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Oxidative stress plays an important role during inflammatory diseases and recent therapies have focused on antioxidant administration to diminish oxidative stress and to arrest inflammatory processes. In this study, we investigated the impact of the GSH modulating effects of curcumin, a naturally derived polyphenol, on inflammatory processes in myelomonocytic U937 cells. One hour after administration of 10 micromol/l curcumin reactive oxygen species (ROS) production was significantly increased in undifferentiated U937 cells (+43%). Twenty-four hour after addition of curcumin, a significantly decreased ROS concentration was found (-32%), whereas GSH (+110%) and GSSG (+88%) content increased. A higher concentration of curcumin (25 micromol/l) caused an even stronger increase of GSH (+145%) and GSSG (+101%), but significantly decreased percentage of living cells to 84%. The increased GSH content of differentiated U937 cells after pre-incubation with curcumin was associated with lowered ROS production, nuclear factor kappa B (NFkappaB) activation (-34%) and tumor necrosis factor alpha (TNF-alpha) secretion (-51%) after LPS exposure. Curcumin inhibited TNF-alpha formation was also seen after GSH depletion by buthionine sulfoximine (BSO). This study shows that the antioxidative effects of curcumin are preceded by an oxidative stimulus, which is time and dose-dependent. Excessive concentrations of curcumin may even harm cells, as cell viability was decreased, in spite of elevated GSH contents. There was no clear relationship between intracellular GSH concentrations and the anti-inflammatory effects of curcumin.

It has been shown that the polyunsaturated fatty acid docosahexaenoic acid (DHA) can sensitize various tumor cells to reactive oxygen species (ROS)-inducing anticancer agents. Recently, we demonstrated that DHA also enhances the apoptotic effect of clinically achievable concentrations (1-2 microM) of arsenic trioxide (As2O3) in several As2O3-resistant human leukemic cell lines via a ROS-dependent mechanism. The aim of the present study was to evaluate whether this combined effect of As2O3 and DHA is also applicable to As2O3-resistant solid tumor cells. We have tested 12 different tumor cell lines, including MDA-MB-468, SK-BR-3, MCF-7 (breast cancer), ES-2, SKOV-3 (ovarian cancer), HT-29, SW-620, LS-174T (colon cancer), PC-3 (prostate cancer), HeLa (cervical cancer), PANC-1 (pancreatic cancer) and one primary melanoma cell line. With the exception of MDA-MB-468 and ES-2, all cells were resistant to treatment with either As2O3 or DHA alone. However, combined treatment with As2O3 and DHA significantly reduced viability in 7 of the 10 As2O3-resistant solid tumors tested. The cytotoxic effect of As2O3 and DHA was associated with the induction of apoptosis and a concomitant increase of intracellular lipid peroxidation products. Importantly, the combined effect of As2O3 and DHA was selectively toxic for malignant cells since no cytotoxic effect was observed in normal skin fibroblasts, human microvascular endothelial cells and peripheral blood mononuclear cells derived from healthy donors. Our data indicate that DHA may help to extend the therapeutic spectrum of As2O3 in the treatment of solid tumors since it may overcome de novo or acquired resistance to As2O3., (Copyright 2004 Wiley-Liss, Inc.)

Purpose of Review: Oxidative stress is caused by a higher production of reactive oxygen and reactive nitrogen species or a decrease in endogenous protective antioxidative capacity. In all types of critical illness, such as sepsis, trauma, burn injury, acute pancreatitis, liver injury, severe diabetes, acute respiratory distress syndrome, AIDS and kidney failure, the occurrence of increased oxidative stress or a reduced antioxidative status is described. Whereas in the past, reactive oxygen and reactive nitrogen species were mainly known as harmful agents, recent investigations have given a new insight into the (patho)physiological importance of these substances as powerful messenger molecules involved in gene regulation, thereby enabling the synthesis of cytokines or adhesion molecules necessary for defending inflammatory processes. As shown in this review, there are numerous possibilities for the quantification of oxidative stress., Recent Findings: Several investigations showed a close association of single or multiple parameters, such as total antioxidative capacity, lipid peroxidation, vitamins C and E, the activation of nuclear factor kappa B, and respiratory burst, with the patient's outcome. However, no recommendation for a single parameter to be measured can be given because the assays described do not allow the definition of an overall "antioxidative status" for patients., Summary: The occurrence of oxidative stress in critically ill patients is associated with a poor prognosis. The measurement of a cluster of assays representative of the quantification of reactive species or of antioxidants may improve the usefulness of therapeutic intervention and increase knowledge of pathophysiological alterations.

Background: In our previous study, we found complexes of the non-coding pMP6 plasmid and cationic liposomes which exerted antitumor activity in the C26 model. We now sought to unravel the underlying protective effector mechanism(s)., Materials and Methods: C26 recipients (i.p.; day 0) were injected once (day -2) or twice (days -2 and 2) with pMP6/liposome complexes. Thus treated mice were evaluated for tumor growth and the occurrence of innate and specific immune responses., Results: A single pMP6/liposome injection prolonged the survival of the animals as compared to non-treated C26 recipients (median survival 28 vs. 19 days). Two injections not only prolonged the survival time (median survival 55 days) but completely prevented tumor development in 50% of C26 recipients. I.p. administration of pMP6/liposome complexes resulted in the expression of the proinflammatory cytokines IL-6, IFN-gamma and TNF-alpha. This was followed by the appearance of activated NK cells within the peritoneal cavity and, somewhat later, by the induction of C26-specific CTLs. T cell depletion studies demonstrated the latter to be critical for the protective effect to occur., Conclusion: Our data suggests that co-application of tumor cells and immunostimulatory pDNA/liposome complexes induces a protective and tumor-specific T cell response. Therefore these complexes may be considered promising agents in the immunological gene therapy of cancer.

