Alternative Medicine Review 2000 (Aug); 5 (4): 290–305 ~ FULL TEXT
Lyn Patrick, ND
The Importance of Redox Homeostasis in HIV
HIV infection and the progression to AIDS involves a long period of latent infection characterized by low levels of viral replication that slowly increase to the point of immunosuppression.  This progression is accelerated if the latent (non-reproducing) provirus in the nuclei of the lymphocyte is activated.  Oxidative stress induces both viral activation of HIV and DNA damage, leading to immunosuppression. [3-5] It is now generally accepted that a central pathologic feature of HIV disease involves oxidative stress, leading to programmed cell death (apoptosis) and depletion of CD4 cells. [6, 7] It has been hypothesized by Montagnier and others [8, 9] that the majority of T-helper (CD4+) cell loss (the cell most susceptible to fatal injury by HIV) actually occurs by apoptosis and not by direct HIV infection. This phenomenon has been seen in in vitro culture and in peripheral blood lymphocytes from HIV-infected patients. 
Evidence of increased oxidation reactions,  depletion of the glutathione-based antioxidant defense system,  and increased levels of oxygen radicals have been demonstrated in the blood and tissues of HIV-infected individuals.6 Elevated levels of hydroperoxides,  malondialdehyde,  and deficiencies of the critical antioxidant enzymes manganese superoxide dismutase, glutathione peroxidase, thioredoxin, and catalase have been demonstrated in plasma, lung lining, erythrocytes, and lymphocytes in HIV-infected individuals. [4, 7, 13] Nutrient malabsorption, glutathione and selenium depletion, and reduction of total thiol (cysteine) levels have all been observed to be associated with the pathology of free radical overload that leads to the cellular apoptosis of T lymphocytes. 
Glutathione: Antioxidant and Antiviral
Glutathione, the most abundant cellular thiol, provides the major antioxidant defense mechanism in all mammalian cells by neutralizing toxic peroxides.  It also helps to maintain levels of ascorbate and tocopherol by acting as a reducing agent.  Glutathione is necessary for maintaining immune mediated T-cell activation and phagocytosis, in addition to cellular and antibody mediated cytotoxicity,  and a normal balance between the T-helper cell 1 (IL-2, IL-12, gamma-interferon) and the T-helper cell 2 (IL-6, IL-4, tumor necrosis factor-alpha, IL-10, IL-1) cytokine response profile.  Glutathione conjugation is also the primary mechanism of eliminating electrophilic xenobiotics (some of which are carcinogens) in the liver. 
Glutathione deficiency has been theorized to be the cause of the increased sensitivity HIV-infected individuals have to high doses of acetaminophen and sulphamethoxazole, a medication used in the prevention of Pneumocystis pneumonia. The metabolic fate of these medications, the hepatic glutathione-S-transferase/mercapturic acid pathway, is less efficient in individuals with glutathione deficiency. [21-24] Plasma glutathione levels in HIV-infected individuals, even in the asymptomatic state, have been found to be depressed as early as three weeks post-infection.  Glutathione levels in lung epithelial fluid have been found to be depressed as much as 60 percent when compared to HIV-negative controls.  Intracellular glutathione levels in both infected CD4 and CD8 lymphocyte subsets are also significantly depressed; levels from 62-69 percent of normal have been found in the CD4 and CD8 lymphocytes of HIV and AIDS patients.  These figures become relevant in light of studies that show glutathione reduction of 10-40 percent is capable of completely inhibiting T-cell activation in vitro.  While not all studies have found depressed glutathione levels in plasma or lymphocytes of infected individuals, [27, 28] levels of reduced glutathione do appear to be disturbed in HIV.  Research assessing ratios of reduced-to-oxidized glutathione in HIV-positive patients found significantly increased levels of oxidized glutathione and subsequently lower levels of reduced glutathione when compared to HIV-negative controls.  These disturbances were greater in patients with more advanced disease, the ratios being higher than have been found in human lymphocytes in any other disease state.  Low serum thiol levels (precursors to glutathione) have been shown in HIV-infected, injecting drug users (IDU) to be associated with an increased risk of mortality: IDU with low serum thiol levels are 5.65 times more likely to experience an accelerated time-to-death. 
