Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress.

Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes' viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H2O2-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H2O2 by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols' benefits in strategies aiming to prevent obesity-related diseases.

Age-dependent cytokine responses: trimethyltin hippocampal injury in wild-type, APOE knockout, and APOE4 mice.

In this study, the hippocampal neurotoxicant trimethyltin (TMT) was used to examine possible differential susceptibility associated with the apolipoprotein E genotype. Mice-wild type (C57BL6J), APOE knockout, and APOE4 transgenic-received either saline or TMT (2 mg/kg, ip) at either 21 days or 8 months of age. At both ages, similar mRNA levels were seen in the hippocampus across genotypes for ICAM-1, A20, and MAC-1. GFAP mRNA was higher in the APOE knockouts and APOE4 as compared to wild-type mice. Within 24 h, TMT produced cell death of hippocampal dentate granule neurons and mild astrogliosis in all animals. In 21-day-old mice, TMT exposure significantly increased mRNA levels for ICAM-1 and MIP-1alpha in all genotypes. EB-22, GFAP, TNFalpha, and TGF-beta1 levels were significantly elevated in both wild-type and APOE knockout mice following TMT. At 8 months of age, genotype specific differences were observed. mRNA levels for GFAP, TNFbeta, TNFalpha, and MIP-1alpha were increased in both APOE knockout and APOE4 mice compared to wild-type mice. TMT exposure significantly increased mRNA levels for GFAP and MIP-1alpha in all animals. TNFalpha mRNA levels were increased in wild-type and APOE4 mice while EB22 mRNA levels were increased in both the APOE knockout and APOE4 mice but not wild-type mice. These data suggest an age-dependent effect on both microglia early inflammatory responses to injury associated with the APOE genotype.

Inhibition of human TNF alpha and LT in cell-free extracts and in cell culture by antisense oligonucleotides.

Antisense oligonucleotides (ODN), complementary to mRNA of human tumor necrosis factor alpha (TNF alpha) and lymphotoxin (LT) were tested for their ability to inhibit TNFs. TNFs production was studied in cell-free systems including wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). All ODN were effective in WGE at low concentration (0.2 microM), except those targeted to the 3' region of TNF alpha mRNA. A short ODN complementary to a common region between TNF alpha and LT inhibited both TNFs. In contrast, high ODN concentration (50 microM) was needed to inhibit LT mRNA translation in RRL, whereas no clear inhibition of TNF alpha was observed unless RNase H was added to the translation mixture. ODN effects on TNFs production by stimulated cell line in culture were also investigated. Three ODN-one located in the 5'-untranslated region, one spanning the AUG initiation codon and one downstream of this AUG-were the most effective sequences to decrease TNF alpha production. Two ODN targeted to the AUG initiation codon of LT were also able to inhibit its production. In conclusion we confirm the role of RNase H in cell free systems, and we found that there is no correlation between ODN efficiency in a cell-free system nor in cell culture. Efficient ODN could be used for in vitro investigation of the role of TNF alpha and LT in mechanism in which they are involved.

Expression and production of cytokines by heterohybrids and their parental B cells in CLL.

Three hybrids derived from CD5+ B cell chronic lymphocytic leukemia (B-CLL) and their parental B cells were studied for phenotypic evolution, immunoglobulin (Ig), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) secretion. When phenotypic evolution was examined, hybrids showed the loss of classical B cell markers, indicating that they follow the same pattern of phenotypic differentiation as normal B cells. Hybrids displayed spontaneous high Ig secretion, which did not appear to be modified through stimulation by phorbol 12-myristate 13-acetate (PMA), recombinant interferon-gamma (rIFN-gamma) and Staphylococcus aureus Cowan I (SAC). Parental cells secreted minimal amounts of Ig spontaneously or through IFN-gamma and SAC stimulation, whereas PMA succeeded in increasing this secretion. An opposite pattern was observed when TNF-alpha and IL-6 secretion an expression at the mRNA level were assessed in hybrids and parental cells. TNF-alpha and IL-6 were spontaneously secreted by parental cells and this secretion was increased after PMA and SAC stimulation, both cytokine secretion and expression at the mRNA level were negative in hybrid cells. The absence of expression of these cytokines could be explained either by chromosomal loss or by down regulation. These results indicate that when parental CLL cells are induced to differentiate in the heterohybrid model, they acquire high spontaneous secretion of Ig, lose the classical B cell phenotypic markers and down regulate the expression of the cytokines studied.

Immunomodulation by cytokine antisense oligonucleotides.

The cytokine network is involved in normal immune reaction and in the progression of several pathologies. Antisense (AS) oligonucleotides, which allow specific inhibition of expression of proteins, offer a new methodology to investigate this complex network. This review focuses on the use of AS to modulate cytokine expression. AS may act in different ways such as blocking fixation or progression of the ribosome along the mRNA, mRNA cleavage by RNase H, or preventing normal RNA maturation. In order to improve AS efficiency, chemical modifications have been developed, and improvement of oligonucleotide uptake has been achieved with different systems of vectorization including liposomes (neutral, cationic, immunoliposome), nanoparticles, or covalent attachment of a carrier. In oncogenesis, intracellular or extracellular autocrine loops have been demonstrated by the use of cytokine AS. Involvement of cytokines in immunological reactions (TH1 and TH2 subset, IgE response, lymphokine activated killer, cytotoxic T lymphocyte...) and in hematopoiesis have also been studied with this approach. Therapeutic application of AS has been suggested by inhibition of inflammatory cytokines in vivo. Clinical trials using AS are under investigation in virological and in oncological diseases. At present, cytokine antisenses primarily represent a tool for dissecting the function of a cytokine in vitro, but they may offer in the future a new way for immunomodulation intervention.