Molecular mechanisms driving Streptococcus mitis entry into human gingival fibroblasts in presence of chitlac-nAg and saliva.

The molecular mechanisms leading to Streptococcus mitis capability of entering oral cells were investigated in a co-culture of S. mitis and Human Gingival Fibroblasts (HGFs) in the presence of saliva. An innovative colloidal solution based on silver nanoparticles (Chitlac-nAg), a promising device for daily oral care, was added to the experimental system in order to study the effects of silver on the bacterial overgrowth and ability to enter non-phagocytic eukaryotic cells. The entry of bacteria into the eukaryotic cells is mediated by a signalling pathway involving FAK, integrin ß1, and the two cytoskeleton proteins vinculin and F-actin, and down-regulated by the presence of saliva both at 3 and 48 h of culture, whereas Chitlac-n Ag exposure seems to influence, by incrementing it, the number of bacteria entering the fibroblasts only at 48 h. The formation of fibrillary extrusion from HGFs and the co-localization of bacteria and silver nanoparticles within the fibroblast vacuoles were also recorded. After longer experimental times (72 and 96 h), the number of S. mitis chains inside gingival cells is reduced, mainly in presence of saliva. The results suggest an escape of bacteria from fibroblasts to restore the microbial balance of the oral cavity.

Features of endothelial dysfunction in umbilical cord vessels of women with gestational diabetes.

BACKGROUND AND AIMS: Gestational diabetes (GDM) is associated with increased oxidative stress and overexpression of inflammatory cytokines, both of which might lead to endothelial dysfunction and vascular disease. As such, GDM could be viewed as a sort of 'short lived' metabolic syndrome. As umbilical cord vessels represent a suitable model for the study of vascular alterations brought about by GDM, the aim of the present work was to characterize the phenotype of human umbilical vein endothelial cells (HUVECs) chronically exposed to hyperglycaemia and to a pro-inflammatory environment during pregnancy so as to identify molecular modifications of cellular homoeostasis eventually impacting on endothelial dysfunction. METHODS AND RESULT: Tissue specimens and HUVECs were obtained from umbilical cords of GDMand control women. As compared to controls, GD-HUVEC exhibited enhanced monocyte adhesion and vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1(ICAM-1) expression and exposure on plasma membrane after tumour necrosis factor-alpha(TNF-α) stimulation (Western blot, flow cytometer). As compared to control cells, GD-HUVEC in basal conditions exhibited enhanced monocyte adhesion, nitric oxide synthase (NOS) expression and activity (eNOS Real-Time polymerase chain reaction, Western Blot for eNOS total protein and monomers/dimers ratio, conversion of [3H]-L-arginine in [3H]-L-citrulline), increased O(-)(2)egeneration together with increased NT levels (immunofluorescence) and reduced NO bioavailability(guanosine 3',5'-monophosphate (cGMP) production, EIA). Furthermore, immunohistochemistry revealed increased eNOS and NT immunoreactivity in GD umbilical cords. CONCLUSION: Endothelial cells exposed in vivo even transiently to hyperglycaemia, oxidative stress and inflammation exhibit durable pro-atherogenic modifications.

Dual role of the caspase enzymes in satellite cells from aged and young subjects.

Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage.

TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway.

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.

The effect of the plasticizer diethylhexyl phthalate on transport activity and expression of P-glycoprotein in parental and doxo-resistant human sarcoma cell lines.

Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 µM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 µM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 µM doxo in the presence of the lowest concentration of DEHP (3 µM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 µM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 µM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.

Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist.