Mechanisms of Apoptosis
The response to antigenic material and the presence of cytokines, hydroxyl radicals or other viruses can trigger the activation of nuclear factor kappaB (NF-kB).  A gene-transcription regulating factor present in lymphocytes, macrophages, and monocytes, NF-kB activates genes in the nuclear material of these cells, resulting in production of cytokines and major histocompatibility (MHC) agents.  NF-kB also binds to HIV proviral gene material in the nucleus of HIV-infected cells and activates HIV replication.  Viral replication, in turn, increases cellular levels of cytokines, like tumor necrosis factor-alpha (TNFa), that promote the production of free radicals and the activation of NFkB, initiating a vicious cycle of viral replication and free radical production.  The presence or absence of reducing thiols (sulfhydryl groups that form the basis for antioxidant enzymes, glutathione, etc.) appears to directly affect this cycle. In cell cultures where glutathione levels or thiol levels have been depleted, the TNFa-stimulated activation of HIV is enhanced.  In cell cultures where N-acetylcysteine (NAC) (an efficient thiol source and glutathione precursor) is added, the activation of NF-kB is blocked (Figure 1).  Glutathione has also been shown to directly inhibit the activity of reverse transcriptase (a major enzyme necessary for HIV replication) by 80-90 percent in cell cultures. 
The attempt to find antioxidants or glutathione "pro-drugs" that recreate normal glutathione states and block production of TNF-a and NF-kB has led to the creation of antioxidant strategies for preventing CD4 cell decline.  Data showing that cysteine, N-acetylcysteine (NAC), reduced glutathione, and ascorbic acid all suppress NF-kB activity and HIV activation in cell lines  has resulted in the production of drugs like L-2-oxothiazolidine-4-carboxylic acid (OTC) or "Procysteine." While OTC has been shown to increase lymphocyte glutathione levels in healthy subjects, it is not itself an antioxidant and has not been shown to be as effective as N-acetylcysteine. 
When OTC was compared to NAC, NAC was more effective at decreasing cytokine-induced HIV replication in different cell lines and replenishing intracellular glutathione levels.  Glutathione, administered orally, has been demonstrated to be absorbed by rat intestine, kidney and lung epithelium,  but intact glutathione cannot be absorbed by T-cells, unless it is given as a glutathione monoester.  Glutathione monoesters, however, have been associated with significant toxicity.  Studies with oral dosing of glutathione in healthy human volunteers have shown no increase in cysteine, glutathione, or glutamate levels after a 3-gram dose,  leading the authors to conclude: "It is not possible to increase glutathione to a clinically beneficial extent by the oral administration of a single dose of 3 grams of glutathione." Using the rate limiting amino acid L-cysteine to increase glutathione production may be inadvisable since it appears to autooxidize rapidly in the bloodstream and increase free radical load. 
N-Acetylcysteine as a Glutathione Regenerator
N-acetylcysteine has been used successfully to treat hepatic and renal failure caused by glutathione depletion secondary to acetaminophen overdose.  It has an extensive history as a mucolytic and has been used in pulmonary diseases including emphysema, tuberculosis, chronic asthma, fibrosing alveolitis, and primary amyloidosis of the lung.  The mechanism of action in these respiratory conditions includes the restoration of reduced and total glutathione levels in lung cell fluid.  N-acetylcysteine has been demonstrated to have heavy metal chelating capacities for toxic metals, as well as for copper, zinc, and boron.  Several studies support evidence that NAC increases glutathione levels in vivo and in vitro; [45-48] there is also evidence NAC may boost cellular immunity directly. 
N-Acetylcysteine in HIV/AIDS
CD4+ and CD8+ T-cells from HIV-infected individuals have an impaired ability to proliferate; they are also unresponsive to recall antigens and unable to secrete normal amounts of interleukin-2, exhibiting properties similar to a state of anergy.  This altered response appears to be the reason for the rebound of viral loads to pre-medication levels following discontinuation of triple anti-viral therapies even after one year of continuous and successful treatment.  NAC appears to be able to help restore CD4 cell function: in a study of 11 asymptomatic HIV-infected individuals with CD4+ counts of over 300/mL, N-acetylcysteine (at 5, 10 and 20 mM) restored normal CD4+ proliferative responses in 8 of 11 patient blood samples. 
N-acetylcysteine appears to be beneficial in HIV as a result of its ability to restore normal glutathione levels in lymphocytes and thereby reduce free radical production.  NAC also, however, acts directly as an antioxidant.  Preincubation with 15 mM concentration of NAC was able to partially protect lymphocytes from asymptomatic HIV-infected patients after exposure to menadione, an oxidizing agent. This study also found a significant relationship between CD4+ counts, plasma peroxidation, and the ability of NAC to preserve the structural characteristics of the lymphocytes. The patients with lower CD4+ counts also had higher levels of lipid peroxidation products and were less protected by the same amount of NAC. As a direct free radical scavenger, NAC reduces hypochlorous acid produced by neutrophils in order to kill target cells.  Because NAC has a direct antioxidant action in lymphocytes, concern has been expressed that NAC supplementation would inhibit the natural mechanism of cytolysis by neutrophils. Cell studies  have shown NAC actually enhances intracellular killing of bacteria by protecting neutrophils and macrophages from free radical damage generated during phagocytosis. Cell studies with HIV-infected monocytes and neutrophils have shown similar results: NAC (at l and 5 mM concentrations), enhanced their cytotoxicity.  Multiple cell studies have shown NAC acts as an antiviral in HIV-infected cell lines; both by direct inhibition of TNF-a52, [55-57] and direct inhibition of viral transcription. 
