Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells.

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

Lack of evidence for superantigen activity of Toxoplasma gondii towards human T cells

Toxoplasma gondii is an obligatory intracellular parasite whose life cycle may include man as an intermediate host. More than 500 million people are infected with this parasite worldwide. It has been previously reported that T. gondii contains a superantigen activity. The purpose of the present study was to determine if the putative superantigen activity of T. gondii would manifest towards human T cells. Peripheral blood mononuclear cells (PBMC) from individuals with no previous contact with the parasite were evaluated for proliferation as well as specific Vß expansion after exposure to Toxoplasma antigens. Likewise, PBMC from individuals with the congenital infection were evaluated for putative Vß family deletions in their T cell repertoire. We also evaluated, over a period of one year, the PBMC proliferation pattern in response to Toxoplasma antigens in patients with recently acquired infection. Some degree of proliferation in response to T. gondii was observed in the PBMC from individuals never exposed to the parasite, accompanied by specific Vß expansion, suggesting a superantigen effect. However, we found no specific deletion of Vß (or Valpha) families in the blood of congenitally infected individuals. Furthermore, PBMC from recently infected individuals followed up over a period of one year did not present a reduction of the Vß families that were originally expanded in response to the parasite antigens. Taken together, our data suggest that T. gondii does not have a strong superantigen activity on human T cells.

Resistance of multicellular aggregates to pharmorubicin observed in human hepatocarcinoma cells

The objective of the present study was to investigate the multicellular resistance of human hepatocarcinoma cells BEL-7402 to pharmorubicin. Cells (1 x 10(4)) and 200 microcarrier Cytodex-3 beads were seeded onto a 24-well plate and cultured in RPMI 1640 medium. After the formation of multicellular aggregates, morphology and cell viability were analyzed by scanning electron microscopy, transmission electron microscopy and flow cytometry, respectively. The IC50 was determined by flow cytometry and MTT assay after the cells cultured in aggregates and monolayers were treated with pharmorubicin. The culture products exhibited structural characteristics somewhat similar to those of trabecular hepatocarcinoma in vivo. Among the microcarriers, cells were organized into several layers. Intercellular spaces were 0.5-2.0 µm wide and filled with many microvilli. The percent of viable cells was 87%. The cells cultured as multicellular aggregates were resistant to pharmorubicin with IC50 4.5-fold and 7.7-fold that of monolayer culture as determined by flow cytometry and MTT assay, respectively. This three-dimensional culture model may be used to investigate the mechanisms of multicellular drug resistance of hepatocarcinoma and to screen new anticancer drugs.

Differential expression of integrin subunits on adherent and nonadherent mast cells

Mast cell progenitors arise in bone marrow and then migrate to peripheral tissues where they mature. It is presumed that integrin receptors are involved in their migration and homing. In the present study, the expression of various integrin subunits was investigated in three systems of adherent and nonadherent mast cells. Mesentery mast cells, freshly isolated bone marrow-derived mast cells (BMMC) and RBL-2H3 cells grown attached to tissue culture flasks are all adherent mast cells and peritoneal mast cells, and cultured BMMC and RBL-2H3 cells grown in suspension represent nonadherent mast cell populations. Pure populations of mast cells were immunomagnetically isolated from bone marrow, mesentery and peritoneal lavage using the mast cell-specific monoclonal antibody AA4. By immunomicroscopy, we could demonstrate that all of these mast cells expressed alpha4, alpha5, alpha6, ß1 and ß7 integrin subunits. The expression of the alpha4 integrin subunit was 25% higher in freshly isolated mesentery mast cells and BMMC. Consistent with the results obtained by immunomicroscopy, mesentery mast cells expressed 65% more mRNA for the alpha4 integrin subunit than peritoneal mast cells. In vitro studies were also conducted using the rat mast cell line RBL-2H3. RBL-2H3 cells grown attached to the tissue culture flasks or as suspension cultures expressed the same integrin subunits identified in bone marrow, mesenteric and peritoneal mast cells ex vivo. Similarly, the expression of alpha4 integrin was higher in adherent cells. Therefore, alpha4 integrins may play a critical role in the anchorage of mast cells to the extracellular matrix in bone marrow and in peripheral tissues.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of ß-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM ß-estradiol inhibited cell proliferation in 30% (0.70 ± 0.03 x 10(5) cells) and increased tyrosinase activity in 50% (7130.5 ± 376.5 cpm/10(5) cells), as compared to untreated cells (1.0 ± 0.05 x 10(5) cells and 4769 ± 25.5 cpm/10(5) cells, respectively). Both responses were completely (100%) blocked by 1 µM tamoxifen. Higher concentrations (up to 1.6 nM) or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-³H]-ß-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 µM, Kd = 0.14 µM, maximal displacement of 93%) or by 10 µM tamoxifen (displacement of 60%). ß-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with ß-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

