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The mechanisms involved in neutrophil `priming' are unknown. `Priming' by substance P and tumour necrosis factor-α (TNF-α) occurred without effecting cytosolic-free Ca2+ signalling and was independent of actin polymerization. We demonstrate here that these two primers, which act on different receptor classes, both cause tyrosine phosphorylation of a number of protein substrates including a prominent 74000 MW protein. This protein was not recognized by anti-c-raf antibodies. The concentration relationship and time-course of tyrosine phosphorylation were consistent with a role in mediating priming. Pretreatment with genistein both inhibited tyrosine phosphorylation and abolished the printing by either substance P or TNF-α. [ABSTRACT FROM AUTHOR]

Introduction The failure to clear persistent Hepatitis B viral (HBV) infection is characterised by an insufficient CD8 T-cell response to several antigens including HBV core (HBc). Generating a strong T-cell response to this antigen to counter immunotolerant mechanisms in chronic HBV is an attractive therapeutic strategy. We have previously shown that lentiviral vaccines (LV) encoding antigen can generate potent CD8 and CD4 responses and these can be dramatically enhanced by co-expression of viral FLICE-like inhibitory protein (vFLIP) from Kaposi sarcoma-associated herpes virus. vFLIP is a potent stimulator of the NFKB pathway which matures and activates dendritic cells, enhancing expression of costimulatory molecules (including CD80, CD86 and ICAM1) and increasing IL-12 secretion. Aim In this study we aimed to assess CD8 T-cell and antibody responses in HLA-A2 transgenic mice vaccinated with LV co-expressing vFLIP and HBc (A), expressing HBc alone (B) or HBc with an inactive vFLIP mutant (vFLIPa57l) (C). Method HLA-A2 transgenic mice were vaccinated with LV 12 days before sacrifice and harvest of splenocytes. These were restimulated overnight with an HLA-A2 restricted HBc peptide (18–27) and/or overlapping HBc peptides. IFN? responses were measured by intracellular cytokine staining and by ELIspot. Antibody responses were assessed by ELISA on serum obtained at sacrifice. Results Vaccination with LV co-expressing vFLIP and HBc (A) results in enhanced CD8-T-cell responses compared with mice vaccinated with LV expressing HBc alone (B) or HBc with an inactive vFLIP mutant (vFLIPa57l) (C). This was demonstrated on intracellular cytokine staining for IFN? of CD8+ve splenocytes re-stimulated overnight with HLA-A2 restricted peptide HBc 18–27 (A:B:C=5.35% :1.44% :0.84%). IFN? Elispot demonstrated twofold greater CD8 T-cell responses after restimulation with overlapping HBc peptides in splenocytes from mice vaccinated 12 days previously with LV co-expressing vFLIP and HBc compared with mice vaccinated with LV encoding HBc alone (p=0.006). LV expressing HBc also generated a strong antibody response comparable to vaccination with recombinant protein HBc virus-like particles (VLP). This was despite the presumed endogenous HBc antigen expression in transduced cells and no known mechanism of HBc secretion. LV encoding a mutant HBc p138g which does not multimerise into VLPs failed to raise an antibody response. Conclusion LV encoding HBc are a potential means of generating both therapeutic CD8 T-cell and antibody responses in chronic HBV. Our data suggest that an intact VLP structure is essential for the generation of an antibody responses to HBc delivered with a lentiviral platform. We are now using this system to explore potential synergy between T-cell and B-cell responses to HBc in HBV infection. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Advanced therapy medicinal products (ATMPs) are potential game changers in modern medical care with an anticipated major impact for patients and society. They are a new drug class often referred to as "living drugs," and are based on complex components such as vectors, cells and even tissues. The production of such ATMPs involves innovative biotechnological methods. In this survey, we have assessed the perception of European citizens regarding ATMPs and health care in Europe, in relation to other important topics, such as safety and security, data protection, climate friendly energy supply, migration, and others. A crucial question was to determine to what extent European citizens wish to support public funding of innovations in healthcare and reimbursement strategies for ATMPs. To answer this, we conducted an online survey in 13 European countries (representative of 85.3% of the entire EU population including the UK in 2020), surveying a total of 7,062 European citizens. The survey was representative with respect to adult age groups and gender in each country. Healthcare had the highest ranking among important societal topics. We found that 83% of the surveyed EU citizens were in support of more public funding of technologies in the field of ATMPs. Interestingly, 74% of respondents are in support of cross-border healthcare for patients with rare diseases to receive ATMP treatments and 61% support the reimbursement of very expensive ATMPs within the European health care system despite the current lack of long-term efficacy data. In conclusion, healthcare is a top ranking issue for European Citizens, who additionally support funding of new technologies to enable the wider application of ATMPs in Europe., Competing Interests: ZA and RO are employed by Pluristem Therapeutics Inc. Haifa, Israel. GD is employed by Innovation Acta S.r.l., Italy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Goldsobel, von Herrath, Schlickeiser, Brindle, Stähler, Reinke, Aberman, Ofir, Dessole, Benvenuti, Neves, Reis, Moll and Volk.)

Purpose: Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations., Experimental Design: Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays., Results: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas., Conclusions: Our work reveals a novel PML-I-TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398-409. ©2016 AACR., (©2016 American Association for Cancer Research.)

Guessing occurs on forced-choice (FC) tests for which responses cannot be based on relevant knowledge. Its importance is in inverse relation to the level of knowledge being measured, so that it becomes an increasing component of test scores as the level of knowledge decreases. It is also used as a benchmark to detect simulated impairment. This investigation examined the role of guessing in a 2-alternative FC face recognition test. Chance groups shown only the test items were asked to guess which were the targets, to measure the variation in scores likely to be found with pure guessing. Controls performed normally, and two simulation groups tried to fake amnesia. Results suggested that simple guessing in the chance group produced variable scores that overlapped both low genuine and "malingering" performance. Low control scores were hidden by the guessing "chance bonus," which ameliorated the apparent level of decline in memory. Simulators told of their role at the outset (before presentation of the target items) produced more convincing "amnesic" scores than those told only just before the test, who produced the expected below-chance level of score. It is suggested that guessing variability should be taken into account in interpreting FC scores.

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1.

Introduction: There is considerable interest in developing a diagnostic test which could differentiate between early Alzheimer's disease (AD) and other causes of memory impairment with more than 80% sensitivity and 80% specificity., Objective: To review the studies that have examined CSF phosphorylated tau as diagnostic test of AD vs clinically representative comparison groups., Method: A critical review of the literature using Embase, Web of Science, Medline and Psychinfo databases supplemented by handsearching and contact with experts in the field., Results: CSF phosphorylated tau is a marker of AD that improves upon the utility of CSF total tau and clinical examination alone. Studies have found high levels of tau phosphorylated at Threonine 231 and/or Serine 199 in AD but not in other causes of dementia, in depression or in healthy elderly controls. Of particular interest, the test appears equally valid in cases of early AD as in moderate or late stages and may also be of use in predicting future decline in subjects with mild cognitive impairment., Conclusion: CSF phosphorylated tau is a promising diagnostic test for AD but this requires replication using pathologically confirmed cases., (Copyright 2003 John Wiley & Sons, Ltd.)

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.

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., (Copyright 2000 S. Karger AG, Basel)

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.

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.

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.

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.