Dendritic cells freshly isolated from human blood express CD4 and mature into typical immtmostimulatory dendritic cells after culture in monocyte-conditioned medium

A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CDS, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The ceils enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines

WALL SHEAR STRESS TOPOLOGICAL SKELETON IDENTIFICATION IN CARDIOVASCULAR FLOWS: A PRACTICAL APPROACH

The observed co-localization of “disturbed” hemodynamics and atherosclerotic lesion prevalence has led to the identification of low and oscillatory Wall Shear Stress (WSS) as a biomechanical localizing factor for vascular dysfunction. However, recent evidences have underlined how consideration of only “low and oscillatory” WSS may oversimplify the complex hemodynamic milieu to which the endothelium is exposed. In this context, recent studies have highlighted the relevance of WSS fixed points, and the stable and unstable manifolds that connect them. These WSS topological features have a strong link with flow features like flow stagnation, separation, and recirculation, which are usually classified as “disturbed” flow. Technically, a fixed point of a vector field is a point where the vector field vanishes, while unstable/stable vector field manifolds identify contraction/expansion regions linking the fixed points. The set of fixed points and their connections form the topological skeleton of a vector field. The presence of WSS fixed points and of WSS contraction/expansion regions, highlighted by WSS manifolds, might induce focal vascular responses relevant for, e.g., early atherosclerosis, or, aneurysm rupture. For these reasons, the topological skeleton analysis of the WSS vector field is of great interest and motivates the study present herein. Lagrangian techniques have been recently proposed to identify WSS manifolds but have certain practical limitations. A Eulerian approach has also been suggested, but only for 2D analytical fields. Here we propose and demonstrate the use of a simple Eulerian approach for identifying WSS topological skeleton on 3D surfaces

A Practical Approach for Wall Shear Stress Topological Skeleton Analysis Applied to Intracranial Aneurysm Hemodynamics

The physiopathological role of Wall Shear Stress (WSS) in intracranial aneurysm development/rupture and the action of contraction/expansion played by shear forces on vessel wall make topological skeleton analysis of the WSS vector field of great interest. Here we present a practical way to analyze WSS topological skeleton through the identification and classification of WSS fixed points and manifolds. The method is based on the calculation of the WSS vector field divergence and Poincarè index, and it is here successfully applied to a dataset computational hemodynamic models of intracranial aneurysms

A Eulerian method to analyze wall shear stress fixed points and manifolds in cardiovascular flows

Based upon dynamical systems theory, a fixed point of a vector field such as the wall shear stress (WSS) at the luminal surface of a vessel is a point where the vector field vanishes. Unstable/stable manifolds identify contraction/expansion regions linking fixed points. The significance of such WSS topological features lies in their strong link with “disturbed” flow features like flow stagnation, separation and reversal, deemed responsible for vascular dysfunction initiation and progression. Here, we present a Eulerian method to analyze WSS topological skeleton through the identification and classification of WSS fixed points and manifolds in complex vascular geometries. The method rests on the volume contraction theory and analyzes the WSS topological skeleton through the WSS vector field divergence and Poincare´ index. The method is here applied to computational hemodynamics models of carotid bifurcation and intracranial aneurysm. An in-depth analysis of the time dependence of the WSS topological skeleton along the cardiac cycle is provided, enriching the information obtained from cycle-average WSS. Among the main findings, it emerges that on the carotid bifurcation, instantaneous WSS fixed points co-localize with cycle-average WSS fixed points for a fraction of the cardiac cycle ranging from 0 to 14.5 % ; a persistent instantaneous WSS fixed point confined on the aneurysm dome does not co-localize with the cycle-average low-WSS region. In conclusion, the here presented approach shows the potential to speed up studies on the physiological significance of WSS topological skeleton in cardiovascular flows, ultimately increasing the chance of finding mechanistic explanations to clinical observations

Two classes of generalized functions used in nonlocal field theory

We elucidate the relation between the two ways of formulating causality in nonlocal quantum field theory: using analytic test functions belonging to the space $S^0$ (which is the Fourier transform of the Schwartz space $\mathcal D$) and using test functions in the Gelfand-Shilov spaces $S^0_\alpha$. We prove that every functional defined on $S^0$ has the same carrier cones as its restrictions to the smaller spaces $S^0_\alpha$. As an application of this result, we derive a Paley-Wiener-Schwartz-type theorem for arbitrarily singular generalized functions of tempered growth and obtain the corresponding extension of Vladimirov's algebra of functions holomorphic on a tubular domain.Comment: AMS-LaTeX, 12 pages, no figure

Internship workplace preferences of final-year medical students at Zagreb University Medical School, Croatia: all roads lead to Zagreb

BACKGROUND: Human resources management in health often encounters problems related to workforce geographical distribution. The aim of this study was to investigate the internship workplace preferences of final-year medical students and the reasons associated with their choices. METHOD: A total of 204 out of 240 final-year medical students at Zagreb University Medical School, Croatia, were surveyed a few months before graduation. We collected data on each student's background, workplace preference, academic performance and emigration preferences. Logistic regression was used to analyse the factors underlying internship workplace preference, classified into two categories: Zagreb versus other areas. RESULTS: Only 39 respondents (19.1%) wanted to obtain internships outside Zagreb, the Croatian capital. Gender and age were not significantly associated with internship workplace preference. A single predictor variable significantly contributed to the logistic regression model: students who believed they would not get the desired specialty more often chose Zagreb as a preferred internship workplace (odds ratio 0.32, 95% CI 0.12–0.86). CONCLUSION: A strong preference for Zagreb as an internship workplace was recorded. Uncertainty about getting the desired specialty was associated with choosing Zagreb as a workplace, possibly due to more extensive and diverse job opportunities

Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm) resonant laser scattering and Raman scattering cross-sections are too small to allow single nanoparticle observation. Nanodiamonds can however be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time-scales. In this work we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells . By blocking selectively different uptake processes we show that nanodiamonds enter cells mainly by endocytosis and converging data indicate that it is clathrin mediated. We also examine nanodiamonds intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule deliver

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses

Dendritic Cells Take up and Present Antigens from Viable and Apoptotic Polymorphonuclear Leukocytes

Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2d) are coinjected in the footpad of mice with autologous DC (H-2b). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC