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Biomedical subjects

K Radosević

Publications and source records attributed to K Radosević.

11 recordsLinked to original sources
PubMed2006

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

Adenoviruses, Human↗
PubMed1998

Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover.

In the normal mouse spleen, two distinct populations of dendritic cells (DC) are present that differ in microanatomical location. The major population of marginal DC is found in the "marginal zone bridging channels" and extends into the red pulp. The interdigitating cells (IDC) are localized in the T cell areas in the white pulp. The aim of the present study was to characterize these two splenic DC populations with regard to their phenotype, in vivo phagocytic function, and turnover. Both marginal DC and IDC are CD11c+ and CD13+, but only IDC are NLDC-145+ and CD8alpha+. Notably, both populations, when freshly isolated, express the macrophage markers F4/80, BM8, and Mac-1. To study the phagocytic capacity of these cells, we employed the macrophage "suicide" technique by injecting liposomes loaded with clodronate i.v. Marginal DC, but not IDC, were eliminated by this treatment. Phagocytosis of DiI-labeled liposomes by DC confirmed this finding. The two DC populations differed significantly with regard to their turnover rates, as studied in a transgenic mouse model of conditional depletion of DC populations with high turnover. In these mice, marginal DC were completely eliminated, but the IDC population remained virtually intact. From these data we conclude that the marginal DC population has a high turnover, in contrast to the IDC population. Taken together, the present results indicate that marginal DC and IDC represent two essentially distinct populations of DC in the mouse spleen. They differ not only in location, but also in phenotype, phagocytic ability, and turnover.

Animals↗
PubMed1995

Occurrence and a possible mechanism of penetration of natural killer cells into K562 target cells during the cytotoxic interaction.

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo) emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism.

Actins↗
PubMed1995

Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: a quantitative study at the single cell level.

This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura-2-AM) and [pH]in (BCECF-AM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell "escapes" immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3-7.6 (approximately 3 min).

Calcium↗
PubMed1994

Changes in actin organization during the cytotoxic process.

Changes in organization of F-actin during the cytotoxic process between NK and K562 cells have been observed and studied using confocal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F-actin content and orientation of F-actin towards the target cell have been observed in conjugated NK cells. The increase in F-actin content probably reflects activation of the NK cell for the killing process. An increase in F-actin content in the conjugated K562 cell, occurring simultaneously with the appearance of filamentous actin structures that often originated/ended at the contact place with the NK cell, was also observed. These changes were delayed compared to the increase in F-actin content in the NK cell and were accompanied by increasing cytotoxic activity. This indicates that they were results of the interaction of the K562 cell with the activated NK cell. The possible role of target cell microfilaments in the cytotoxic process is addressed.

Actin Cytoskeleton↗
PubMed1993

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins.

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

Animals↗
PubMed1993

A flow cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process.

This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity.

Clone Cells↗
PubMed1993

Signal processing in slit-scan flow cytometry of cell conjugates.

The design and implementation of a real-time signal processing system for slit-scan flow cytometry is described. The system is used to measure the separate scatter and fluorescence peak heights of 2 adherent cells. Preliminary measurements of changes in the membrane potential induced by interactions between natural killer (NK) cells and their target cells are presented.

Barbiturates↗
PubMed1993

Flow cytometric method for simultaneous detection of lymphocyte-K562 conjugates and immunophenotyping of the conjugate forming cells.

A flow cytometric method for the simultaneous quantification and immunophenotyping of conjugates formed by human peripheral blood lymphocytes (PBL) and K562 cells has been developed. The method uses three fluorescent probes. One of the fluorescent probes (F-18) is used for labeling of PBL prior to incubation with K562 cells. After incubation the cells are treated with monoclonal antibodies labeled with phycoerythrin and Red613, respectively. The combination of F-18 fluorescence and light scattering signals enables identification and quantification of the conjugates while the fluorescence of the monoclonal antibodies provides information about the phenotype of the conjugate forming cells. Results obtained using different monoclonal antibodies are presented. The highest conjugate forming capacity has been found in the CD56+CD8+ population while the CD4+CD8- population has shown the lowest capacity to form conjugates. The influence of a washing step on the conjugate formation is discussed. The possibility to use the method in combination with a cytotoxicity assay is indicated.

Antibodies, Monoclonal↗
PubMed1990

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells.

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.

Cell Line↗