**2.1. Free radical production and Reactive Oxygen Species (ROS)**

Free radicals are chemical species capable of independent existence and which contain one or more unpaired electrons [1]; they are like loose cannons or rather hot pepper ready to cause biological damage. They consistently react with proteins, lipids, polysaccharides and nucleic acids of biological components and thus damage cell membranes, organelles and importantly DNA [2, 3] with the resultant disease condition [4]. It has been estimated that the average person produces about 10,000–20,000 free radicals attacking each body cell daily [5]. In biological systems, oxygen radicals are known collectively as reactive oxygen species (ROS) possessing high chemical reactivity, with original formation in the mitochondria and smooth endoplasmic reticulum as oxygen reduces along the electron transport chain [6,7]. ROS are formed in other processes that include white blood cells such as neutrophils, which specialise in producing oxygen radicals used in host defence against invading pathogens, cellular exposure to abnormal environment such as hypoxias [8], drugs/xenobiotics [9] and ionising radiation [10]. Hypoxic conditions are created when oxygen is limited and mitochondria pump out ROS which alert the cell to the shortage, but how cells sense hypoxia is a subject of much debate; Bell et al. [11] described these extensively. Reactive oxygen species (ROS), namely, hydroxyl radicals (HO'), superoxide anions (02 - ), nitric oxide (NO) and hydrogen peroxide (H202), are constantly generated in aerobic organisms in response to both external and internal stimuli [12]; therefore, metabolic activity initiated by immune cells against stimuli creates electron-deficient free radicals. As highly reactive molecules, they damage other molecules by abstracting electrons from them. High doses and/or inadequate removal of ROS results in oxidative stress which causes severe metabolic malfunctions and damage to biological macromolecules. ROS therefore are the main cause of oxidative stress.

### **2.2. ROS in HIV infection: Do they differ in the different stages of HIV?**

Oxidative stress (OS) means any perturbation in the pro-oxidant to antioxidant balances in favour of oxidation, thus resulting in damage to cells. OS increases the replication of HIV and the amount of certain cytokines, among them is tumour necrosis factor-alpha (TNF-α) through the activation of nuclear factor-kappa binding (NF-κB) and indirectly by activation of genes that further promote OS [13]. Oxidant production in HIV infection, however, is through the stimulatory effects of gp125 (an HIV glycoprotein) and tat, the viral-transactivating protein [14]. Mycoplasmas as well enhance the replication of HIV by increasing oxidative stress since they are known to produce H202. HIV coinfection with mycoplasma therefore results in the release of H202 from T-cells.

It has been proposed that CD4+ T-cells are depleted by apoptosis and that T-cells are primed to undergo apoptosis upon cross-linking of CD4 by gp120 of the virus [15]. Subsequent activation perhaps by conventional antigens or superantigens induces apoptosis in these Tcells and increases the progression rate. OS have been implicated to play a rather devastating role in the progression of HIV disease. In this case, certain questions are raised, namely, does the OS condition in HIV infection differ in the various stages of the disease? Does the threshold of OS correspond to the worsening clinical manifestation of the disease as the infection progresses? What is the OS threshold that is associated with the stages of infection?

Generally, chronic OS, with constant generation of free radicals, affects the immune system's fight against HIV through the following mechanisms:


**2. Free radicals in HIV disease progression**

184 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

hydroxyl radicals (HO'), superoxide anions (02

release of H202 from T-cells.

**2.1. Free radical production and Reactive Oxygen Species (ROS)**

Free radicals are chemical species capable of independent existence and which contain one or more unpaired electrons [1]; they are like loose cannons or rather hot pepper ready to cause biological damage. They consistently react with proteins, lipids, polysaccharides and nucleic acids of biological components and thus damage cell membranes, organelles and importantly DNA [2, 3] with the resultant disease condition [4]. It has been estimated that the average person produces about 10,000–20,000 free radicals attacking each body cell daily [5]. In biological systems, oxygen radicals are known collectively as reactive oxygen species (ROS) possessing high chemical reactivity, with original formation in the mitochondria and smooth endoplasmic reticulum as oxygen reduces along the electron transport chain [6,7]. ROS are formed in other processes that include white blood cells such as neutrophils, which specialise in producing oxygen radicals used in host defence against invading pathogens, cellular exposure to abnormal environment such as hypoxias [8], drugs/xenobiotics [9] and ionising radiation [10]. Hypoxic conditions are created when oxygen is limited and mitochondria pump out ROS which alert the cell to the shortage, but how cells sense hypoxia is a subject of much debate; Bell et al. [11] described these extensively. Reactive oxygen species (ROS), namely,


(H202), are constantly generated in aerobic organisms in response to both external and internal stimuli [12]; therefore, metabolic activity initiated by immune cells against stimuli creates electron-deficient free radicals. As highly reactive molecules, they damage other molecules by abstracting electrons from them. High doses and/or inadequate removal of ROS results in oxidative stress which causes severe metabolic malfunctions and damage to biological

