Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity.

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 µg/mL and between 15.61 and 31.25 µg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction, oxidative stress markers, and sperm parameters in response to bacterial lipopolysaccharide in infertile men.

PURPOSE: To study the role of Toll-like receptor 4 (TLR4) in human spermatozoa and to assess sperm parameters, oxidative stress markers, and acrosome reaction in response to the stimulation of TLR4 by its ligand, the lipopolysaccharide (LPS), as a major endotoxin of Gram-negative bacteria. METHODS: Our study was carried out in 73 sperm samples from patients undergoing semen analysis for couple infertility investigations. The studied patients were divided into three groups: normozoospermic fertile patients (n = 13), patients with abnormal and leukospermic semen (n = 13), and patients with abnormal and non-leukospermic semen (n = 47). TLR4 expression in human spermatozoa was initially analyzed by western blot. Sperm samples were incubated in the presence of LPS (200 ng/ml) for 18 h. Then, sperm motility and vitality were evaluated by microscopic observation and oxidative stress markers as malondialdehyde (MDA) and carbonyl groups (CG) were spectrophotometrically assessed in neat and selected sperm. A triple-stain technique was also performed to evaluate acrosome reaction in 15 sperm samples from infertile patients. RESULTS: TLR4 expression was confirmed in human spermatozoa with a molecular weight of 69 kDa. In the normozoospermic group, no significant differences in sperm parameters and oxidative stress markers were shown after incubation with LPS in neat and selected sperms. Regarding samples from the non-leukospermic group, LPS reduced spermatozoa motility and vitality rates in selected sperm (P = 0.003; P = 0.004, respectively). A significant increase of MDA and CG levels was also detected (P = 0.01; P = 0.02, respectively). However, only the MDA levels were significantly increased (P = 0.01) in neat LPS-stimulated sperm. The same results were shown within the leukospermic group. The comparison between the two groups, leukospermic and non-leukospermic, in selected sperms showed a more important LPS effect in the leukospermic group significantly on motility and MDA rates (P = 0.006; P = 0.009, respectively). Furthermore, a significant decrease in reacted spermatozoa rate was detected in response to LPS in selected sperm samples from infertile men (P = 0.03). CONCLUSIONS: These findings indicate that human spermatozoa express TLR4 and respond to LPS stimulation with alterations in viability, motility, and the acrosome reaction implicating reactive oxygen species (ROS) production in sperm samples from infertile patients.

Reactive oxygen species production and antioxidant enzyme expression after Epstein-Barr virus lytic cycle induction in Raji cell line.

In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48 h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24 h (p < 0.05) and reached a peak at 48 h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12 h, as compared with untreated cells, reaching a peak at 48 h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

Transcription of the Epstein-Barr virus lytic cycle activator BZLF-1 during oxidative stress induction.

While latent Epstein-Barr virus infection can be in vitro reactivated by various reagents such as 12-0-tetradecanoylphorbol-13-acetate and calcium ionophore, relatively little is known about in vivo physiological and biochemical factors implicated in this reactivation. Previous studies have described an association between oxidative stress and Epstein-Barr virus infection. In this present study, we investigated the effect of oxidative stress inductors: H2O2 and FeSO4 on reactivation of EBV through BZLF-1 gene expression. Oxidative stress was induced in Raji cell line with 0.2 mM H2O2 or with 0.1 mM FeSO4, and assessed by malondialdehyde level determination, as well as superoxide dismutase and catalase genes expression. Simultaneously, the expression of Epstein-Barr virus immediate-early gene BZLF-1 was analyzed by RT-PCR after 6, 12, 24, 36, and 48 h after H2O2 or FeSO4 treatment. Oxidative stress was evidenced in the Raji cell line by high MDA level as well as superoxide dismutase and catalase genes up-regulation. The transcripts of BZLF-1 were detected from 6 h after 30 min of H2O2 or FeSO4 treatment and maintained until 48 h. These results strongly suggest that oxidative stress contributes to the reactivation of EBV lytic cycle, through induction of BZLF-1 gene expression, a process that may play an important role in the pathogenesis of EBV-associated diseases.

High levels of autoantibodies against catalase and superoxide dismutase in nasopharyngeal carcinoma.

INTRODUCTION: Autoantibodies against the antioxidant enzymes have been described in Epstein-Barr virus-associated diseases. Here, we hypothesize that Epstein-Barr virus (EBV), which is associated with nasopharyngeal carcinoma (NPC), induces anticatalase and/or antisuperoxide dismutase autoantibodies that inhibit catalase and/or superoxide dismutase activities and thereby contribute to the oxidative stress status described in this pathology. METHODS: Using a standard enzyme-linked immunosorbent assay (ELISA), the levels of immunoglobulin G (IgG), and M (IgM) directed against catalase and superoxide dismutase in the sera of 30 NPC patients and 30 healthy control individuals were evaluated. The antioxidative profile was tested among the same patients by measuring serum catalase and superoxide dismutase activities. To investigate the implication of EBV in the establishment of autoantibody production in NPC patients, a correlation study between serological testing for EBV viral capsid antigen immunoglobulin G (VCA IgG) and autoantibodies against both enzymes was undertaken. FINDINGS: The levels of IgG against superoxide dismutase and catalase were found to be increased in sera patients compared to controls (P<0.001). NPC patients exhibited decreased catalase (P<0.001) and superoxide dismutase activities (P<0.001) in their sera. However, a positive correlation between superoxide dismutase IgM antibody and IgG antibody titers to VCA (P<0.05, r=0.483, n=21) was found. A positive correlation between catalase (IgM) antibodies and IgG antibody titers to VCA (P<0.05, r=0.546, n=30) was also found. CONCLUSION: High levels of anticatalase and antisuperoxide dismutase antibodies in the sera of NPC patients were found.

