Rapid Quantum Magnetic IL-6 Point-of-Care Assay in Patients Hospitalized with COVID-19

Interleukin-6 (IL-6) has been linked to several life-threatening disease processes. Developing a point-of-care testing platform for the immediate and accurate detection of IL-6 concentrations could present a valuable tool for improving clinical management in patients with IL-6-mediated diseases. Drawing on an available biobank of samples from 35 patients hospitalized with COVID-19, a novel quantum-magnetic sensing platform is used to determine plasma IL-6 concentrations. A strong correlation was observed between IL-6 levels measured by QDTI10x and the Luminex assay (r = 0.70, p-value p-value < 0.001). To validate the non-inferiority of QDTI to Luminex in terms of the accuracy of IL-6 measurement, two clinical parameters—the need for intensive care unit admission and the need for mechanical intubation—were chosen. IL-6 concentrations measured by the two assays were compared with respect to these clinical outcomes. Results demonstrated a comparative predictive performance between the two assays with a significant correlation coefficient. Conclusion: In short, the QDTI assay holds promise for implementation as a potential tool for rapid clinical decision in patients with IL-6-mediated diseases. It could also reduce healthcare costs and enable the development of future various biomolecule point-of-care tests for different clinical scenarios

Diagrams of the signaling proteins investigated in this study for each treatment case in R38+ and R38− compared to the RA-treated WT HL60 cells.

<p>For a given protein, solid black arrows indicate increased expression while white arrows indicate increased phosphorylation. Dashed black lines indicate an interaction that has previously been demonstrated in this lab by immunoprecipitation and/or FRET, with the exception of Fgr:Slp76 and Fgr:c-Raf. These two interactions are implied by the known binding of Slp76 and c-Raf to Src-family kinase members. Dashed black lines with arrowheads indicate phosphorylation (kinase) events known in the literature. Solid-filled factors vs. white-filled factors serve to clarify the expression indicated by the black arrows. Gradient-filled factors indicate expression but to a lower level than the RA-treated WT case. Downstream effects (CD11b expression, p47^phox expression, etc) are written in black if they occur, grey if they do not occur, or in gradient if they occur but to a lesser extent than the RA-treated WT HL60 case. A: In RA-treated WT HL60, CD38 is upregulated, along with its intracellular binding partners Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK show increased phosphorylation, while c-Raf is upregulated and shows increased phosphorylation at S259, S621 and S289/296/301. Differentiation markers that occur include CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production. B: In RA-treated R38+ HL60, CD38 is upregulated, but not Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. C: In RA-treated R38− HL60, CD38 is not upregulated, nor Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. D: In PP2-treated R38+ HL60, CD38 is partially upregulated (indicated by the gradient CD38), and Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. E: In PP2-treated R38− HL60, CD38 is not upregulated, but Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. F: In PP2+RA-treated R38+ HL60, CD38 is upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production. G: In PP2+RA-treated R38− HL60, CD38 is partially upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include partial CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production.</p

The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

<div><p>Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-<i>trans</i> retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47^phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.</p> </div

48 h PE fluorescence (CD38 surface expression) histograms for RA-resistant HL60 cells over 84 days.

<p>RA-resistant HL60 cells were labeled with PE-conjugated anti-CD38 and analyzed by flow cytometry. RA-resistant HL60 grown continuously in RA initially display elevated CD38 expression (histogram located in the gate P2). A growing subpopulation that does not express CD38, despite continual RA exposure, is indicated (by an arrow) as a low signal peak (top left graph). Over the course of 84 days, the number of cells that do not express CD38 continues to increase. The two distinct RA-resistant HL60 cells lines (bottom right graph), one with RA-inducible CD38 expression and one that has lost RA-inducible CD38 expression, were separated using Fluorescent Activated Cell Sorting (FACS) and the separated cell lines were termed R38+ and R38−, respectively.</p

Wright’s stain cytology for control and treated WT, R38+ and R38− HL60 cell lines.

<p>Control WT cells are round; RA-treated R38+ and R38− also retain a round, stem-like appearance. WT HL60 cells showed morphological changes consistent with differentiation toward granulocytes when treated with RA, PP2, or PP2+RA. Meanwhile the two RA-resistant HL60 cell lines, R38+ and R38−, both showed morphological changes consistent with differentiation only during PP2 and PP2+RA treatment, but not RA treatment alone.</p

48 h Western blot data for control and RA-treated WT, R38+ and R38− HL60 cells.