Oxidative stress (OS) means any perturbation in the pro-oxidant to antioxidant balances in favour of oxidation, thus resulting in damage to cells. OS increases the replication of HIV and the amount of certain cytokines, among them is tumour necrosis factor-alpha (TNF-α) through the activation of nuclear factor-kappa binding (NF-κB) and indirectly by activation of genes that further promote OS [13]. Oxidant production in HIV infection, however, is through the stimulatory effects of gp125 (an HIV glycoprotein) and tat, the viral-transactivating protein [14]. Mycoplasmas as well enhance the replication of HIV by increasing oxidative stress since they are known to produce H202. HIV coinfection with mycoplasma therefore results in the

It has been proposed that CD4+ T-cells are depleted by apoptosis and that T-cells are primed to undergo apoptosis upon cross-linking of CD4 by gp120 of the virus [15]. Subsequent activation perhaps by conventional antigens or superantigens induces apoptosis in these Tcells and increases the progression rate. OS have been implicated to play a rather devastating role in the progression of HIV disease. In this case, certain questions are raised, namely, does the OS condition in HIV infection differ in the various stages of the disease? Does the threshold

macromolecules. ROS therefore are the main cause of oxidative stress.

**2.2. ROS in HIV infection: Do they differ in the different stages of HIV?**

), nitric oxide (NO) and hydrogen peroxide


Clinical and immunological classification systems of HIV infection use a four-stage system for both adults and children. Higher numbers indicate advanced degrees of deterioration in clinical and/or immunological status. The recent WHO clinical staging or case definition recognises four (1–4) stages (clinical stage 1, asymptomatic; clinical stage 2, mild symptoms; clinical stage 3, advanced symptoms; clinical stage 4, severe symptoms) [16] as well as the US Centers for Disease Control and Prevention (CDC) which provided a revised classification system for paediatric HIV infection; here, the immunological status staging is used for HIV surveillance. The ranking of the CDC HIV symptoms seems different from the WHO listing. The US CDC clinical staging categories are N, not symptomatic; A, mildly symptomatic; B, moderately symptomatic; and C, severely symptomatic [17]. Similarly, the current CDC HIV surveillance and staging recognises five infection stages in which a confirmed case can be classified as 0, 1, 2, 3 or U [18]. Zero (0) indicates a negative HIV test within six months of the first HIV infection diagnosis. Stages 1–3 are determined by the CD4 test immunologic criteria based primarily on the CD4+ T-lymphocyte count as indicated below: stage 1, ≥1,500 in oneyear-olds/≥500 in adults; stage 2, 750–1,499/200–499; and stage 3, 750/<200 [18]. If none of the above apply (e.g. because of missing information on CD4 test results), the stage is U (un‐ known). These four stages may be referred to, respectively, as the seroconversion and primary HIV infection stage, chronic HIV infection, HIV infection with symptoms and AIDS.

An earlier study by Peterhans showed that viruses could generate ROS from phagocytic cells [19]. Currently, it is known that other viruses (DNA, RNA) could cause cell death through generation of ROS in the infected cell [20,21]. There is confirmed increased free radical production in stage 2 of HIV infection than in stage 4 [22]. Invariably, the antioxidant compo‐ nent is decreased in some cells by half of its amount in stage 2 of the infection [22]. Gaman and her colleagues [23] reported similar patterns of OS increase in stage B/C of chronic lymphocytic leukaemia (CLL) patients. Ibeh et al. using serodiscordant HIV patients showed a high OS condition in serodiscordant-seropositive individuals against their seronegative partners [24]. In a further study, he observed an increased oxidative stress condition in different stages of HIV disease in patients undergoing antiretroviral therapy in Nigeria, where over 50% of the nontreatment group was in stage 2 of the infection [25]. These reports indicate differing OS condition in the various stages of HIV infection and possibly in other viral infections and its capability to serve as a potent surveillance tool (Fig. 1). Further studies should focus on threshold of OS generated and/or needed to determine entrance to the various infection stages. A possible explanation to the observed consecutive more intense overproduction of ROS in the various stages especially in stage 2 is associated partly with changes in the expression of the antiapoptotic/antioxidant compounds Bcl-2 and thioredoxin along the course of the disease by hydrogen peroxide H2O2 [26]. It is known that the free radical H2O2 plays a central role in activating NF-κB (NF-κB activates HIV replication) through the activation of a factor that binds to a DNA-binding protein; NF-κB in turn stimulates HIV gene expression by acting on the promoter region of the viral long terminal repeat (LTR). NF-κB regulates cellular responses as a 'rapid-acting' primary transcription factor. This makes it to be a first responder to harmful cellular stimuli such as H2O2. Known inducers of NF-κB activity include reactive oxygen species (ROS), tumour necrosis factor alpha (TNF-α) and interleukin-1-beta (IL-1β) [27]. If there is no adequate levels of antioxidants, the activity of NF-κB increases in excess amounts and accelerates HIV replication. It is estimated that more than one billion T4 cells are killed and over 50 million HIV replenished on a daily basis in AIDS; this characteristic causes an increase in cytokine synthesis and free radical damage of cells [28].
