Evaluation of mRNA Biomarkers to Identify Risk of Hospital Acquired Infections in Children Admitted to Paediatric Intensive Care Unit

Objectives

Hospital-acquired infections (HAI) are associated with significant mortality and morbidity and prolongation of hospital stay, adding strain on limited hospital resources. Despite stringent infection control practices some children remain at high risk of developing HAI. The development of biomarkers which could identify these patients would be useful. In this study our objective was to evaluate mRNA candidate biomarkers for HAI prediction in a pediatric intensive care unit.

Design

Serial blood samples were collected from patients admitted to pediatric intensive care unit between March and June 2012. Candidate gene expression (IL1B, TNF, IL10, CD3D, BCL2, BID) was quantified using RT-qPCR. Comparisons of relative gene expression between those that did not develop HAI versus those that did were performed using Mann Whitney U-test.

Patients

Exclusion criteria were: age <28 days or ≥16 years, expected length of stay < 24 hours, expected survival < 28 days, end-stage renal disease and end-stage liver disease. Finally, 45 children were included in this study.

Main Results

The overall HAI rate was 30% of which 62% were respiratory infections. Children who developed HAI had a three-fold increase in hospital stay compared to those who did not (27 days versus 9 days, p<0.001). An increased expression of cytokine genes (IL1B and IL10) was observed in patients who developed HAI, as well as a pro-apoptosis pattern (higher expression of BID and lower expression of BCL2). CD3D, a key TCR co-factor was also significantly down-modulated in patients who developed HAI.

Conclusions

To our knowledge, this is the first study of mRNA biomarkers of HAI in the paediatric population. Increased mRNA expressions of anti-inflammatory cytokine and modulation of apoptotic genes suggest the development of immunosuppression in critically ill children. Immune monitoring using a panel of genes may offer a novel stratification tool to identify HAI risk.     

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.     

Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol

Among oxysterols oxidized at C7 (7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), 7β-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1β secretion. TNF-α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α- or β-hydroxyl radical position plays a key role in apoptosis and IL-1β secretion.