N-Acetylcysteine in Clinical Trails
The basic question underlying NAC clinical trials is whether the repletion of available thiol groups will normalize lymphocyte glutathione levels, minimize cytokine-induced viral proliferation, and stop the CD4+ cell depletion associated with glutathione deficiency.15 Clinical trials to date contain only partial answers to these crucial questions.
DeQuay et al  found significant reductions in cysteine and glutathione in all of nine HIV-infected subjects. Low baseline levels of cysteine in blood and in CD4, CD8 T lymphocytes, B-cells, and monocytes were returned to normal after a single oral dose of N-acetylcysteine (30 mg/kg body weight) (Figure 2). Glutathione levels were elevated four hours later in five of the nine subjects. The remaining four, who had the lowest baseline glutathione levels, each had less than 100/mm3 CD4+ cells and did not exhibit increased glutathione levels with NAC treatment. The authors commented that glutathione production in CD4 and CD8 cells was slow, and felt a longer period of administration would be necessary to adequately assess glutathione production. The study included an HIV-positive patient whose lymphocyte glutathione levels doubled after seven days of 600 mg NAC three times daily.
However, NAC clinical studies have not been consistent. In one trial examining 45 HIV-positive men and women, taking 800 mg daily for four months, NAC was successful in normalizing plasma cysteine levels and significantly reducing TNF-a, but there were no changes in glutathione levels.  Although none of the individuals was on an antiretroviral regimen, the median CD4+ count remained the same after four months while the control group had a significant CD4+ cell decline (p<0.005). The dosage was considerably lower in this trial (800 mg vs 30 mg/kg), which may account for the inability of NAC to affect glutathione repletion.
Leonore Herzenberg and her group at Stanford  studied glutathione repletion in 27 HIV-positive men who were taking 3200-8000 mg NAC daily (median 4400 mg). This high dose was chosen because it was below a previously determined maximum-tolerated dose, and it was based on a prior erroneous study that indicated NAC had low bioavailability. At baseline, the average glutathione levels in uninfected controls were 28-percent higher than the HIV-positive group with CD4+ counts over 200. The NAC treatment group had a significant elevation in whole blood glutathione after eight weeks while the control group remained unchanged. The average increase in the NAC group was 113 percent, an increase that brought the treatment group close to the baseline of uninfected controls (Figure 3). The trial was continued for two years, even though the treatment group was only given NAC for 8-32 weeks (median 24). After two years, the NAC group (25 subjects) had a significantly greater chance of surviving than the group who never took NAC (p=0.002). The most beneficial information derived from this study, however, was the data relating glutathione levels and survival in those who had not taken NAC. In those with CD4+ counts less than 200, 85 percent of the 28 subjects in the high lymphocyte glutathione group were still alive after 2.5 years. Only 18 percent of the 69 subjects in the low glutathione group survived. Although the number of subjects on NAC treatment was too small to attach significance to treatment outcomes, there was a very significant relationship of glutathione levels to survival.
Oliver  gave 15 HIV-positive individuals 600-1200 mg NAC daily for over six months. Peripheral blood lymphocyte apoptosis profiles were done at the onset and after six months of treatment. All of the HIV-infected subjects had evidence of significant apoptosis at baseline. The NAC subjects, however, had significantly less evidence of cell death compared to baseline, HIV-infected controls, and HIV-negative controls after six months.
A study combining the effects of NAC (600 mg three times daily) and selenium in the form of sodium selenite (500 mcg daily) was designed to answer questions about the effects of these antioxidants on glutathione production, lymphocyte subsets, and viral load.  Twenty-four HIV-positive, anti-retroviral-naive men and women (CD4+ counts 200-500/mm3) were randomized into two groups, one treated for 24 weeks and the other treated for the last 12 weeks of the study. A control group consisted of 25 healthy HIV-negative men. Baseline serum selenium and plasma glutathione (reduced and oxidized) levels were significantly reduced in the HIV-positive group. After six weeks of treatment, serum selenium concentrations increased by 53 percent and, although they dropped a little, remained at 45 percent above baseline for the duration of the treatment. Glutathione levels did not change in either group, but they were only measured at weeks 6 and 12. CD4+ percent (measured as a percentage of total lymphocytes) increased significantly in the first group at week 6 and week 24. Suppressor cell (CD8+) levels fell significantly within six weeks, (closer to control levels) and remained there for 24 weeks. Although the reduction of CD8+ cells is difficult to interpret, the increase in CD4+ percentage was significant; a falling CD4+ percent has been shown to be an indicator of faster progression to AIDS.  It is important to note that viral load was unaffected by this treatment.