ABSTRACT
AIMS: To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms. METHODS AND RESULTS: This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay. CONCLUSIONS: A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens.
Subject(s)
Bacteremia/diagnosis , Blood/microbiology , Parasitemia/diagnosis , Real-Time Polymerase Chain Reaction/standards , Viremia/diagnosis , Blood/parasitology , Blood/virology , Humans , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.
Subject(s)
Chemotaxis , Immediate-Early Proteins/metabolism , Lysophospholipids , Platelet-Derived Growth Factor/pharmacology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Animals , Arrestins/metabolism , Becaplermin , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Chemotaxis/drug effects , Gene Deletion , Humans , Immediate-Early Proteins/genetics , Mice , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins c-sis , Receptor Cross-Talk , Receptors, Lysophospholipid , Receptors, Platelet-Derived Growth Factor/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , Sphingosine/pharmacology , Transfection , beta-ArrestinsABSTRACT
Sphingolipid signaling pathways have been implicated in many critical cellular events. Sphingosine-1-phosphate (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg1 gene in mice. Edg1(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E12.5 and E14.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-1 mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-1 to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.
Subject(s)
Cardiovascular System/embryology , GTP-Binding Proteins/metabolism , Immediate-Early Proteins/genetics , Lysophospholipids , Muscle, Smooth, Vascular/embryology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Animals , Blood Vessels/embryology , Cell Movement , Fibroblasts/cytology , Fibroblasts/drug effects , Heart/embryology , Homozygote , Mice , Mice, Knockout , Phenotype , Receptors, Lysophospholipid , Signal Transduction , Sphingosine/metabolism , Sphingosine/pharmacologyABSTRACT
Sphingosine-1-phosphate (SPP), formed by sphingosine kinase, is the ligand for EDG-1, a GPCR important for cell migration and vascular maturation. Here we show that cytoskeletal rearrangements, lamellipodia extensions, and cell motility induced by platelet-derived growth factor (PDGF) are abrogated in EDG-1 null fibroblasts. However, EDG-1 appears to be dispensable for mitogenicity and survival effects, even those induced by its ligand SPP and by PDGF. Furthermore, PDGF induced focal adhesion formation and activation of FAK, Src, and stress-activated protein kinase 2, p38, were dysregulated in the absence of EDG-1. In contrast, tyrosine phosphorylation of the PDGFR and activation of extracellular signal regulated kinase (ERK1/2), important for growth and survival, were unaltered. Our results suggest that EDG-1 functions as an integrator linking the PDGFR to lamellipodia extension and cell migration. PDGF, which stimulates sphingosine kinase, leading to increased SPP levels in many cell types, also induces translocation of sphingosine kinase to membrane ruffles. Hence, recruitment of sphingosine kinase to the cell's leading edge and localized formation of SPP may spatially and temporally stimulate EDG-1, resulting in activation and integration of downstream signals important for directional movement toward chemoattractants, such as PDGF. These results may also shed light on the vital role of EDG-1 in vascular maturation.
Subject(s)
Cell Movement/physiology , Immediate-Early Proteins/physiology , Lysophospholipids , Protein-Tyrosine Kinases/metabolism , Pseudopodia/physiology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Receptors, Platelet-Derived Growth Factor/metabolism , Sphingosine/analogs & derivatives , src-Family Kinases/metabolism , 3T3 Cells , Animals , Apoptosis/drug effects , Biological Transport/drug effects , Cell Division/drug effects , Cells, Cultured , Chemotaxis/drug effects , Cytoskeleton/drug effects , DNA/biosynthesis , DNA/drug effects , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Enzyme Activation , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genotype , Green Fluorescent Proteins , Immediate-Early Proteins/genetics , Luminescent Proteins/drug effects , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Knockout , Microscopy, Confocal , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Platelet-Derived Growth Factor/pharmacology , Receptors, Lysophospholipid , Receptors, Platelet-Derived Growth Factor/physiology , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Sphingosine/pharmacology , Time FactorsABSTRACT
An amended version of the Short Form 36 health survey questionnaire (SF-36), which has been suggested as being more suitable for self-completion in older adults, was evaluated among a group of elderly subjects with Parkinson's disease (PD). In the subjects who were able to fully complete the modified SF-36 unaided, the severity of PD was broadly reflected by low reported levels of functioning and well-being with this measure. However, the modified questionnaire still did not overcome the problem of missing responses from elderly subjects. A total of 24% of respondents missed one or more of the 36 statements and 80% of missing responses were concentrated in the three statements which had been modified. The SF-36 in its original form would need to be combined with a disease-specific measure to adequately evaluate the health status of older adults with PD.