Toll-like receptors (TLR) play a pivotal role in the innate immune response, and the expression levels of these receptors may reflect the sensitivity of immune cells to infections. The binding of lipopolysaccharide (LPS) to TLR-4 triggers human monocytes to produce cytokines, which play a dominant role in the inflammatory response, as can be observed during sepsis and after polytrauma. Here, we evaluated TLR-4 expression of isolated monocytes in the presence of tumor necrosis factor (TNF)-alpha, interleukin (IL) 6, IL-8, and IL-10, and we investigated cellular activation of this treatment. TNF-alpha significantly down-regulated TLR-4 mRNA expression after 6 h (100% vs. 38.5% +/- 4%; P < 0.05). This down-regulation was followed by a dose- and time-dependent diminished expression of TLR-4 surface protein (100% vs. 8.0% +/- 5%; P < 0.01). Forty-eight hours after TNF-alpha treatment, a reduced nuclear factor (NF)-kappaB translocation and a diminished IL-6 secretion after LPS stimulation were found (100% vs. 42.0% +/- 23%; P < 0.05). In contrast, IL-6 incubation upregulated TLR-4 cell surface protein (100% vs. 165.8% +/- 24%; P < 0.05) and increased the ability to activate NF-kappaB and AP-1 after LPS stimulation. Stimulation with IL-8 or IL-10 had no significant effects. We conclude that not only LPS but also TNF-alpha and IL-6 have the potency to regulate the immune response via TLR-4. Down-regulation of TLR-4 by TNF-alpha is associated with LPS hyporeactivity for NF-kappaB formation, whereas upregulation of TLR-4 via IL-6 can increase the responsiveness of mononuclear phagocytes.

Glutamine (GLN) is the most abundant free amino acid (AA) in the human body. Under GLN-free conditions, which can be obtained when cells are cultivated in vitro, tissue cells cannot grow. Therefore, when classifying GLN as a "non-essential" AA, one must consider that in the human body GLN is synthesized from essential AAs and is continuously delivered from skeletal muscle to other organs. It is fascinating that a relatively simple AA like GLN can stimulate a large variety of cellular reactions. GLN stimulates not only the growth of cells but also the expression of surface antigens, the formation of cytokines, and the synthesis of heat shock proteins. Further, a GLN deficiency leads to a cell cycle arrest in G(0) to G(1) and reduces apoptosis. Interestingly, many of these biological activities also are associated with the cellular reduced oxygen potential, which depends mainly on the ratio of reduced to oxidized glutathione. Experimental animal studies have shown that the administration of GLN increases tissue concentrations of reduced glutathione. This review describes the relation of GLN to reduced glutathione metabolism and discusses the alteration of reduced glutathione metabolism under a variety of clinical conditions such as reperfusion injury, myocardial infarction, respiratory insufficiency, cancer, diabetes, liver disease, and clinical protein catabolism.

Reactive oxygen species, formed in various biochemical reactions, are normally scavenged by antioxidants. Glutathione in its reduced form (GSH) is the most powerful intracellular antioxidant, and the ratio of reduced to oxidised glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. Several clinical conditions are associated with reduced GSH levels which as a consequence can result in a lowered cellular redox potential. GSH and the redox potential of the cell are components of the cell signaling system influencing the translocation of the transcription factor NF kappa B which regulates the synthesis of cytokines and adhesion molecules. Therefore, one possibility to protect cells from damage caused by reactive oxygen species is to restore the intracellular glutathione levels. Cellular GSH concentration can be influenced by exogenous administration of GSH (as intravenous infusion or as aerosol), of glutathione esters or of GSH precursors such as glutamine or cysteine (in form of N-acetyl-L-cysteine, alpha-lipoic acid). The modulation of GSH metabolism might present a useful adjuvant therapy in many pathologies such as intoxication, diabetes, uremia, sepsis, inflammatory lung processes, coronary disease, cancer and immunodeficiency states.

A crude extract of demineralized bone matrix caused an altered differentiation of limb bud cells which was seen within 5 days in culture. Using this bioassay system we purified two factors to homogeneity and found that according to their N-terminal sequences they corresponded to TGF-beta 1 and TGF-beta 2 isolated from platelets. Biochemical analyses and biological studies (molecular mass determination, inactivation by reducing agents and proteases, antibody neutralization, competitive binding to TGF-beta receptors and influence on protein expression) provided additional evidence that the two proteins isolated from demineralized bone matrix were apparently identical to TGF-beta 1 and TGF-beta 2. Proteoglycan content, alkaline phosphatase activity and response of the cells to PTH stimulated adenylate cyclase were quantitatively changed by the factors. Culturing limb bud cells on polycarbonate membranes resulted in a rapid and extensive growth and differentiation of the cells to palpable tissue pieces. Relative to controls distinct cell and tissue morphology was observed macroscopically and in histological sections of these tissue pieces.