Clinical impact and diagnostic accuracy of 2-[18F]-fluoro-2-deoxy-d-glucose positron-emission tomography/computed tomography (PET/CT) brain imaging in patients with cognitive impairment: a tertiary centre experience in the UK.

AIM: To evaluate the clinical impact of combined 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) positron-emission tomography/computed tomography (PET/CT) brain imaging performed in selected patients with cognitive impairment at a tertiary referral centre in the UK, and to assess the accuracy of FDG PET/CT to correctly establish the diagnosis of Alzheimer's dementia (AD) in "real-world" clinical practice. METHODS AND MATERIALS: Using an institutional radiology database, 136 patients were identified for inclusion in the study. FDG PET/CT was performed using a standard technique and interpreted by dual-trained radiologists and nuclear medicine physicians. Standardised questionnaires were sent to the referring clinicians to establish the final clinical diagnosis and to obtain information about the clinical impact of FDG PET/CT. RESULTS: There was a 72% questionnaire return (98/136), with mean patient follow-up of 471 (standard deviation 205) days. FDG PET/CT had an impact on patient management in 81%, adding confidence to the pre-test diagnosis in 43%, changing the pre-test diagnosis in 35%, reducing the need for further investigations in 42%, and resulting in a change in therapy in 32%. There was substantial correlation between the PET/CT diagnosis and final clinical diagnosis with a correlation (k) coefficient of 0.78 (p<0.0001). The accuracy of FDG PET/CT in diagnosis of AD was 94% (95% confidence interval [CI]: 87-99), with a sensitivity of 87% (95% CI: 75-92) and a specificity of 97% (95% CI: 87-99). CONCLUSION: FDG PET/CT brain imaging has a significant clinical impact when performed selectively in patients with cognitive impairment and shows high accuracy in the diagnosis of AD in "real-world" clinical practice.

Schedule-dependent effects of haloperidol and amphetamine: multiple-schedule task shows within-subject effects.

A two-lever, multiple-schedule task was used to evaluate the effects of haloperidol (HA) and amphetamine (AM) on responding controlled by continuous reinforcement (CRF) and progressive ratio (PR) schedules of reinforcement. Rats were trained to press one lever for food delivered on a CRF schedule and the other lever for food delivered on a PR schedule. The operative schedule was signaled by the illumination of a cuelight mounted above the appropriate lever. Following 30 sessions of training, dose-response functions were determined for HA (0.0075, 0.015, 0.03, and 0.06 mg/kg) and AM (0.0625, 0.125, 0.25, 0.50, 0.75, and 1.00 mg/kg). Both drugs produced dose- and schedule-dependent effects. For example, administration of 0.03 mg/kg HA did not affect responding under the CRF schedule but did reduce responding during PR components, whereas administration of 0.06 mg/kg reduced responding under both schedules of reinforcement. Some doses of AM produced increased responding under the CRF schedule and, within the same session, decreased responding under the PR schedule. The results with HA are consistent with the view that interfering with dopaminergic function affects the allocation and maintenance of responding and that this effect depends on properties of the schedule of reinforcement. The results with AM emphasize that statements about the effects of the drug on positively reinforced behavior cannot be made without reference to specific schedules of reinforcement.

Vasodilator-stimulated phosphoprotein is involved in stress-fiber and membrane ruffle formation in endothelial cells.

Vasodilator-stimulated phosphoprotein (VASP) is highly expressed in vascular endothelial cells, where it has been implicated in cellular reorganization during angiogenesis, as well as in endothelial retraction and changes in vessel permeability. However, the cellular functions of VASP are not known. In this study, we have expressed wild-type and mutant forms of VASP in endothelial cells to determine in what aspects of cytoskeletal behavior this protein participates. Expression of wild-type VASP induces marked membrane ruffling and formation of prominent stress fibers in bovine aortic endothelial cells. Deletion of the proline-rich domain of VASP abolishes its ability to bind profilin but does not affect ruffling or stress fiber formation. Further deletions reveal a sequence within the carboxy-terminal domain that is responsible for in vivo bundle formation. Ruffling occurs only on the expression of forms of VASP that possess bundling activity and the capacity to bind zyxin/vinculin-derived peptide. The ability of distinct subdomains within VASP to bind adhesion proteins and induce F-actin bundling in vivo suggests that this protein could function in the aggregation and tethering of actin filaments during the formation of endothelial cell-substrate and cell-cell contacts. These data provide a mechanism whereby VASP can influence endothelial migration and organization during capillary formation and modulate vascular permeability via effects on endothelial cell contractility.

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2.

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

Tie-1 receptor tyrosine kinase endodomain interaction with SHP2: potential signalling mechanisms and roles in angiogenesis.

