Novel Cytokine Multiplex Assay for Tear Fluid Analysis in Sjogren's Syndrome.

Purpose: Sjögren's syndrome (SS) is an autoimmune disease associated with ocular surface inflammation. The goals of this study were to establish a novel bead-based protein microarray for simultaneous analysis of 11 cytokines from tear fluid collected with Schirmer strips from patients with SS.Methods: Three to ten microliter of tear fluid was collected with Schirmer strips from both eyes of 13 healthy controls and 12 SS patients. Tear fluid was eluted from the Schirmer strips, total protein and concentrations of 11 different cytokines were analyzed.Results: The multiplex assay demonstrated high assay sensitivity with LoDs between 1.4 and 55.3 pg/µl with mean CVs between 3.7% and 9.7%. Statistically significant upregulation (p < .005) of eight cytokines was observed in SS patients compared to controls. Additionally, four patient cytokine values showed a significant inverse correlation (r<-0.7) with Schirmer strip readings.Conclusion: The assay offers analytical reliability for quantification of biomarkers in small amounts of tear fluid with potential utility for treatment monitoring of SS and other types of Dry Eye Disease.

First siRNA library screening in hard-to-transfect HUVEC cells.

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.

Neutral sphingomyelinase 1 deficiency in the mouse causes no lipid storage disease.

Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. To elucidate the function of the first cloned Mg(2+)-dependent, neutral sphingomyelinase (nSMase 1) in sphingomyelin catabolism and its potential role in signaling processes in a genetic and molecular approach, we have generated an nSMase 1-null mutant mouse line by gene targeting. The nSMase 1-deficient mice show an inconspicuous phenotype and no accumulation or changed metabolism of sphingomyelin or other lipids, despite grossly reduced nSMase activity in all organs except brain. We also addressed the recent proposal that nSMase 1 possesses lysophospholipase C activity. The unaltered metabolism of lysophosphatidylcholine or lyso-platelet-activating factor excludes the proposed role of nSMase 1 as a lysophospholipase C.