Caspase-8 and p38MAPK in DATS-induced apoptosis of human CNE2 cells

Nasopharyngeal carcinoma is a common malignancy in Southern China of uncertain etiologic origin. Diallyl trisulfide (DATS), one of the major components of garlic (Allium sativum), is highly bactericidal and fungicidal. In this study, we investigated the function of p38 mitogen-activated protein kinase (MAPK) and caspase-8 in DATS-induced apoptosis of human CNE2 cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], flow cytometry assay, and Western blotting. After CNE2 cells were treated with DATS (50, 100, or 150 μM) for 24 h, cell viability rates were 75.9, 63.4 and 39.6%, and apoptosis rates were 24.5, 36.9, and 62.4%, respectively. The data showed that DATS induced CNE2 cell death in a dose-dependent manner. After human CNE2 cells were treated with 100 μM DATS and inhibitors (10 μM SB203580 and Z-LETD-FMK for p38MAPK and caspase-8, respectively), changes in cell viability and apoptosis and in p38MAPK and caspase-8 activity were detected. Cell viability rates were 66.5 and 68.1% and decreased 9.9 and 11.5% compared with inhibitor treatment alone. Apoptosis rates were 31.53 and 29.98% and increased 9.1 and 10% compared with inhibitor treatment alone. The results indicated that DATS activates p38MAPK and caspase-8, but both inhibitors have an effect on P38MAPK and caspase-8 activity. In conclusion, our data indicate that p38MAPK and caspase-8 are involved in the process of DATS-induced apoptosis in human CNE2 cells and interact with each other.

A phthalide derivative isolated from endophytic fungiPestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniaeisolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation ofp73, JunB, FKHR, andBim. The results indicate that MP may be a potential treatment for cervical cancer.

Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

Permissivity of human HeLa cells to bovine adenovirus type 2 (BaV2 infection

Infection of hUlnan cells by bovine adenovirlls type 2 (BAV2) is abortive. To obtain a better understanding of this pllenomel1011, and in particular to identify Wllich steps in the viral replicative cycles are altered dllring this virlls-host cells interaction, we have llndertaken a detailed study of BAV2 infections of the nonpennissive hUlnan IIeLa cells. Using autoradiography and 3H-thymidine-labeled vvhole virus particles for infection of HeLa cells, vve determined that viral attachluent appears normal. Furthermore, Southern analysis revealed that internalization and transport to the nuclells occurs in BAV2 infected HeLa cells. To investigate viral DNi\ synthesis, infectivity assays involving hydroxyllrea, a viral DN-A synthesis inhibitor, were carried out. The results revealed that Bft:LV2 DNA synthesis does not occur in HeLa cells. Fllrtller investigations into viral early gene expression by northern blotting analyses indicated that HeLa cells fail to support expression of EIA. This suggested that abortive infection by BAV2 could be attributed to faiiure of EIA to express. To test the possibility that the failure to express ElA was due to the inability of the host cell to recognize the E lA prOlTIoter, ,ve carried out transient expression transfection experiments using plaslnids \vith the bacterial lacZ linder the control of either BAV2 or i\d5 EIA promoter. X-gal histochelIlical assays sho\ved expression of lacZ from the Ad5 ElA prOlnoter but no expression of lacZ [rOln the BAV2 EIA prOlTIoter. This further suggests that the abortive infection b:y BAV2 could be attributed to failure of EIA to express dlle to a nonfllnctional prOlTIoter in hlunan cells. Thus we speClllated that abortive infection of HeLa cells by adenoviruses may be averted by providing EtA functions in trans. To demonstrate this, we coinfected HeLa cells with Ad5 and BAV2, reasoning that Ad5 could cOlnpensate for EIA deficiency in BAV2. OUf results showed that BAV2 DNA synthesis was indeed Sllpported in HeLa cells coinfected with Ad5dlE3 as revealed by Southern analysis. In contrast, coinfection of HeLa cells \vith BAV2 and Ad5dlElE3 mutallt did not support BLt\V2 DNA synthesis. Interestingly, BAV2 failed to replicate in 293 cells which are constitlltively expressing the El genes. This could ilnply that El is necessary but not sufficient to avert the failllre ofBAV2 to undergo productive infection ofhulnan cells.

Investigation of the Expression of Glucose Transporter Proteins in Human Cancer Cells

Cancer cells are known to display increased glucose uptake and consumption. The glucose transporter (GLUT) proteins facilitate glucose uptake, however, their exact role in cancer metabolism remains unclear. The present study examined mRNA and protein expression of GLUT1, GLUT3, GLUT4 and GLUT12 in lung, breast and prostate cancer cells and corresponding noncancerous cells. Additionally, GLUT expression was determined in tumours from mice xenografted with human cancer cells. Differences in the mRNA and protein expression of GLUTs were found between cancerous and corresponding noncancerous cells. These findings demonstrate abundant expression of GLUT1 in cancer and highlight the importance of GLUT3 as it was expressed in several cancer cells and tumours. GLUT expression patterns in vitro were supported by the in vivo findings. The study of GLUT protein expression in cancer is important for understanding cancer metabolism and may lead to identification of biomarkers of cancer progression and development of target therapies.

Dynamic epigenetic changes in immune responses to infection in human dendritic cells

La méthylation de l'ADN est une marque épigénétique importante chez les mammifères. Malgré le fait que la méthylation de la cytosine en 5' (5mC) soit reconnue comme une modification épigénétique stable, il devient de plus en plus reconnu qu'elle soit un processus plus dynamique impliquant des voies de méthylation et de déméthylation actives. La dynamique de la méthylation de l'ADN est désormais bien caractérisée dans le développement et dans le fonctionnement cellulaire des mammifères. Très peu est cependant connu concernant les implications régulatrices dans les réponses immunitaires. Pour se faire, nous avons effectué des analyses du niveau de transcription des gènes ainsi que du profilage épigénétique de cellules dendritiques (DCs) humaines. Ceux-ci ont été faits avant et après infection par le pathogène Mycobacterium tuberculosis (MTB). Nos résultats fournissent le premier portrait génomique du remodelage épigénétique survenant dans les DCs en réponse à une infection bactérienne. Nous avons constaté que les changements dans la méthylation de l'ADN sont omniprésents, identifiant 3,926 régions différentiellement méthylées lors des infections par MTB (MTB-RDMs). Les MTB-RDMs montrent un chevauchement frappant avec les régions génomiques marquées par les histones associées avec des régions amplificatrices. De plus, nos analyses ont révélées que les MTB-RDMs sont activement liées par des facteurs de transcription associés à l'immunité avant même d'être infecté par MTB, suggérant ces domaines comme étant des éléments d'activation dans un état de dormance. Nos données suggèrent que les changements actifs dans la méthylation jouent un rôle essentiel pour contrôler la réponse cellulaire des DCs à l'infection bactérienne.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.