The endothelial receptor tyrosine kinase plays an essential role in vascular development where it is thought to be required for vessel maturation and stabilization. The ligands responsible for activating Tie-1, its signalling pathways and specific cellular functions are however not known. As with some other receptor tyrosine kinases, Tie-1 is subject to extracellular proteolytic cleavage generating a membrane bound receptor fragment comprising the intracellular and transmembrane domains. Here we examine the signalling potential of this Tie-1 endodomain. We show that the Tie-1 endodomain has poor ability to induce tyrosine phosphorylation. However, on formation the endodomain physically associates with a number of tyrosine phosphorylated signalling intermediates including the tyrosine phosphatase and adaptor protein SHP2. The assembly of this multimolecular complex is consistent with the endodomain having a ligand-independent signalling role in the endothelial cell. The potential roles of ectodomain cleavage and cleavage activated signalling in regulating microvessel stability in angiogenesis, vessel remodelling and regression are considered.

Characterization and regulation of the receptor tyrosine kinase Tie-1 in platelets.

The receptor tyrosine kinase Tie-1 is expressed predominantly on endothelial cells where it has an essential role in blood vessel formation. Targeted disruption of the Tie-1 gene results in a lethal phenotype with severe disruption to the normal integrity of the vasculature. In an examination of Tie-1 in vivo, we observed a significant pool of the receptor present in the circulation associated with the platelet fraction. Western blotting reveals the platelet form of Tie-1 to be a protein of approximately 110 kDa, this contrasts with the 135/125-kDa doublet found in endothelial cells. Platelet activation results in increased surface expression of Tie-1. The closely related receptor tyrosine kinase Tie-2/Tek is not present in platelets. Endothelial Tie-1 undergoes metalloprotease-mediated ectodomain cleavage in response to phorbol ester and other agonists. Tie-1 cleavage leads to release of the extracellular domain and generation of a cell-associated intracellular domain with signalling capacity. The potential for cleavage was investigated in platelets. In contrast to endothelial Tie-1, phorbol ester does not stimulate truncation of the platelet receptor, suggesting these cells lack one or more components of the regulated metalloprotease system controlling Tie-1. These data demonstrate the Tie-1 receptor tyrosine kinase is present on platelets and its surface expression is regulated. Furthermore, platelet Tie-1 differs significantly from the endothelial receptor. Platelet Tie-1 has the potential to modulate endothelial function by competing for any Tie ligands and may have signalling roles important in controlling aspects of platelet behaviour.

The genetics of Alzheimer's disease.

Since the first description of Alzheimer's disease (AD) at the beginning of the century until relatively recently, it was customary to define Alzheimer's disease as occurring in the presenium. The same neuropathological changes occurring in brains over the age of 65 were called "senile dementia." Because there have been no clinical or pathological features to separate the two groups, this somewhat arbitrary distinction has been abandoned. Although AD is currently considered to be a heterogeneous disease, the most consistent risk factor to be implicated other than advancing age is the presence of a positive family history. This potential genetic vulnerability to AD has been recognized for some time. Some of the earliest evidence suggestive of a genetic contribution to AD came from Kallmann's 1956 study (1) demonstrating a higher concordance rate in monozygotic twins for "parenchymatous senile dementia" compared with dizygotic twins and siblings. This monozygotic excess has been confirmed in studies applying more rigorous diagnostic criteria although there may be widely disparate ages of onset between twins (2). The most convincing evidence for a genetic contribution to AD has come form the study of pedigrees in which the pattern of disease segregation can be clearly defined. Thus, the abandonment of the early and late-onset dichotomy has occurred at a time when, at the genetic level, important differences have been identified through the discovery of specific gene defects in early onset cases.

Angiogenesis and the atherosclerotic carotid plaque: an association between symptomatology and plaque morphology.

PURPOSE: Symptomatic carotid disease resulting from generation of thromboemboli has been associated with plaque instability and intraplaque hemorrhage. These features of the lesion could be influenced by the fragility and position of neovessels within the plaque. The purpose of this study was to determine whether any association exists between neovessel density, position, morphology, and thromboembolic sequelae. METHODS: Carotid endarterectomy samples were collected from 15 asymptomatic patients with greater than 80% stenoses and from 13 highly symptomatic patients who had suffered ipsilateral carotid stenotic events within 1 month of surgery. Both groups were matched for gender, age, risk factors, degree of carotid artery stenosis, and plaque size. Samples were stained with hematoxylin/eosin and van Geison. Immunohistochemistry was performed by using an endothelial specific antibody to CD31. Plaques were assessed for histologic characteristics, and neovessels were counted and characterized by size, site, and shape. RESULTS: There were significantly more neovessels in plaques (P <.00001) and fibrous caps (P <.0001) in symptomatic compared with asymptomatic plaques. Neovessels in symptomatic plaques were larger (P <.004) and more irregular. There was a significant increase in plaque necrosis and rupture in symptomatic plaques. Plaque hemorrhage and rupture were associated with more neovessels within the plaque (P <.017, P <.001) and within the fibrous cap (P <.046, P <.004). Patients with preoperative and intraoperative embolization had significantly more plaque and fibrous cap neovessels (P <.025, P <.001). CONCLUSION: Symptomatic carotid disease is associated with increased neovascularization within the atherosclerotic plaque and fibrous cap. These vessels are larger and more irregular and may contribute to plaque instability and the onset of thromboembolic sequelae.