Subject(s)
Geriatric Assessment , Health Status , Health Surveys , Parkinson Disease/psychology , Surveys and Questionnaires/standards , Activities of Daily Living , Age Factors , Aged , Humans , Reproducibility of Results , Severity of Illness IndexABSTRACT
OBJECTIVE: To determine the utility of an observer-based rating scale to detect depression in patients without aphasia. DESIGN: Correlation analysis between the Stroke Aphasia Depression Questionnaire, shortened version (SADQ-10) and a validated self-rating measure of depression, the Geriatric Depression Scale (GDS). The sensitivity and specificity of the SADQ-10 were also calculated. SETTING: Stroke rehabilitation unit. SUBJECTS: Sixty-five stroke patients without significant aphasia undergoing rehabilitation. INTERVENTIONS: All patients were assessed with the GDS-15 and the SADQ-10. RESULTS: The SADQ-10 at a cut-point of 14 out of 30 had a sensitivity of 70% and a specificity of 77% to detect depression. This measure demonstrated good internal consistency but showed only a modest correlation with the GDS-15 (r = 0.40, p < 0.001). CONCLUSION: In the population under study the SADQ-10 did not appear to be a valid measure of depression compared with the GDS and, therefore, may not be suitable for use in patients without significant aphasia.
Subject(s)
Aphasia/etiology , Depression/etiology , Stroke Rehabilitation , Aged , Female , Humans , Male , Sensitivity and Specificity , Stroke/complications , Surveys and QuestionnairesABSTRACT
OBJECTIVE: To develop a brief, valid and reliable self-report scale for the assessment of activities of daily living in Parkinson's disease (PD). DESIGN: Self-report questionnaire development. SUBJECTS: One hundred and seventy subjects with a diagnosis of clinically probable PD living in the community. MEASURES: The self-rating scale--Parkinson's Disease Activities of Daily Living Scale (PADLS), Webster Scale, CAMCOG neuropsychological test,15-item Geriatric Depression Scale (GDS-15) and the self-rated Parkinson's Disease Quality of Life (PDQL) questionnaire. METHODS: The PADLS was initially validated and test-retest reliability assessed in a group of PD patients (n = 38). Next a convenience sample of 132 patients was drawn from a community-based PD register. Subjects were invited to complete the PADLS, PDQL, GDS-15, Webster scale and CAMCOG test. RESULTS: The PADLS correlated significantly with increasing age, duration of illness, disease severity, increasing depression, impaired cognition and poorer health-related quality of life. CONCLUSION: The PADLS was found to be a reliable and valid measure of ADL, demonstrating acceptable internal consistency and strong associations with existing measurers of disease severity, depression, cognitive screening and health-related quality of life. The PADLS allows patients to subjectively report the impact that PD has upon daily activities and will complement existing formal clinical measures in PD.
