[PMC free article] [PubMed] [Google Scholar] 35. unable to show a concomitant decrease in mRNA expression during relapses. This could be explained by down-regulation of gene expression at the translational rather than transcriptional level. In conclusion, it is conceivable that up-regulation of T-cell IL-13 production in children with active nephrotic relapse was associated with suppression of monocyte CD14 expression, down-regulating pro-inflammatory cytokine production, and could account for the increased susceptibility to bacterial sepsis seen in nephrotic children in active relapse. and and IL-8 in lipopolysaccharide-activated monocytes [16,19]. In addition, it down-regulates the expression of monocyte CD14 by suppressing CD14 at both the mRNA level and protein levels [16,20]. CD14 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein expressed primarily on monocytes and to a lesser extent on granulocytes . CD14 interacts with different components of both Gram-negative and Gram-positive bacteria, as well as fungi, defining it as a central pattern recognition molecule in innate immunity . It triggers monocyte activation in conjunction with serum lipopolysaccharide (LPS)-binding protein and serves as a receptor for both LPS and peptidoglycan [23,24], two of the most abundant constituents in the bacterial cell wall. Engagement of LPS to membrane-bound CD14 promotes an array of leucocyte function, including cellular activation, leading to the production of inflammatory cytokines such as IL-1 and tumour necrosis factor-(TNF-and IL-8 in children with SRNS in relapse and remission. In addition, the protein expression of membrane CD14 on monocytes, and the serum concentration of CD14 in its soluble form were assessed. MATERIALS AND METHODS Patient population Forty-one children (23 boys and 18 girls) with steroid-responsive nephrotic syndrome were included in a cross-sectional study, and examined in clinical relapse and/or remission. Their mean age was 9 5 years (range 2C22 years). In addition, 29 normal healthy controls were also studied. Relapse was defined as increased urinary protein excretion (Albustix 2+ for at least 3 consecutive days or 40 mg/m2 per h) and serum albumin 25 g/l. Remission was defined as serum albumin 35 g/l and normal urinary protein excretion (Albustix trace or negative for at least 3 consecutive days or 5 mg/m2/h). None of the patients included in the study had systemic SHR1653 lupus erythematosus, SHR1653 liver disease and other vasculities. Informed consent was obtained from the parents before blood sampling. The study was approved by the Ethics Committee of the National University SHCC Hospital. Heparinized blood was obtained from the study subjects. Children with SRNS were either not on any treatment during the time of blood sampling, or on prednisolone during relapse and remission, as they were steroid-dependent. None of them were on angiotensin-converting enzyme inhibitors, non-steroidal anti-inflammatory drugs, cyclosporin or cyclophosphamide. In addition, paired data both in nephrotic relapse and remission were available from 14 patients for mRNA expression studies and from 11 children for intracellular cytokine studies. Each patient pair was matched for prednisolone therapy; that is, they were either on or off prednisolone during the time of blood sampling. Cytoplasmic cytokine assay by flow cytometry One ml of freshly sampled blood was incubated with 1 was calculated based on the formula: (AS-AIC)-(US-UIC), where AS = activated sample, AIC-activated isotype SHR1653 control, US = unstimulated sample and UIC = unstimulated isotype control. The geometric mean fluorescence intensities were also determined. Isolation of monocytes Peripheral blood mononuclear cells (PBMCs) were isolated from patients with SRNS and healthy controls by standard density gradient centrifugation with Lymphoprep (Nycomed AS, Olso, Norway). The MACS-Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to obtain the pure monocyte fraction. This is based on an indirect magnetic labelling system for the isolation of monocytes from human PBMC by magnetic depletion of T cells, NK cells, B cells, dendritic cells and basophils. Briefly, the cells are indirectly magnetically labelled using a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56 and anti-IgE antibodies and MACS Microbeads coupled to an antihapten MoAb. The magnetically labelled cells are depleted by retaining them on a MACS column in the magnetic field of the MidiMACS. The cells were resuspended and pelleted in buffer in a.