ABSTRACT: Neonatal gram-negative sepsis is often characterized by a fulminat clinical course compared to adults resulting in higher morbidity and mortality. Genome-wide gene expression analysis can provide insights into the molecular alterations in sepsis. To evaluate in vitro activation of the neonatal and adult immune system, gene expression patterns were compared in mononuclear cells from cord (CBMNC) and adult peripheral blood (APBMNC). To better understand the influence of early molecular signals on the effects of sepsis, Affymetrix gene profiling (8475 genes) was done on RNA isolated from CBMNC and APBMNC without and after incubation with 100 ng/ml lipopolysaccharide (LPS). We demonstrated significant alterations in the expression of 108 CBMNC and APBMNC genes compared with basal levels, 188 significant changes in CBMNC, and 97 in APBMNC, including cytokines, chemokines, and immunoregulatory genes. Furthermore, we found five genes showing a significant interaction effect between cell-type and LPS-stimulation, including tumor necrosis factor receptor superfamily, member 6 (FAS), absent in melanoma 2 (AIM2), malic enzyme 1 (ME1), hemoglobin epsilon 1 (HBE1), and trans-prenyltransferase (TPRT). These results provide further support for a marked difference in the pathogenesis of neonatal and adult sepsis and may stimulate additional studies to investigate some of the altered genes as potential new targets for diagnostic tools and therapeutic strategies. Experiment Overall Design: The statistical analysis was carried out using the software package R, version 1.9.1, together with libraries gcrma and limma of the Bioconductor Project, version 1.4. The data preprocessing steps, background-adjustment, normalization and computation of GCRMA gene expression measures, were performed according to Wu et al. For the statistical analysis empirical Bayes inference for linear models with factors treatment (basal and LPS stimulated) and cell type (CBMNC and APBMNC) and their interaction was used. From there moderated t-statistics based on shrinkage of the estimated sample variance towards a pooled estimate and corresponding p-values were calculated for comparisons LPS stimulated vs. basal expression for adult and neonatal MNC, and neonatal vs. adult MNC with and without LPS-treatment, respectively, as well as for the interaction effect of the factors treatment and cell type. P-values for each comparison were adjusted according to Benjamini and Hochberg to control the false discovery rate. We used a threshold of 0.01 for the adjusted p-values and selected only those probe sets which showed a fold change => 2
