Application of flow cytometry in the study of apoptosis in neonatal rat cardiomyocytes.

Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1 and 10 microM for 48 h. NE concentrations of 1 and 10 microM decreased the number of viable cardiomyocytes by 18% (*p < 0.05) and 24% (**p = 0.01), respectively. ISO in a concentration of 1 microM increased the number of viable cardiomyocytes by 13% while 10 microM decreased the number of viable cardiomyocytes by 43% (***p < 0.001). Apoptotic cells were detected by flow cytometry and confocal microscopy. NE in concentrations of 1 and 10 microM increased the percentage of apoptotic cells by 12.2% and 34.3%, respectively, while ISO alone in a concentration of 10 microM increased the percentage of apoptotic cells by 11.3%. The results demonstrated that these two methods are reliable and suitable for the detection and study of apoptosis in cultures of neonatal cardiomyocytes.

Aujeszky's disease virus production in disposable bioreactor.

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky's disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82x10(6) cells ml-1, the reactor was inoculated with 9 ml of gE- Bartha K-61 strain ADV suspension (10(5.9) TCID50) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10(7.0) TCID 50 ml-1, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

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.

A model system for optimising the selection of membrane antigen-specific human antibodies on intact cells using phage antibody display technology.

The functional expression of human antibody fragments on the surface of filamentous bacteriophage, and selection of phage antibodies (PhAbs) with antigens, has provided a powerful tool for generating novel antibodies. Applications of phage antibody display technology have increased over the past decade. Successful isolation of phage antibodies has been reported mostly using purified antigens. Isolation has proven to be more complicated with complex mixtures of antigens, such as intact cells. A given cell type contains thousands of different epitopes, each capable in theory of binding phage antibodies. Often antigens are not known or cannot be purified without disrupting their conformational integrity. To overcome problems involving phage antibody selections on intact cells, we have developed an experimental model system that allows for optimisation and comparison of various selection strategies. The model system comprises labelling of intact cells with the fluorescently labelled phospholipid fluorescein-DHPE. Upon incubation, this phospholipid is readily incorporated in the membrane of any cell type. Labelling intensity is regulated by varying the phospholipid concentration. After optimisation of key steps in the selection procedure, we were able to isolate fluorescein-DHPE specific phage from a synthetic library using intact cells. This model system can be applied to any cell type and we demonstrate that it can be used to efficiently compare and optimise selection strategies.

Splenic dendritic cells from the non-obese diabetic mouse induce a prolonged proliferation of syngeneic T cells. A role for an impaired apoptosis of NOD T cells?

In this study we have tried to detect abnormalities in the immunophenotype and/or function of dendritic cells from the non-obese diabetic mouse (NOD DC), that might be related to islet autoimmunity. The immunophenotype of NOD splenic DC did not show significant abnormalities as compared with the immunophenotype of splenic DC from C57BL/10 mice. Furthermore, NOD splenic and lymph node DC stimulated proliferation of syngeneic T cells as efficiently as DC from C57BL/10 and BALB/c mice. The allogeneic response induced by NOD DC was similar to or only slightly lower than the response induced by C57BL/10 DC. Both a normal immunophenotype of NOD DC and efficient T cell stimulation were observed regardless of the stage of diabetes development. However, the syngeneic T cell proliferation induced by NOD splenic DC, but not by C57BL/10 splenic DC, was significantly prolonged, and it was accompanied by an increased proportion of activated/memory CD4(+)cells. We demonstrated that during the interaction of NOD cells fewer apoptotic cells were generated as compared with the interaction of C57BL/10 cells. Thus, the prolonged T cell response during the syngeneic interaction between NOD DC and T cells might be due to an impaired apoptosis induction. The impaired apoptosis might be of critical importance in the development of islet autoimmunity in the NOD mouse.

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.

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.

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).

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.

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.

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.

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.

Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process.

We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca(2+)-specific dye Fluo-3, while the other cell type is labeled with the pH-sensitive dye SNARF-1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding--and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2-0.3 pH units). TC do not reveal a significant change in pHi.

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.

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.