Potential roles of metalloprotease mediated ectodomain cleavage in signaling by the endothelial receptor tyrosine kinase Tie-1.

The orphan receptor tyrosine kinase Tie-1 is expressed predominantly in endothelial cells. Expression of this receptor is increased in physiologic angiogenesis and pathologic situations including tumor growth and arteriovenous malformations. Tie-1 is essential for vascular development where it acts in later stages of angiogenesis to suppress endothelial activation and stabilize the newly formed vessel. Stimulation of protein kinase C in endothelial cells results in endoproteolytic cleavage of Tie-1, releasing the extracellular ligand-binding domain of the receptor. We show that this is mediated by a metalloprotease. Immunoprecipitation and immunoblotting of lysates prepared from human placentas confirm that Tie-1 truncation occurs in vivo. We propose cleavage of this receptor may be a mechanism for inducing vessel destabilization by preventing ligand-activated signaling through Tie-1. Using an antibody that recognizes the carboxy terminus of the intracellular domain, we show that the Tie-1 endodomain formed on cleavage persists as a cell-associated fragment for several hours. Subcellular fractionation reveals this tyrosine kinase containing receptor fragment to be localized in the membrane fraction of the cell. Immunoprecipitation with antibodies recognizing phosphotyrosine demonstrates that cleavage of Tie-1 stimulates association of newly generated endodomain with cellular phosphoproteins. Furthermore, there was a marked induction of tyrosine phosphorylation of several proteins after PMA-induced endodomain generation. These data indicate that ectodomain cleavage may be a mechanism for down-regulating ligand-induced signaling through Tie-1 while activating an alternative ligand-independent signaling pathway in endothelial cells. Ectodomain cleavage occurs in some other receptor tyrosine kinases. We suggest that rather than solely being a means of down-regulating receptor activity, ectodomain cleavage may be a novel way for a receptor to switch between two alternative signaling pathways.

Association between bleomycin hydrolase and Alzheimer's disease in caucasians.

A recent study showed modest evidence for an increased frequency of the bleomycin hydrolase (BH) V/V genotype in Alzheimer's disease (AD) patients compared with non-demented controls. To test this hypothesis, we examined this polymorphism in 621 rigorously evaluated patients and 502 control subjects (all caucasian) but were unable to detect an association between BH and AD even after controlling for age, gender, and apolipoprotein E (ApoE) genotype. We conclude that this polymorphism does not account for inherited susceptibility to AD in the populations represented in this sample.

Absence of association between Alzheimer disease and the -491 regulatory region polymorphism of APOE.

A novel polymorphism (-491 A/T) within the regulatory region on the apolipoprotein E gene has recently been reported to be associated with risk for Alzheimer disease (AD). To test this association in an independent data set, we have examined this polymorphism in a sample of 88 well-characterized AD cases and compared the allele frequency and genotype frequencies for this polymorphism with those observed in 112 cognitively normal subjects drawn from the same ethnic group. These results suggest that in the current data set at least, the -491 A/T polymorphism is not associated with risk for AD, but may be in partial linkage disequilibrium with the APOE epsilon2/epsilon3/epsilon4 polymorphism.

Evidence for an Alzheimer disease susceptibility locus on chromosome 12 and for further locus heterogeneity.

