Monitoring HSVtk suicide gene therapy: the role of [18F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [F-18] FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [F-18] FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [F-18] FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour- bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2- chloroadenosine did not significantly affect the [F-18] FHPG uptake in vitro. Thymidine and uridine significantly decreased [F-18] FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [F-18] FHPG uptake in C6tk and C6 tumours decreased from 3.070.5 to 1.070.2 after infusion of adenine. Thus, in our tumour model, [F-18] FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier

Radiologist experience and CT examination quality determine metastasis detection in patients with esophageal or gastric cardia cancer

We aimed to separate the influence of radiologist experience from that of CT quality in the evaluation of CT examinations of patients with esophageal or gastric cardia cancer. Two radiologists from referral centers ('expert radiologists') and six radiologists from regional non-referral centers ('non-expert radiologists') performed 240 evaluations of 72 CT examinations of patients diagnosed with esophageal or gastric cardia cancer between 1994 and 2003. We used conditional logistic regression analysis to calculate odds ratios (OR) for the likelihood of a correct diagnosis. Expert radiologists made a correct diagnosis of the presence or absence of distant metastases according to the gold standard almost three times more frequently (OR 2.

Regulation of Endothelial Cell Adhesion Molecule Expression by Mast Cells, Macrophages, and Neutrophils

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases

Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter and 3-10 microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold.In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future

CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(−/−) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(−/−) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)–activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α–activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration

High-resolution imaging of human atherosclerotic carotid plaques with micro18F-FDG PET scanning exploring plaque vulnerability

FDG-PET can be used to identify vulnerable plaques in atherosclerotic disease. Clinical FDG-PET camera systems are restricted in terms of resolution for the visualization of detailed inflammation patterns in smaller vascular structures. The aim of the study is to evaluate the possible added value of a high-resolution microPET system in excised carotid plaques using FDG. In this study, 17 patients with planned carotid endarterectomy were included. Excised plaques were incubated in FDG and subsequently imaged with microPET. Macrophage presence in plaques was evaluated semi-quantitatively by immunohistochemistry. Plaque calcification was assessed additionally with CT and correlated to FDG uptake. Finally, FDG uptake and macrophage infiltration were compared with patient symptomatology. Heterogeneous distributions and variable intensities of FDG uptake were found within the plaques. A positive correlation between the distribution of macrophages and the FDG uptake (r = 0.68, P <.01) was found. A negative correlation was found between areas of calcifications and FDG uptake (r = -0.84, P <.001). Ratio FDG(max) values as well as degree of CD68 accumulation were significantly higher in CVA patients compared with TIA or amaurosis fugax patients (P <.05) and CVA patients compared with asymptomatic patients (P <.05). This ex vivo study demonstrates that excised carotid plaques can be visualized in detail using FDG microPET. Enhancement of clinical PET/CT resolution for similar imaging results in patients is needed

Neighborhood deprivation and biomarkers of health in Britain: The mediating role of the physical environment

Background: Neighborhood deprivation has been consistently linked to poor individual health outcomes; however, studies exploring the mechanisms involved in this association are scarce. The objective of this study was to investigate whether objective measures of the physical environment mediate the association between neighborhood socioeconomic deprivation and biomarkers of health in Britain. Methods: We linked individual-level biomarker data from Understanding Society: The UK Household Longitudinal Survey (2010-2012) to neighborhood-level data from different governmental sources. Our outcome variables were forced expiratory volume in 1 s (FEV1%; n=16,347), systolic blood pressure (SBP; n=16,846), body mass index (BMI; n=19,417), and levels of C-reactive protein (CRP; n=11,825). Our measure of neighborhood socioeconomic deprivation was the Carstairs index, and the neighborhood-level mediators were levels of air pollutants (sulphur dioxide [SO2], particulate matter [PM10], nitrogen dioxide [NO2], and carbon monoxide [CO]), green space, and proximity to waste and industrial facilities. We fitted a multilevel mediation model following a multilevel structural equation framework in MPlus v7.4, adjusting for age, gender, and income. Results: Residents of poor neighborhoods and those exposed to higher pollution and less green space had worse health outcomes. However, only SO2exposure significantly and partially mediated the association between neighborhood socioeconomic deprivation and SBP, BMI, and CRP. Conclusion: Reducing air pollution exposure and increasing access to green space may improve population health but may not decrease health inequalities in Britain

Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method

The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Dendritic Cell Based Tumor Vaccination in Prostate and Renal Cell Cancer: A Systematic Review and Meta-Analysis

BACKGROUND: More than 200 clinical trials have been performed using dendritic cells (DC) as cellular adjuvants in cancer. Yet the key question whether there is a link between immune and clinical response remains unanswered. Prostate and renal cell cancer (RCC) have been extensively studied for DC-based immunotherapeutic interventions and were therefore chosen to address the above question by means of a systematic review and meta-analysis. METHODOLOGY/PRINCIPAL FINDINGS: Data was obtained after a systematic literature search from clinical trials that enrolled at least 6 patients. Individual patient data meta-analysis was performed by means of conditional logistic regression grouped by study. Twenty nine trials involving a total of 906 patients were identified in prostate cancer (17) and RCC (12). Objective response rates were 7.7% in prostate cancer and 12.7% in RCC. The combined percentages of objective responses and stable diseases (SD) amounted to a clinical benefit rate (CBR) of 54% in prostate cancer and 48% in RCC. Meta-analysis of individual patient data (n = 403) revealed the cellular immune response to have a significant influence on CBR, both in prostate cancer (OR 10.6, 95% CI 2.5-44.1) and in RCC (OR 8.4, 95% CI 1.3-53.0). Furthermore, DC dose was found to have a significant influence on CBR in both entities. Finally, for the larger cohort of prostate cancer patients, an influence of DC maturity and DC subtype (density enriched versus monocyte derived DC) as well as access to draining lymph nodes on clinical outcome could be demonstrated. CONCLUSIONS/SIGNIFICANCE: As a 'proof of principle' a statistically significant effect of DC-mediated cellular immune response and of DC dose on CBR could be demonstrated. Further findings concerning vaccine composition, quality control, and the effect of DC maturation status are relevant for the immunological development of DC-based vaccines