Intermediate monocytes exhibit higher levels of TLR2, TLR4 and CD64 early after congenital heart surgery.

INTRODUCTION: Congenital heart surgery with cardiopulmonary bypass (CPB) initiates an immune response which frequently leads to organ dysfunction and a systemic inflammatory response. Complications associated with exacerbated immune responses may severely impact the postoperative recovery. The objective was to describe the characteristics of monocyte subpopulations and neutrophils at the level of pattern recognition receptors (PRR) and the cytokine response after CPB in infants. METHODS: An observational cohort study was conducted between June 2016 and June 2017 of infants < 2 years of age, electively admitted for surgical correction of acyanotic congenital heart defects using CPB. Fourteen blood samples were collected sequentially and processed immediately during and up to 48 h following cardiac surgery for each patient. Flow cytometry analysis comprised monocytic and granulocytic surface expression of CD14, CD16, CD64, TLR2, TLR4 and Dectin-1 (CLEC7A). Monocyte subpopulations were further defined as classical (CD14++/CD16-), intermediate (CD14++/CD16+) and nonclassical (CD14+/CD16++) monocytes. Plasma concentrations of 14 cytokines, including G-CSF, GM-CSF, IL-1ß, IL-1RA, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, TNF-α, IFN-γ, MIP-1ß (CCL4) and TGF-ß1, were measured using multiplex immunoassay for seven points in time. RESULTS: Samples from 21 infants (median age 7.4 months) were analyzed by flow cytometry and from 11 infants, cytokine concentrations were measured. Classical and intermediate monocytes showed first receptor upregulation with an increase in CD64 expression four hours post CPB. CD64-expression on intermediate monocytes almost tripled 48 h post CPB (p < 0.0001). TLR4 was only increased on intermediate monocytes, occurring 12 h post CPB (p = 0.0406) along with elevated TLR2 levels (p = 0.0002). TLR4 expression on intermediate monocytes correlated with vasoactive-inotropic score (rs = 0.642, p = 0.0017), duration of ventilation (rs = 0.485, p = 0.0259), highest serum creatinine (rs = 0.547, p = 0.0102), postsurgical transfusion (total volume per kg bodyweight) (rs = 0.469, p = 0.0321) and lowest mean arterial pressure (rs = -0.530, p = 0.0135). Concentrations of IL-10, MIP-1ß, IL-8, G-CSF and IL-6 increased one hour post CPB. Methylprednisolone administration in six patients had no significant influence on the studied surface receptors but led to lower IL-8 and higher IL-10 plasma concentrations. CONCLUSIONS: Congenital heart surgery with CPB induces a systemic inflammatory process including cytokine response and changes in PRR expression. Intermediate monocytes feature specific inflammatory characteristics in the 48 h after pediatric CPB and TLR4 correlates with poorer clinical course, which might provide a potential diagnostic or even therapeutic target.

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway-suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

A Microcavity Array-Based 4D Cell Culture Platform.

(1) Background: We describe a 4D cell culture platform with which we tried to detect and to characterize migration dynamics of single hematopoietic stem cells in polymer film microcavity arrays integrated into a microtiter plate. (2) Methods: The system was set up with CD34-expressing KG-1a cells as a surrogate for hematopoietic stem cells. We then evaluated the system as an artificial hematopoietic stem cell niche model comprised of a co-culture of human hematopoietic stem cells from cord blood (cord blood CD34+ cells, hHSCs) and human mesenchymal stromal cells (hMSCs) from bone marrow over a period of 21 days. We used a software-based cell detection method to count single hematopoietic stem cells (HSCs) in microcavities. (3) Results: It was possible to detect single HSCs and their migration behavior within single microcavities. The HSCs displayed a pronounced migration behavior with one population of CD34-expressing cells located at the bottom of the microcavities and one population located in the middle of the microcavities at day 14. However, at day 21 the two populations seemed to unite again so that no clear distinction between the two was possible anymore. (4) Conclusions: Single cell migration detection was possible but microscopy and flow cytometry delivered non-uniform data sets. Further optimization is currently being developed.