Lipid peroxidation and antioxidant system in the tumor and in the blood of patients with nasopharyngeal carcinoma.

Reactive oxygen species play a key role in cancer development by inducing and maintaining the oncogenic phenotypes of cancer cells. In this study, we examined lipid peroxidation and antioxidant enzymes activities in the blood and in the tumor of nasopharyngeal carcinoma patients. Plasma malondialdehyde, conjugated dienes, erythrocytes catalase, and superoxide dismutase activities have been measured in 30 untreated nasopharyngeal carcinoma patients and 30 controls on one hand. On the other hand, tumor malondialdehyde level, catalase, and superoxide dismutase activities have been measured in five nasopharyngeal carcinoma patients and compared with four controls. The lipid peroxidation was confirmed in the plasma by the high levels of malondialdehyde and conjugated dienes (p<0.001, respectively). Additionally, significantly higher concentrations of malondialdehyde were found in biopsies compared to the control group (p<0.001). In erythrocytes, superoxide dismutase activity was higher in patients than in controls (p<0.05), while it was unchanged in the tumor (p>0.05). Both erythrocytes and tumor catalase activities were significantly lower in patients than in controls (p<0.001, respectively). Statistical studies have shown a positive correlation between malondialdehyde level and IgA antibodies level against Epstein­Barr virus capsid antigen (p<0.05). In conclusion, we reported the presence of an oxidative stress in the blood and in the biopsies of nasopharyngeal carcinoma patients where Epstein­Barr virus seems to play a role.

Induction of Epstein-Barr virus (EBV) lytic cycle in vitro causes oxidative stress in lymphoblastoid B cell lines.

Here, we investigated the effect of induction of the Epstein-Barr virus (EBV) viral lytic cycle on the oxidant/antioxidant balance in three lymphoblastoid cell lines: B95-8, Raji, and LCL C1. The induction of the EBV lytic cycle was done by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate (8 nM). Oxidative stress was assessed by measuring malondialdehyde as a parameter of lipid peroxidation, the levels of glutathione, and the activities of three antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase). After 48 h (peak of lytic cycle), a significant decrease in superoxide dismutase activity was observed in B95-8, Raji, and LCL C1 cells (P < 0.05). In addition, in B95-8 cells also a significant decrease of catalase activity was detected (P < 0.05). The glutathione peroxidase activity and the glutathione level were not significantly modified by the induction in any of the cell lines. We found a significant rise in malondialdehyde levels in B95-8, Raji, and LCL C1 cells after the induction of the lytic cycle compared to controls (P < 0.05). In conclusion, induction of EBV lytic cycle in lymphoblastoid cells causes increased oxidative stress in the host cells within 48 h, a process that could be involved in malignant transformations.

Epstein-Barr virus induces an oxidative stress during the early stages of infection in B lymphocytes, epithelial, and lymphoblastoid cell lines.

The study investigates the direct effect of Epstein-Barr virus infection on the oxidative profile of in vitro cultivated human cells. For this purpose, a panel of human EBV target cells presenting heterogeneity in their cellular and culture types (epithelial cells or lymphocytes; primary culture or continuous cell culture) was selected. These cells are purified human B lymphocytes, DG75, 293, and HepG2 cell lines. The oxidative stress was evaluated during the early stages of infection (2, 12, and 24 h) by measuring malondialdehyde, the end product of the lipid peroxidation, as well as the activities of two antioxidant enzymes: catalase and superoxide dismutase. The obtained results were compared with those of the untreated cells and the K562 cell line which has no interaction with EBV. The incubation of the different target cells with EBV induced an oxidative stress in the purified B lymphocytes, DG75, and 293, but not in HepG2 and K562. This oxidative stress was highlighted by an increase in MDA level (P < 0.05), which began 2 h after the addition of the virus and persisted after 12 and 24 h. Simultaneously, a decrease in catalase and superoxide dismutase activities was observed (P < 0.05), suggesting an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species (ROS). The efficiency of EBV infection, assessed by viral DNA PCR amplification, was confirmed in 293 and DG75 but not in HepG2, which was in total concordance with their oxidative profiles. In conclusion, the EBV infection of B and epithelial cells leads to the establishment of an oxidative stress which can play a key role during the viral transformation.

Date seed oil limit oxidative injuries induced by hydrogen peroxide in human skin organ culture.

The skin is chronically exposed to pro-oxidant agents, leading to the generation of reactive oxygen species (ROS). To protect the skin against an over-load of oxidant species, we studied the chemoprotective effect of one new natural product: "date seed oil: DSO". This oil may serve as a potential source of natural antioxidants such as phenols and tocopherols. Here, the antioxidative potential of DSO was compared that of to extra virgin olive oil. Adult human skin was maintained in organ culture in the presence of the DSO and extra virgin olive oil before the addition of hydrogen peroxide (H2O2), in order to prevent the tissue from its oxidizing effects. Skin specimens were collected for histology and for melanin studies. In the investigated model system, DSO protects skin against oxidative injuries. It has a significant chemoprotective effect, by inhibition of damage caused by H_{2}O_{2} compared with specimens without such addition endowing with a radical scavenging ability. The various components from DSO were much more potent antioxidant and more free radical scavengers of the H2O2 than those of olive oil. Our study shows that topical DSO treatment of the skin stimulates events in the epidermis leading to repair skin damage possibly due to antioxidant synergisms.