Introduction

The term "free radicals" designates a family of compounds characterized by great reactivity due to the impaired electron in the outer orbital. To this group belong reactive oxygen species (ROS), such as superoxide anion, hydroxyl radical and hydrogen peroxide, as well as reactive nitrogen species (RNS) which include nitric oxide and peroxynitrite. Although structurally different, free radicals share similar mechanisms to harm body's cells and tissues through damage on proteins, DNA and lipids [1]. The alterations of membrane functions occurring as a consequence of phospholipid modifications represent a relevant, radical species-dependent injury, either when considering the organism as a whole, or a specific integrated function, such as the immune response [2]. The potential therapeutic applications of antioxidants in free radical-related diseases led to the hypothesis of their use to slow down or reverse, for example, symptoms associated with with neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), or spongiform encephalopathies. Such effect could occur through a block of proinflammatory cytokines action and the resulting oxidative damage [3-7]. However, several clinical studies demonstrated that not only malnutrition, but also the excess of certain nutrients (e.g. iron, alphatocopherol, beta-carotene, ascorbic acid) may set into motion oxidation phenomena and, therefore, cell injury [8,9]. Thus, it is of relevance that prior to considering introducing antioxidant therapy into mainstream medicine, significant advances in basic cell biology, pharmacology and clinical bioanalysis will be required. 

Oxidative Stress The body is normally under a dynamic equilibrium between free radical generation and quenching. The physiological defense systems to counteract free radicals encompass endogenous enzyme systems, such as catalase, glutathione reductase and superoxide dismutase, as well as glutathione, urate and coenzyme Q, or exogenous factors (β-carotene, vitamin C, vitamin E and selenium) [10]. All these molecules have an antioxidant effect due to their ability to transform ROS into stable and harmless compounds or by scavenging both ROS and RNS with a redoxbased mechanism [10]. Very recently, a main role in the fight against oxidative stress has been assumed by enzymes such as heme oxygenase (HO) and biliverdin reductase (BVR). Heme oxygenase is a microsomal enzyme which metabolizes heme into ferrous iron, carbon monoxide and biliverdin (BV); the latter is then reduced by BVR into bilirubin (BR), a molecule endowed with strong antioxidant and antinitrosative activities [11- 14]. Interestingly, all these protective factors act in a concerted way, enhancing the antioxidant defense system of the cell. When the balance between ROS/RNS and antioxidants turns in favor of the former, oxidative/nitrosative stress occurs. Although oxidative stress is associated with most diseases, routine assay methods are not nowadays available in the clinical practice. A strategy widely used to determine oxidative stress is measurement of malonyldialdehyde, F2-isoprostanes, or 8-hydroxydesoxyguanosine. Actually, these molecules are regarded as the most reliable markers available [15]. 

A classic example of an oxidation product apparently leading to disease, is oxidized cholesterol in low-density lipoprotein (LDL), which displays a higher atherogenic potential than native LDL, and mainly involved in the pathogenesis of atherosclerosis and coronary heart disease (CHD) [16]. At the cellular level, a large body of data clearly demonstrated that ROS, when produced in low amounts and in a controlled manner, are physiological components of the signalling generated by cytokines, growth factors and neurotrophic peptides [17-22], although they may also activate apoptotic cell death [23]. Extracellularly generated ROS can diffuse through anion channels into the cytoplasm; the resulting variation in the cell redox state leads to modulation of an array of transcription factors (eg. NFkB, AP-1), protein kinases (e.g. AKT, JNK, p38), and receptor activated MAP kinases involved in apoptosis [17,24- 26]. Moreover, the proapoptotic molecules Fas and Fas ligand (FasL) undergo positive transcriptional regulation after exposure to oxidants [27]. Interestingly, Krammer and Colleagues demonstrated that in vitro administration of vitamin E suppresses FasL mRNA expression and protects T cells of HIV-1 infected individuals from Fas mediated apoptosis [28]. Moreover, it was demonstrated that administration of combinations of vitamin E and C to cultures of human umbilical vein endothelial cells (HUVEC) treated with lipopolysaccharide could prevent apoptosis by upregulation of Bcl-2 [29]. Antioxidants, The Immune System And Related Disorders The protective function against external pathogens carried out by the immune system is by itself a source of ROS, since activated neutrophils, produce free radicals to a significant extent [30]. 

Moreover, during the inflammatory process, activation of phagocytes through the interaction of proinflammatory mediators, or bacterial products with specific receptors results in the assembly of the multicomponent flavoprotein NADPH oxidase which catalyzes the production of large quantities of the superoxide anion radical (O2 - ) [31]. In addition to classical reactive oxygen metabolites, activated neutrophils and monocytes release the hemoprotein myeloperoxidase (MPO) into the extracellular space, where it catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HClO) [32]. HClO is a non-specific oxidizing and chlorinating agent that reacts rapidly with a variety of biological compounds, such as sulphydryls, polyunsatured fatty acids, DNA, pyridine nucleotides, aliphatic and aromatic aminoacids and nitrogen-containing compounds [33-35]. Moreover, apart from their direct toxic effects, neutrophil-derived oxidants may promote tissue injury indirectly by altering the protease/antiprotease equilibrium that normally exists within the intestinal interstitium. The oxidative inactivation of important protease inhibitors, coupled to the oxidantmediated activation of latent proteases, creates a favorable environment for neutrophils that allows degradation of the interstitial matrix through elastases, collagenases and gelatinases, as well as injury to epithelial cells [36,37]. 

However, not only immune cell produce ROS necessary for the microbicidal activity, but they are also sensitive to external ROS, due to their high polyunsaturated fatty acids (PUFA) content. Immune cells are atypical, as compared with other somatic cells, in that they contain high levels of antioxidant vitamins, presumably providing protection against lipid peroxidation and immunosuppression, both of which are well known risks posed by high PUFA content [38]. The reactivity of immune cells to exogenous ROS has been shown to be age-dependent. In fact, lymphocytes from elderly individuals appear to be more sensitive to exposure to hydrogen peroxide than those from young adults [39]. Moreover, it has been demonstrated that a micronutrient deficiency can be the cause of suppression of immune function affecting both innate Tcell-mediated immune response and adaptive antibody response, thus altering the balanced host response. Therefore, an adequate intake of vitamins and antioxidant elements seems to be essential for an efficient function of the immune system. Micronutrient deficiency occurs in various conditions, such as eating disorders, tobacco smokers, chronic diseases, aging. During aging, changes in the immune system are frequent and associated with increased susceptibility to infections. Antioxidant vitamins and trace elements contribute to maintain an effective immune response [40].

 For example, administration of vitamin E supplement to healthy elderly patients produced an increased antibody titer to both hepatitis B and tetanus vaccine [41], thus enhancing T-cell mediated functions. In conclusion, maintaining adequate antioxidant status may provide a useful approach in attenuating cell injury and dysfunction observed in some inflammatory/ autoimmune disorders [42,43]. Autoimmunity has been for decades considered the result of a breakdown in self-tolerance. At the present, it is known that autoimmunity is a physiological process [44]. This phenomenon becomes pathological when the number of autoreactive cells, and particularly the avidity of their receptors for autoantigens, increases [44]. Triggering of the disease usually depends both on the increase in immunogenicity of the target cell, which may be secondary to a viral infection (Chediak-Higashi syndrome and Griscelli syndrome by EBV), and on the individual's own capacity to recognize the autoantigens (HLA, or T cell repertoire in Familial hemophagocytic lymphohistiocytosis [FHL]) [45]. Moreover, apart from the genetic defects that may predispose to autoimmune diseases, one must take into account the environmental factors that are implicated in the development of such pathologies. Among them, an important role is played by xenobiotics such as chemicals, drugs and metals [46]. Iron, aluminum, and manganese readily cross the blood brain barrier via specific or nonspecific carriers, and contribute to the nervous tissue damage [47,48]. The toxic effects of metals are mediated through free radical formation, or enzyme inhibition [49- 53]. 

In addition, metals may act as immunosuppressants (cytostatically), or as immunoadjuvants (through nonspecific activation of the immune response) [54,55]. Several mechanisms are proposed on how metals may act within the immune system to induce autoimmunity. Patients suffering from scleroderma develop autoantigens with metal-binding sites. After metal binding, free radical species are generated which fragment auto-antigens thereby exposing cryptic epitopes, which may then trigger autoimmunity [56,57]. Taken together, these findings underlie the importance of exogenous factors in the pathogenesis of autoimmunity. Nevertheless, all these elements do not appear sufficient to provoke chronic autoimmune diseases such as Multiple Slerosis (MS), myasthenia gravis, Insulin Dependent Diabetes Mellitus (IDDM) or Hashimoto's thyroiditis, and the passage to chronic disease is usually secondary to a defect in immunoregulation. Several classes of regulatory T cells, such as Th2, CD25+ and natural killer (NK) T cells, are implied in autoimmune pathologies. In an animal model of a Th2-dominated autoimmune syndrome, the administration of the antioxidant N-acetyl-cysteine (NAC) induced a decrease in mast-cell expression of both IgE and IL-4 [58]. Of major interest is the discovery of the therapeutic potential of a new benzoquinone-containing product derived from wheat germ fermentation. This latter has been shown to have immune restorative properties because it affects the Th1/Th2 network by inhibiting the Th2 response [59]. Another beneficial effect of this molecule is its anti-metastatic activity, shown in various human malignancies and Jurkat leukemia cell line [60].