<p>A representative blot is displayed above its respective bar graph, and each bar graph (error bars represent standard error) presents the fold change respective to each control. The fold change was calculated after performing densitometry across three or more repeated blots. Note that the scale of the y-axis for each bar graph differs. A: There was no change in total ERK or MEK levels for any cell line. RA induced MEK and ERK phosphorylation in all three cell lines. Only RA-treated WT HL60 cells showed upregulation of c-Raf expression. Also, only RA-treated WT HL60 cells exhibited increased c-Raf phosphorylation at S259, S621 and S289/296/301. Neither R38+ nor R38− displayed increased c-Raf expression or phosphorylation after RA treatment. B: RA-treated WT HL60 cells showed upregulation of Lyn, Fgr, Vav1, and c-Cbl expression. RA-inducible Slp76 expression was evident in R38+ and R38−. Immunoprecipitation of c-Cbl followed by blotting of CD38 reveals that there is little CD38 and c-Cbl interaction in RA-treated R38+ compared to RA-induced WT HL60. GAPDH (not shown) served as loading control; c-Cbl (not shown) served as control for c-Cbl immunoprecipitation.</p

48 h Western blot data for control, PP2, RA and PP2+RA treated R38+ and R38

<p>−<b>.</b> A representative blot is displayed above its respective bar graph, and each bar graph (error bars represent standard error) presents the fold change respective to each control. The fold change was calculated after performing densitometry across three or more repeated blots. Note that the scale of the y-axis for each bar graph differs. A: There was no change in total ERK or MEK levels during any treatment for either R38+ or R38−. Interestingly, PP2 (alone and when co-treated with RA) decreased MEK and ERK phosphorylation in both R38+ and R38−. However PP2 and PP2+RA treatment induced upregulation of c-Raf expression and c-Raf phosphorylation at S259, S621 and S289/296/301 in both R38+ and R38−. B: PP2 and PP2+RA induced upregulation of Lyn, Vav1, c-Cbl and Slp76 expression. Y416 phosphorylation was reduced with PP2 treatment. C: Fgr can be detected with combined RA and PP2 treatment, but not with PP2 alone in both R38+ and R38−. Although the blots of Fgr were repeated three or more times, the blot that additionally displays the band for RA-treated WT HL60 (in the blot shown) was performed once. GAPDH (not shown) served as loading control.</p

CD38 and CD11b expression, cell cycle progression, and p47^phox expression in control, PP2- and/or RA-treated R38+ and R38− HL60 cells.

<p>Error bars represent standard error of at least three repeats. P values were calculated using a student’s t test and are compared between untreated respective control unless otherwise indicated. A: Control, RA-treated and/or PP2-treated R38+ and R38− HL60 cells were labeled with PE-conjugated CD38 and APC-conjugated CD11b and analyzed by flow cytometry. To measure the percent positive signal, controls were set to exclude 95% of the live cell population peak. After 48 h, PP2 induced significant (p<0.001) CD38 expression (∼50%) in R38+. Combined PP2+RA treatment in R38+ significantly increased (p<0.001) CD11b to levels comparable with those of RA-treated WT HL60 cells (∼40%). In R38−, co-treatment resulted in diminished yet still significant increases CD38 (p<0.05, nearly 40%) and CD11b (p<0.01, more than 20%) expression. PP2 treatment alone had no significant effect on CD38 or CD11b expression in R38−. B: To assess cell cycle progression, control and RA-treated WT, R38+ and R38− HL60 cells were stained with propidium iodide-hypotonic solution and analyzed by flow cytometry. Controls were gated to approximately 45% G1/G0 phase, 35% S phase and 20% G2/M phase. PP2 induced growth arrest (more than 60%) in both R38+ (p<0.01 alone and with RA) and R38− (p<0.01 with PP2 alone, p<0.05 with co-treatment). C: p47^phox expression was upregulated in both R38+ and R38− with combined PP2+RA treatment. PP2 alone did not increase p47^phox expression greater than RA treatment alone in either R38+ or R38−.</p