A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

* To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

Microglia are dispersed throughout the central nervous system. Under physiological circumstances they display a 'ramified' resting phenotype. In different neuropathologies microglia reversibly transform into the activated form, an amoeboid phagocyte with a broad spectrum of immune effector functions. In this study, a coculture of porcine microglia and the pig renal epithelial cell line LLC-PK1 was used to investigate microglial cell biology. The morphology of the cocultures was elucidated as well as the functionality of the microglia cells by proliferation, superoxide and phagocytosis assays. Our results demonstrate that direct intercellular contact between the two cell types was necessary for microglia to acquire a ramified morphology. Moreover, the conditioned medium of the renal cells promoted proliferation of microglia, inhibited giant cell formation and stimulated microglia to retain their capability to generate superoxide and to perform phagocytosis. In conclusion, we have constructed a cell culture system showing differentiation of microglia in vitro and keeping them in optimal conditions.

Leukemic cells exert immunosuppressive effects that interfere with dendritic cell (DC) function and hamper effective antileukemic immune responses. Here, we sought to enhance the immunogenicity of leukemic cells by loading them with the double-stranded (ds) RNA Toll-like receptor 3 (TLR3) ligand polyriboinosinic polyribocytidylic acid (poly(I:C)), mimicking viral infection of the tumor cells. Given the responsiveness of DC to TLR ligands, we hypothesized that the uptake of poly(I:C)-loaded leukemic cells by immature DC (iDC) would lead to DC activation. Primary acute myeloid leukemia (AML) cells and AML cell lines markedly responded to poly(I:C) electroporation by apoptosis, upregulation of TLR3 expression, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules and by production of type I interferons (IFN). Upon phagocytosis of poly(I:C)-electroporated AML cells, DC maturation and activation were induced as judged by an increased expression of MHC and costimulatory molecules, production of proinflammatory cytokines and an increase of T helper 1 (T(H)1)-polarizing capacity. These immune effects were suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of poly(I:C) transfection over pulsing. Our results demonstrate that poly(I:C) electroporation is a promising strategy to increase the immunogenicity of AML cells and to convert iDC into activated mature DC following the phagocytosis of AML cells.

Background Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented.Methods Patients with angiographically proven coronary artery disease (n = 18) and age and sex-matched controls (n = 18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively.Results Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722 +/- 601/ml and 0.08 +/- 0.01%) than in controls (12640 +/- 1289/ml and 0.21 +/- 0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11857 +/- 11895/ml versus 15226 +/- 928/ml and 0.17 +/- 0.03% versus 0.26 +/- 0.01 % in controls.Conclusions The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H2O2 resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H2O2 was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300μM H2O2 for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Background: Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.Results: In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E7(11-20) peptide in order to induce an in vitro CD8(+) T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8(+) T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-gamma ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-gamma-secreting CD8(+) T cells in 5/5 healthy donors.Conclusion: We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Proc Amer Assoc Cancer Res, Volume 47, 2006 576 Both in clinical and preclinical studies it is shown that gemcitabine has clear radiosensitizing properties. This radiosensitizing effect is dependent of the cell cycle effect, an early S phase block followed by a G2/M block, and an increased induction of apoptosis. This study was performed to clarify the apoptotic pathway responsible for radiosensitization by gemcitabine. We investigated the activation of caspase 8 and 9, both initiator caspases of the extrinsic or intrinsic pathway, respectively. The cell lines used for this study were ECV304 (mt-p53), a human bladder cancer cell line and H292 (wt-p53), a human mucoepidermoid lung cancer cell line. The cells were treated with gemcitabine (IC25 and IC90) during 24 h, radiotherapy (γ-Co60, 0-6 Gy, room temperature), or with the combination, i.e. gemcitabine immediately followed by radiation. 72h after treatment, apoptotic cell death is determined using TUNEL assay, Annexin V staining and caspase 3 activity assay. Caspase 8 and 9 activation is determined by Western blot at different time points (0-72h) after gemcitabine or radiotherapy and 72 h after the combination. Using the mitochondria-selective probe (tetramethylrhodamine methylester) and flow cytometry, the mitochondrial membrane potential (Ψ) was determined. 72 h after treatment, the combination of gemcitabine and radiotherapy resulted in a clear increase of apoptosis in both cell lines. At that moment, both procaspase 8 and 9 cleaving was observed with Western blot. In addition, Ψ was reduced when the amount of annexin V positive cells increases. To determine the order of caspase 8 and 9 activation, caspase cleaving was determined at different time points after gemcitabine or radiotherapy. In both settings, caspase 8 activation was observed at the different time points after treatment, while no cleaving products of caspase 9 could be shown. In conclusion, treatment of ECV304 and H292 cells under radiosensitizing conditions resulted in an increased induction of apoptosis with caspase 8 and 9 cleaving and a reduction of Ψ. This means that both the extrinsic and the intrinsic pathway is activated. However, treatment with gemcitabine or radiotherapy alone resulted only in activation of the extrinsic pathway. Further research is necessary to investigate the caspase 9 activation under radiosensitizing conditions. This could be a result of the increased apoptosis by the combination of gemcitabine and radiation or by Bid activation, the link between the intrinsic and the extrinsic pathway. The Belgian Federation against cancer supported this work .

Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.

Dendritic cells (DC), the professional antigen presenting cells of the immune system, exert important functions both in induction of T cell immunity as well as of tolerance. Previously, it was accepted that the main function of immature DC (iDC) in their in vivo steady state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. However, a growing body of experimental evidence now indicates that traditional DC maturation can no longer be used to distinguish between tolerogenic and immunogenic properties of DC. In this study, we compared the in vitro stimulatory capacity of immature DC (iDC), cytokine cocktail-matured DC (CC-mDC) and poly I:C-matured DC (pIC-mDC) in the absence and presence of antigen. All investigated DC types could induce at least 2 subsets of regulatory T cells. We observed a significant increase in both the number of functionally suppressive transforming growth factor (TGF)-beta+ interleukin (IL)-10+ T cells as well as of CD4+CD25+FOXP3+ T cells within DC/T cell co-cultures as compared to T cell cultures without DC. The induction of these regulatory T cells correlates with in vitro T cell non-responsiveness after co-culture with iDC and CC-mDC, while stimulation with pIC-mDC resulted in reproducible cytomegalovirus pp65 or influenza M1 matrix peptide-specific T cell activation as compared to control cultures in the absence of DC. In addition, the T cell non-responsiveness after stimulation with iDC was shown to be mediated by TGF-beta and IL-10. Moreover, the suppressive capacity of CD4+ T cells activated by iDC and CC-mDC was shown to be transferable when these CD4+ T cells were added to an established T cell response. In contrast, addition of CD4+ T cells stimulated by pIC-mDC made responder T cells refractory to their suppressive activity. In conclusion, we hypothesize that DC have a complementary role in inducing both regulatory T cells and effector T cells, where the final result of antigen-specific T cell activation will depend on the activation state of the DC. This emphasizes the need for proper DC activation when T cell immunity is the desired effect, especially when used in clinical trials.

The quantitative measurement of individual cells and their characteristics by means of flow cytometry is already for many years of great value for clinical studies. However, its potential as a tool in (eco)toxicology has only recently been discovered. Analysis of cell cycle kinetics with DNA-staining dyes can offer a valuable alternative to detect effects of chemicals on cell proliferation, an important endpoint in screening estrogen-like properties of chemicals. In the present study, flow cytometric cell cycle analysis in growth arrested MCF-7 cells exposed to five xenoestrogens correspond well with cell proliferation results of the conventionally used E-screen assay. Moreover, re-induction of proliferation in MCF-7 cells, indicated by the percentage of cells in S(ynthesis)-phase, is most pronounced after 24 h exposure, thus allowing a faster screening of xenoestrogens. This flow cytometric proliferation assay confirms that the estrogenic activity of structurally analogous parabens is mediated by the estrogen receptor pathway and is proportional to the alkyl chain length. Moreover, the ER-mediated mode of action of two fluorotelomer alcohols (6:2 FTOH and 8:2 FTOH), recently reported as xenoestrogenic, could be elucidated. These results support the potential of flow cytometric cell cycle kinetics as a screening assay for estrogen-like properties of chemicals.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells (eg. dendritic cells (DC), B-lymphocytes) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from untreated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA coding for consensus HxB-2 (hHXB-2) Gag protein. These DC elicited a strong HIV-1 Gag-specific interferon (IFN)-γ response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DC also triggered IFN-γ secretion by autologous peripheral blood mononuclear cells (PBMC), CD8+ T-cells and CD4+ T-cells from all patients tested. Similar observations were made with CD40-activated cultured autologous B-cells (from HIV-1-seropositive patients) electroporated with hHXB-2 gag mRNA. Gag mRNA-electroporated, but not mock-electroporated, DC or B-cells secreted Gag protein. Next, a novel strategy was developed, using autologous virus sequences. Proviral DNA was amplified by polymerase chain reaction (PCR) from PBMC and viral cDNA was amplified by reverse transcriptase PCR (RT-PCR) from plasma virus. Proviral and viral mRNA were then obtained by in vitro transcription of proviral DNA and plasma viral cDNA, respectively. Significant specific IFN-γ T-cell responses were induced in all patients tested by DC electroporated with patients' autologous proviral and plasma viral mRNA, coding for Gag or Env. The stimulatory effect was seen on PBMC, CD8+ T-cells and CD4+ T-cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin (IL)-2 T-cell response was induced by DC electroporated with hHxB-2 or proviral gag mRNA. Sequence analysis in 4 randomly chosen patients showed that they were infected by 4 different subtypes. In a heteroduplex mobility assay (HMA) up to 50% of the cloned amplified sequences exhibited a differential migration pattern; by sequencing a high degree of variation was demonstrated, particularly between clones derived from proviral DNA and plasma viral cDNA, with mutations in an immunodominant epitope (Gag) or mutations and deletions in non-immunodominant epitopes (Env). The stimulatory effect of autologous DC electroporated with autologous viral sequences opens a major perspective for the development of patient-specific immunotherapy for HIV-1 disease, that might be necessary to control the virus, in view of the major inter-patient and intra-patient sequence variability.