 Intriguingly, the combined treatment with wheat germ and vitamin C profoundly inhibited metastasis formation in various tumor models of different origin (Lewis lung carcinoma, B16 melanoma and human colon carcinoma xenografts [HCR25]) [61]. On the contrary, wheat germ had no toxicity on peripheral blood leukocytes (PBLs) at doses that affected tumor cells. The crude powder extract of fermented wheat germ inhibits nucleic acid ribose synthesis primarily through the non-oxidative steps of the pentose cycle [60]. Curiously, another quinone compound, carnosic acid quinone, like wheat germ, recovers potent antioxidant activity upon standing [62]. Keeping in mind the importance of oxidative stress in the regulation/dysregulation of immune system, the use of antioxidants in such diseases has been reasonably proposed. Rheumatoid arthritis (RA) is a classic example of autoimmune disease. Joint inflammation in rheumatoid arthritis (RA) is characterized by invasion of T cells in the synovial space and proliferation of activated macrophages and fibroblasts in the synovial intima [63]. Therefore, in the rheumatic joint there is an increased activity of fibroblasts and leucocytes which produce ROS [64,65]. Very recently, antioxidants have been successfully used as adjuvant therapy in RA [66,67]. Although the results obtained with RA seemed to be very promising, the indiscriminate use of antioxidants in autoimmune disorders is not recommended. 

In fact, autoimmune lymphoproliferative syndrome (ALPS), MS, type 1 diabetes and multiple autoimmune syndrome, have been linked to decreased Fas functionality [68] and, as discussed previously, antioxidants may up-regulate Fas and FasL in vitro. Increasing evidence provides support that oxidative stress and apoptosis are closely related physiological phenomena and are implicated in diseases including autoimmune diseases. Therefore molecules that target both apoptosis-related signal transduction and oxidative stress, like antioxidants, are likely to result in the improvement of these pathologies. A novel possible approach to modulate immune system thus preventing autoimmunity or transplant rejection is the activation of cytoprotective and antioxidant enzymes such as HO-1. Heme oxygenase-1, the inducible isoform of HO, is a key protein in the cell stress response and its up-regulation is a common event during pro-inflammatory conditions [11,69-72]. Recent work clearly demonstrated that regulatory T cells overexpress HO-1 and release CO under pro-oxidant conditions. Carbon monoxide may inhibit the proliferation of effector T cells, thus reducing the immune response and prevent autoimmunity and/or graft reaction [73,74]. Dietary antioxidants, in particular polyphenols, has been shown to increase HO-1 expression in different in vitro systems [3,75,76] and the potential use of this natural substances to regulate immune response should be carefully addressed. Antioxidants, Cancer And Neurodegenerative Disorders It is well known that the dietary consumption of fruits, vegetables, herbs, or their phytochemical constituents aid in cancer prevention [77-79]. 

It is believed that the antioxidant properties of such foods protect cells from ROSmediated DNA damage that can result in mutation and subsequent carcinogenesis. ROS-induced DNA damage can take many forms, ranging from specifically oxidized purine and pyrimidine bases, to DNA lesions such as strand breaks, sister chromatid exchanges (SCEs), and the formation of micronuclei [80]. However, the equation "antioxidant = benefit" is not always true. In vivo experiments demonstrated that retinol increases both the humoral and the cell-mediated immune response and could enhance immune surveillance against tumorigenesis [81-83]. Retinol may influence the immune response by quenching free radicals, which could lower the level of immunosuppressing lipid peroxides, alter arachidonic acid metabolism, etc. [82,84]. In the last few years many studies have been conducted to investigate the effects of vitamins on disease prevention. The first results have been encouraging and a wide number of people are taking antioxidant supplements with the aim to improve their health. These studies, initially, have shown that a high consumption of fruit and vegetables decreases risks of lung cancer in healthy individuals and a combination of β-carotene, vitamin E and selenium reduced stomach cancer mortality in China [85,86]. Conversely, supplemental β-carotene alone or in combination with retinol or vitamin E did not have any effect on cancer risk, or increased the development of lung cancer in smokers [87,88].