Subject(s)
Activities of Daily Living , Disability Evaluation , Parkinson Disease/physiopathology , Aged , Female , Humans , MaleABSTRACT
AIM: to compare the two new executive function tests of the revised Cambridge Cognitive Examination (CAMCOG-R), a bedside measure of cognitive function, with existing neuropsychological assessments of executive function in elderly stroke survivors. METHODS: we assessed 83 stroke survivors at 1 and 3 months post-stroke with the new CAMCOG-R, the Weigl colour form sorting test and Raven's coloured progressive matrices. We assessed functional recovery with the Barthel index and depression with the self-report 15-item geriatric depression scale. We used descriptive statistics, Pearson correlation coefficients, paired t-tests and principal axis factor analyses to interpret the data. RESULTS: the new CAMCOG-R executive functioning tests showed moderate correlation with the Weigl and Raven tests (P<0.01). Improved functional outcome as measured by the Barthel index was significantly associated with higher executive function test scores (P<0.05). Depression was significantly associated with poorer performance on all tasks of executive function (P<0.05). A factor analysis of the scores on all of the neuropsychological tests revealed a single strong factor that accounted for 66% of the variance. The CAMCOG-R and the executive functioning subscales used in this population established sensitivity to change over time. CONCLUSION: although the new executive tests of the CAMCOG-R compared reasonably well with the Weigl and Raven neuropsychological tests, the extra time taken to administer the CAMCOG-R may not be justified. The new CAMCOG-R executive function tests were vulnerable to the effects of depression. Finally, the executive function tests might have provided more of a global measure of cognitive function, raising doubts about their construct validity in our patient population.
Subject(s)
Cognition Disorders/diagnosis , Neuropsychological Tests/standards , Stroke/psychology , Aged , Female , Follow-Up Studies , Humans , Male , Sensitivity and Specificity , SurvivorsABSTRACT
EDG-1, encoded by the endothelial differentiation gene-1, is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP) that has been shown to stimulate angiogenesis and cell migration in cultured endothelial cells. Unexpectedly, EDG-1 knockout embryos had a normal blood vessel network, vasculogenesis and angiogenesis, but died in utero owing to massive haemorrhaging as a result of failure of smooth muscle cells and pericytes to migrate around the circumference and reinforce endothelial tubes [Liu, Wada, Yamashita, Mi, Deng, Hobson, Rosenfeldt, Nava, Chae, Lee, et al. (2000) J. Clin. Invest. 106, 951-961]. This vascular maturation defect is similar to the phenotype of mice homozygous for disrupted alleles of platelet-derived growth factor B-subunit homodimer (PDGF-BB) or its receptor PDGFR-beta. We found that fibroblasts from EDG-1 null embryos did not migrate toward PDGF or SPP, and inhibition of motility correlated with defective activation of the small guanosine triphosphatase Rac, which is required for lamellipodia formation and directional locomotion [Hobson, Rosenfeldt, Barak, Olivera, Poulton, Caron, Milstien, and Spiegel (2001) Science 291, 1800-1803]. Moreover, we showed that PDGF-directed cell migration requires both sphingosine kinase activation and expression of EDG-1, suggesting a functional link between PDGF signalling and EDG-1. Indeed, treatment of wild-type cells with PDGF transactivated EDG-1 as determined by translocation of beta-arrestin and phosphorylation of EDG-1. These findings reveal a new paradigm for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility. Our observations might also clarify the role of EDG-1 in vascular maturation and angiogenesis.
Subject(s)
Immediate-Early Proteins/metabolism , Immediate-Early Proteins/physiology , Lysophospholipids , Platelet-Derived Growth Factor/metabolism , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Cell Line , Cell Movement , Chemotaxis , Humans , Models, Biological , Receptors, Lysophospholipid , Signal Transduction , Sphingosine/metabolism , Transcriptional ActivationABSTRACT
EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [(32)P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [(32)P]SPP was saturable, demonstrating high affinity (K(D) = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a G(i/o) protein, resulting in the activation of growth-related signaling pathways. (Blood. 2000;95:2624-2629)
Subject(s)
Lysophospholipids , Receptors, Cell Surface/metabolism , Sphingosine/analogs & derivatives , Animals , CHO Cells , Cricetinae , Gene Transfer Techniques , Humans , Ligands , Protein Binding , Radioligand Assay , Receptors, Cell Surface/genetics , Signal Transduction/drug effects , Sphingosine/metabolism , Sphingosine/pharmacologyABSTRACT
Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.