality , Survival Analysis
2.
Crit Care ; 23(1): 131, 2019 Apr 17.
Article in English | MEDLINE | ID: mdl-30995946

ABSTRACT

BACKGROUND: Sepsis is the leading cause of mortality for critically ill patients worldwide. Patients develop T lymphocyte dysfunctions leading to T-cell exhaustion associated with increased risk of death. As interleukin-7 (IL-7) is currently tested in clinical trials to reverse these dysfunctions, it is important to evaluate the expression of its specific CD127 receptor on the T-cell surface of patients with septic shock. Moreover, the CD127lowPD-1high phenotype has been proposed as a T-cell exhaustion marker in chronic viral infections but has never been evaluated in sepsis. The objective of this study was first to evaluate CD127 and CD127lowPD-1high phenotype in septic shock in parallel with functional T-cell alterations. Second, we aimed to reproduce septic shock-induced T-cell alterations in an ex vivo model. METHODS: CD127 expression was followed at the protein and mRNA levels in patients with septic shock and healthy volunteers. CD127lowPD-1high phenotype was also evaluated in parallel with T-cell functional alterations after ex vivo activation. To reproduce T-cell alterations observed in patients, purified T cells from healthy volunteers were activated ex vivo and their phenotype and function were evaluated. RESULTS: In patients, neither CD127 expression nor its corresponding mRNA transcript level was modified compared with normal values. However, the percentage of CD127lowPD-1high T cells was increased while T cells also presented functional alterations. CD127lowPD-1high T cells co-expressed HLA-DR, an activation marker, suggesting a role for T-cell activation in the development of this phenotype. Indeed, T-cell receptor (TCR) activation of normal T lymphocytes ex vivo reproduced the increase of CD127lowPD-1high T cells and functional alterations following a second stimulation, as observed in patients. Finally, in this model, as observed in patients, IL-7 could improve T-cell proliferation. CONCLUSIONS: The proportion of CD127lowPD-1high T cells in patients was increased compared with healthy volunteers, although no global CD127 regulation was observed. Our results suggest that TCR activation participates in the occurrence of this T-cell population and in the development of T-cell alterations in septic shock. Furthermore, we provide an ex vivo model for the investigation of the pathophysiology of sepsis-induced T-cell immunosuppression and the testing of innovative immunostimulant treatments.


Subject(s)
Shock, Septic/blood , T-Lymphocytes/physiology , Aged , Female , France , Humans , Interleukin-7/analysis , Interleukin-7/blood , Interleukin-7/physiology , Interleukin-7 Receptor alpha Subunit/analysis , Interleukin-7 Receptor alpha Subunit/blood , Lymphocyte Count/methods , Male , Middle Aged , Phenotype , Programmed Cell Death 1 Receptor/analysis , Programmed Cell Death 1 Receptor/blood , Shock, Septic/physiopathology
SELECTION OF CITATIONS
SEARCH DETAIL
...