Morphological features of granulosa cells can reflect their functional status. The present study was aimed at comparing possible differences in the fine structure of human granulosa cells exposed to gonadotrophin-releasing hormone (GnRH) agonist or antagonist treatment during ovarian stimulation. Cells were obtained from follicular aspirates of 21 women treated with recombinant follicle-stimulating hormone (rFSH) plus either a GnRH agonist or a GnRH antagonist. Conventional light microscopy procedures and computerized image analysis systems were used to identify different cell type morphological patterns and to quantify different cells distribution. Two morphologically distinct granulosa cell populations, defined as large/pale and small/dark cells, were identified and a different distribution in the two groups of women under investigation was found: a significantly higher percentage in large/pale cells was detected in the agonist-treated women (P<0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P<0.05). Ultrastructural observations showed the presence in both cell populations of typical hallmarks of steroidogenic cells, highlighting signs of functional activity in the large/pale cell population. Further investigations are needed to define the possible clinical significance of these morphological findings.

Regulation of CREB activation by p38 mitogen activated protein kinase during human primary erythroblast differentiation.

Among the molecular events underlying erythroid differentiation, we analyzed the signalling pathway leading to cAMP response element binding (CREB) nuclear transcription factor activation. Normal donor blood light density cells differentiated to pro-erythroblasts during the proliferative phase (10 days) of the human erythroblast massive amplification (HEMA) culture, and to orthochromatic erythroblasts, during the differentiation phase (4 additional days) of the culture. Since erythropoietin was present all over the culture, also pro-erythroblasts left in proliferative medium for 14 days continued their maturation without reaching the final steps of differentiation. p38 mitogen activated protein kinase (p38 MAPK) and CREB maximal activation occurred upon 4 days of differentiation induction, whereas a lower activation was detectable in the cells maintained in parallel in proliferative medium (14 days). Interestingly, when SB203580, a specific p38 MAPK inhibitor, was added to the culture the percentage of differentiated cells decreased along with p38 MAPK and CREB phosphorylation. All in all, our results evidence a role for p38 MAPK in activating CREB metabolic pathway in the events leading to erythroid differentiation.

PI-3-kinase/NF-kappaB mediated response of Jurkat T leukemic cells to two different chemotherapeutic drugs, etoposide and TRAIL.

Jurkat T leukemic cells respond to Etoposide, antineoplastic agent which targets the DNA unwinding enzyme, Topoisomerase II, and TNF-Related-Apoptosis-Inducing-Ligand (TRAIL), 34 kDa transmembrane protein, which displays minimal or no toxicity on normal cells and tissues, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an higher number of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1 and, in parallel, IkappaB alpha on both serine and tyrosine residues. On the other hand, in Etoposide Jurkat exposed cells Ser 32-36 phosphorylation of IkappaB alpha is not sufficient to overbalance the apoptotic fate of the cells, since Bax increase, IAP decrease, and caspase-3 activation determine the persistence of the apoptotic state along with the occurrence of cell death by necrosis. Thus, the existence of a balance between apoptotic and rescue response in 20% of cells surviving to TRAIL suggests the possibility of pushing it in favor of cell death in order to improve the yield of pharmacological strategies.

Caspase-3 is dually regulated by apoptogenic factors mitochondrial release and by SAPK/JNK metabolic pathway in leukemic cells exposed to etoposide-ionizing radiation combined treatment.

Ionizing radiation induces a series of multiple intracellular events which can lead to activation of caspases, cytoplasmic proteases involved in the occurrence of apoptosis. The response of leukemic cells to ionizing radiation is amplified when they have been pre-treated with the anticancer drug etoposide, therefore the aim of this work has been to establish the lowest etoposide concentration combined with the lowest ionizing radiation dose to obtain the best antineoplastic response. Two leukemic cell lines, HL-60 and Jurkat, employed in this study demonstrated different sensitivities to ionizing radiation and to etoposide treatment, with Jurkat T cells requiring a higher dose (1 microM) to display cell cycle perturbation and apoptotic DNA damage similar to those seen in HL-60. We hypothesize that this kind of response could be mediated by mitochondrial release of apoptogenic factors and by SAPK/JNK metabolic pathway activation, both leading to caspase-3 cleavage. All in all these results provide insight into the sensitivity or resistance of leukemic cells to antineoplastic agents and identify molecular targets for rational therapeutic intervention strategies.