G-Trap Assay II: Characterization of blood Leukocyte Functionality differentiates immune activation and immune suppression in bacteremia patient samples

Abstract

Sepsis is a severe organ dysfunction syndrome caused by a dysregulation of the immune system’s response to infection. Unfortunately, most infection-causing pathogens aren’t routinely detectable in real-time to enable targeted and lifesaving treatment. Thus, clinicians frequently have limited data on which to base treatment decisions. A complete blood count with differential is available within 24 h, and positive culture is only available in ∼30% of cases. Furthermore, a blood culture, the traditional gold standard for accurate diagnosis of bacteremia, may take up to five days for results, long after a clinical decision for sepsis management is required. Circulating leukocytes can sense chemotactic signals released by bloodborne pathogens or focal infections not in the bloodstream. Our earlier study showed that pathogen and host immune factors released in the bloodstream stimulated GTP binding of Ras homology (Rho) GTPases such as Rac1 in quiescent endothelial and human leukocytes after exposure to blood plasma from infected patients.1 In this study, we measured Rac1•GTP as a biomarker of immune functionality of peripheral blood monocytes and polymorphonuclear cells extracted from blood samples drawn for diagnostic use in blood culture assays; from 120 non-infected control patients and serial blood test samples from 28 patients with a confirmed diagnosis of bloodstream infection. 18 cases presented with Rac1•GTP elevation of ≥3 fold above that of control samples. Ten patients with normal or below-normal GTPase activity, accompanied by neutrophilia or pancytopenia. We used Principal Component Analysis to differentiate the 2D spatial distribution of infected patients and negative controls. Measuring differential leukocyte functionality in infected and control patients’ blood samples with the G-Trap assay may provide an innovative process for a real-time distinction between infection and non-infectious etiologies.

G-Trap Assay I: Small GTPases as sensitive immune response biomarkers for active bacteremia

“…We have initiated further work targeting the immune-performance status of infected patients' leukocytes to assess the functionality of leukocytes in infected patients and the net effect of empiric antibiotic treatment. [71] MATERIALS AND METHODS Effector Proteins. Glutathione S-transferase (GST) tagged-effector chimeras used for the studies are as follows: p21 activated kinase protein binding domain (PAK-1 PBD), a Rac1 effector, was obtained from Millipore Sigma, Rhotekin-RBD, a Rho effector protein, was purchased from Cytoskeleton (Denver, CO), RalGDS-RBD, a Rap1 effector protein expressed and purified from a plasmid, kindly provided by Dr. Burridge (UNC-Chapel Hill) [72], and GST-RILP was prepared as previously described.…”

“…We have initiated further work targeting the immune-performance status of infected patients’ leukocytes to assess the functionality of leukocytes in infected patients and the net effect of empiric antibiotic treatment. [71]…”