RESUMEN
BACKGROUND: Biomarker assays are often conducted on whole blood samples in the course of drug development studies. Because bacterial lipopolysaccharide (LPS) (endotoxin) contamination is known to cause spontaneous cytokine production by monocytes, contamination of blood collection tubes may interfere with biomarker assay results. METHODS: Whole blood from healthy donors was collected into plastic or glass sodium (Na(+))-heparin Vacutainer() blood collection tubes and heparinized syringes. Samples were analyzed for phosphoprotein response, cytokine production, and RNA expression. Tubes were tested for endotoxin contamination by use of the limulus amoebocyte lysate assay. RESULTS: Results of phospho-flow cytometry, branched DNA (bDNA), and ELISA assays indicated that a specific lot (#5339582) of plastic Na(+)-heparin Vacutainer tubes was highly contaminated with an endotoxinlike substance, and contamination was confirmed by the limulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changed expression was predictive of LPS stimulation were increased when whole blood was incubated in contaminated tubes for 6 or 18 h. Two additional lots of plastic tubes tested had detectable amounts of endotoxin sufficient to strongly alter phospho-flow cytometry analyses, as determined by the fold change in phosphorylation of p38 mitogen-activated protein kinase in response to tumor necrosis factor alpha and LPS. In contrast, 3 lots of glass tubes had substantially lower levels of spontaneous blood activation. CONCLUSIONS: Endotoxin contamination associated with tubes from 3 lots of a particular type of plastic Na(+)-heparin Vacutainer tube dramatically affected biomarker assay measurements. Prescreening these tubes is suggested before their use in clinical sample analysis.
Asunto(s)
Anticoagulantes , Recolección de Muestras de Sangre/instrumentación , Endotoxinas/análisis , Contaminación de Equipos , Heparina , Biomarcadores/sangre , Proteína C-Reactiva/biosíntesis , Proteína C-Reactiva/genética , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , Quimiocina CCL7/sangre , Quimiocina CCL7/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Interleucina-1beta/sangre , Interleucina-6/sangre , Interleucina-6/genética , Lipopolisacáridos/farmacología , Fosforilación , Plásticos , ARN Mensajero/sangre , Componente Amiloide P Sérico/biosíntesis , Componente Amiloide P Sérico/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/sangreRESUMEN
T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigen-presenting cells from PDL2-/- mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2-/- mice exhibited increased activation of CD4(+) and CD8(+) T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.
Asunto(s)
Tolerancia Inmunológica/fisiología , Activación de Linfocitos , Péptidos/metabolismo , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/inmunología , Pollos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Péptidos/genética , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1RESUMEN
In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.