CONTEXT: Alzheimer disease (AD) susceptibility genes have been identified on chromosomes 1, 14, 19, and 21, and a recent study has suggested a locus on chromosome 12. OBJECTIVE: To confirm or refute the existence of a familial AD susceptibility locus on chromosome 12 in an independent sample of familial AD cases. DESIGN: Retrospective cohort study. DNA data for 6 chromosome 12 genetic markers were evaluated using parametric lod score and nonparametric linkage methods and linkage heterogeneity tests. The latter include the admixture test of homogeneity in the total group of families and the predivided sample test in families stratified by the presence or absence of an apolipoprotein E (APOE) epsilon4 allele among affected members. Parametric analyses were repeated assuming autosomal dominant inheritance of AD and either age- and sex-dependent penetrance or zero penetrance for the analysis of unaffected relatives. SETTING: Clinical populations in the continental United States, Canada, Argentina, and Italy. PATIENTS: Fifty-three white families composed of multiple members affected with AD, from whom DNA samples were obtained from 173 patients with AD whose conditions were diagnosed using established criteria and from 146 nondemented relatives. MAIN OUTCOME MEASURE: Presence of an APOE epsilon4 allele among affected family members. RESULTS: Using parametric methods, no evidence for linkage to the region spanned by the chromosome 12 markers could be detected if familial AD is assumed to arise from the same genetic locus in all 53 families. However, significant evidence for linkage was detected in the presence of locus heterogeneity using the admixture test (odds ratio, 15, 135:1). The estimated proportion of linked families within the 53 families examined varied between 0.40 and 0.65, depending on the genetic model assumed and APOE status. The precise location of the AD gene could not be determined, but includes the entire region suggested previously. Nonparametric linkage analysis confirmed linkage to chromosome 12 with the strongest evidence at D12S96 (P<.001). CONCLUSIONS: Our data provide independent confirmation of the existence of an AD susceptibility locus on chromosome 12 and suggest the existence of AD susceptibility genes on other chromosomes. Screening a larger set of families with additional chromosome markers will be necessary for identifying the chromosome 12 AD gene.

Regulation of insulin-like growth factor-1 expression in vascular endothelial cells by the inflammatory cytokine interleukin-1.

Insulin-like growth factor-1 (IGF-1) has important roles in vascular physiology and pathologies such as atherosclerosis. However, the factors that control expression of this growth factor in human vascular cells are largely unknown. In this study the effects of the inflammatory cytokine interleukin-1 (IL-1) on IGF-1 mRNA and peptide synthesis was examined in human endothelial cells. Cultured human umbilical vein endothelial (HUVE) cells were challenged with interleukin-1 and IGF-1 Ea and Eb mRNA levels were determined by nuclease protection assays and reverse transcription/polymerase chain reaction. IL-1 caused a dramatic increase in IGF-1 Ea mRNA in HUVE cells. Transcript levels peaked between 2 and 4 h of IL-1 treatment and declined over the subsequent 4 h. Consistent with its effect on mRNA, the inflammatory cytokine causes a 3-fold stimulation in production of IGF-1 peptide from endothelial cells. These results demonstrate increased IGF-1 mRNA and peptide in vascular endothelial cells stimulated with IL-1. This suggests that under conditions of local inflammation at the vessel wall the direct action of IL-1 on endothelium can lead to induction of IGF-1 which could promote the intimal hyperplasia often seen in these circumstances.

The focal adhesion phosphoprotein, VASP.

Vasodilator-stimulated phosphoprotein (VASP) is associated with focal adhesions and areas of dynamic membrane activity, where it is thought to have an important role in actin filament assembly and cell motility. VASP contains a central proline-rich sequence which recruits the G-actin binding protein profilin. Localization of VASP to the leading edge of a migrating cell can lead to local accumulation of profilin, which in turn can supply actin monomers to growing filament ends. VASP binds to the focal adhesion proteins vinculin and zyxin and this probably directs the phosphoprotein to focal adhesions and the leading edge of stimulated cells. VASP functions as a binding intermediate between profilin and focal adhesion proteins. Intracellular pathogens, including Listeria monocytogenes, have coat proteins which bind VASP. This is one way in which these pathogens use VASP, and other proteins from the host cell, to assemble the actin filaments they require to move around the cytoplasm of infected cells and enter neighbouring cells. Understanding the role of VASP and other proteins in cell and bacterial motility is likely to lead to development of new therapeutic strategies for diseases including atherosclerosis and tumour growth, and for limiting the spread of intracellular pathogens.

Analysis of the butyrylcholinesterase gene and nearby chromosome 3 markers in Alzheimer disease.

The K-variant of butyrylcholinesterase (BCHE-K) recently has been reported to be associated with Alzheimer disease (AD) in carriers of the epsilon4 allele of the apolipoprotein E (APOE) gene. We have re-examined the frequency of the BCHE-K allele in a large data set of both sporadic and familial cases of AD disease, and we have also examined the segregation of three genetic markers on chromosome 3 near BCHE . Our data neither support an association of BCHE-K with sporadic or familial AD, nor do they suggest the existence of another gene nearby on chromosome 3 as a common cause of familial AD.

The endothelial receptor tyrosine kinase tie-1 is upregulated by hypoxia and vascular endothelial growth factor.

The receptor tyrosine kinase tie-1 is essential for angiogenesis where it appears to have a role in vessel maturation. Here we have examined the effects of hypoxia and vascular endothelial growth factor (VEGF) on the level of tie-1 protein expressed in bovine aortic endothelial cells. Both hypoxia (2% O2) and VEGF were found to increase tie-1 in a time-dependent manner. Hypoxic induction was direct and effects of hypoxia and VEGF were not additive. Experiments with actinomycin D indicate that these activators regulate tie-1 at the transcriptional level.