Serum level of S100A8/A9 as a biomarker for establishing the diagnosis and severity of community-acquired pneumonia in children

BackgroundS100A8/A9, which is a member of S100 proteins, may be involved in the pathophysiology of Community-acquired pneumonia (CAP) that seriously threatens children’s health. However, circulating markers to assess the severity of pneumonia in children are yet to be explored. Therefore, we aimed to investigate the diagnostic performance of serum S100A8/A9 level in determining the severity of CAP in children.MethodsIn this prospective and observational study, we recruited 195 in-hospital children diagnosed with CAP. In comparison, 63 healthy children (HC) and 58 children with non-infectious pneumonia (pneumonitis) were included as control groups. Demographic and clinical data were collected. Serum S100A8/A9 levels, serum pro-calcitonin concentrations, and blood leucocyte counts were quantified.ResultsThe serum S100A8/A9 levels in patients with CAP was 1.59 ± 1.32 ng/mL, which was approximately five and two times higher than those in healthy controls and those in children with pneumonitis, respectively. Serum S100A8/A9 was elevated parallelly with the clinical pulmonary infection score. The sensitivity, specificity, and Youden’s index of S100A8/A9 ≥1.25 ng/mL for predicting the severity of CAP in children was optimal. The area under the receiver operating characteristic curve of S100A8/A9 was the highest among the indices used to evaluate severity.ConclusionsS100A8/A9 may serve as a biomarker for predicting the severity of the condition in children with CAP and establishing treatment grading

Efficient production of sTNFRII-gAD fusion protein in large quantity by use of the modified CHO-S cell expression system.

TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system

Western blot analysis of sTNFRII-gAD in culture supernatants using monoclonal antibody against adiponectin (A) and sTNFRII (B).

<p>Lane 1, molecular weight markers (kDa); Lane 2, reducing conditions; Lane 3, nonreducing conditions.</p

Ultracentrifuge analysis of the trimerized sTNFRII-gAD.

<p>It gave a molecular weight of 165 kDa.</p

The metabolism of glucose and lactate of sTNFRII-gAD-expressing CHO-S cells in 3L and 5L fed-batch cultures in shaker bottles and AP30 bioreactor.

<p>(F: feed medium F001).</p

Schematic diagram of the recombinant plasmid pMH3-sTNFRII-gAD.

<p>GC-rich element: GC-rich non-coding DNA fragments; sTNFRII-gAD: soluble TNF receptor II and globular domain of adiponectin; actin promoter: chick beta-actin promoter; polyA: rabbit globulin polyA signal.</p

Efficient Production of sTNFRII-gAD Fusion Protein in Large Quantity by Use of the Modified CHO-S Cell Expression System - Figure 5

<p>The first-step purification of sTNFRII-gAD by anion exchange chromatography (A). The second-step purification of sTNFRII-gAD by a size exclusion chromatography (B). Peak 1: multimeric sTNFRII-gAD; peak 2: trimeric sTNFRII-gAD; peak 3: monomeric sTNFRII-gAD.</p

Biacore sensorgram of sTNFRII-gAD/sTNFRII-Fc binding to TNFα immobilized on a CM5 chip.

<p>Binding measurements were performed using serial dilutions of sTNFRII-gAD and sTNFRII-Fc. The concentrations of sTNFRII-gAD were 130, 65, 32.5, 16.25, 8.125, and 4.0625 nM (A), while the concentrations of sTNFRII-Fc were 520, 260, 130, 65, 32.5, 16.25, and 8.125 nM (B).</p

Dot blot analysis of sTNFRII-gAD protein in 96-well plates at 24 h with monoclonal antibody against TNFRII.

<p>Lane 1∶100, 75, 50, 25, 12.5 and 6.25 µg/ml of sTNFRII-Fc; Lane 2, 3, 4∶1/1, 1/3 and 1/6 dilutions of the supernatants from the 1st, 2nd and 3rd hyper-expression clones.</p