Vitamin C, E, & NAC and FIV

Oxidative stress is a term for damage to animal or plant cells caused by reactive oxygen species (ROS). ROSs form as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling. However, during times of environmental stress ROS levels can increase dramatically, which can result in significant damage to cell structures. This creates a situation known as oxidative stress, an imbalance between pro-oxidants and anti-oxidants, with the former prevailing. Oxidative stress is suspected to play a key role in many neurodegenerative and cardiovascular diseases[1]. It has also been found to play a key role in the advancement of HIV and FIV infection. In the last decade several common antioxidants have been shown in scientific studies to significantly impact basic measures of disease status and progression in these diseases, including viral load (number of virus per ml of plasma), CD4+ cell counts (the cells whose loss is most directly related to immune deficiency), apoptosis (cells destroyed by misdirected action of the immune system itself), and lymphocyte (B cell, T cell, and natural killer cell) proliferation. This discussion focuses on three antioxidants that have received attention from researchers: Vitamin C, Vitamin E, and NAC (n-acetylcysteine).

In 1998, a ground-breaking three-month study of 49 HIV+ individuals published in the journal AIDS concluded that daily Vitamin C (1000mg) and Vitamin E (800mg) produced "a significant trend toward viral load reduction"[2]. Vitamin C is the chief water soluble antioxidant ; Vitamin E is the most potent fat-soluble antioxidant. The two together are known to interact cooperatively in that Vitamin C is important to regenerating Vitamin E during the antioxidant defense process. At the end of three months, the treated group showed a decrease in viral load of .45+/-.39 log. The untreated control group showed an increase of .50 +/- .40 log. (In viral load parlance "log" is short-hand for a factor of 10. A decrease in viral load from 50,000 to 5,000 would be a decrease of 1 log. +/- is a statistical means of representing deviations from the average figure in both directions.)

In the same year, an in vitro (i.e., outside the body in cultured cell lines) Japanese study demonstrated that NAC and Vitamin C given concurrently could produce significant improvement in important measures of FIV disease status and progression[3]. Acetylcysteine is the N-acetyl derivative of the amino acid L-cysteine and is a precursor in the formation of the antioxidant glutatione in the body, which protects cells from oxidative stress. The study found significant inhibition of viral replication and immune-cell apoptosis, as well as of tumor necrosis factor-alpha (TNF-α), a major proinflammatory cytokine as having a harmful role in both areas. The study concluded, "On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH levels [intracellular glutathione] might be effective for inhibition of viral replication and cell death associated with the development of immunedeficiency." There has never been a follow-up study in live animals.

Two subsequent studies of NAC and Vitamin C confirmed their potential value as supplements for those with HIV. A 2000 Norwegian study of high-dose NAC and Vitamin C for six days recorded a significant rise in CD4+ cells, a .8 log reduction in viral load, and enhanced lymphocyte proliferation in those with advanced disease[4]. A 2004 study of NAC alone for 180 days found significant decrease in viral load, TNF-α production, and cellular apoptosis, and an increase in CD4+ cells and lymphocyte viability[5].

In vitro results do not guarantee in vivo results, and results with HIV do not automatically apply to FIV. For instance, it has been theorized that Vitamin C and Vitamin E block activation of a host transcription factor called Nuclear Factor kappa-B (NF-κB), which HIV co-opts to induce viral replication [2]. ("Transcription factors" are proteins the body manufactures in order to switch genes on and off.) Not all strains of FIV have a binding site for NF-κB, so it is thought that this particular transcription factor might be less important than some others for activation of FIV implanted in host genes [6]. And since cats manufacture their own Vitamin C internally it may have a different level of importance in feline immune response. But the in vitro study of NAC and Vitamin C offers hope that the results of HIV studies are applicable to cats with FIV.

[Further Information on NAC]

Several troublesome issues surround research into NAC as HIV and FIV therapy.

Two studies of NAC from 1994 and 1997 by the same research team conclude that while NAC has an antioxidant effect in CD4+ lymphocytes, it has a prooxidant effect in macrophages and leads to an increased rate of viral replication in these cells [7]

Briefly, FIV and HIV are specifically "tropic" (attracted to) for two immune cells ( although they infect numerous other types): CD4+ lymphocytes (T helper cells) and macrophages. Macrophages are cells of the nonspecific immune system that reside in tissue and are opportunistic scavengers of potentially harmful pathogens. But they also have a role in the specific immune system by engulfing and breaking up pathogens, then presenting fragments of antigen to CD4+ cells to "teach" them to recognize them so that the appropriate signals can be put out to other cells in the immune system, telling them exactly how to configure to attack the pathogen.

Macrophages are the primarily-infected cells early in infection. As infection progresses, the infection of CD4+ cells plays a larger and larger role. However, infection of macrophages always remains a feature of the disease, and macrophages are responsible for spreading infection to the central nervous system and are one of the two cells of the CNS impacted by the virus. ("Microglial" cells of the CNS are a specialized type of macrophage.) So even though HIV and FIV are essentially diseases of CD4+ loss, macrophages still figure prominently in the overall pathology of HIV/FIV infection.

While the team that performed the two studies mentioned recommends that NAC not be used as HIV therapy because of their finding with regard to it, virtually no one else coming after them appears to make this recommendation and, in fact, virtually no one takes note of these studies. Why they have gone unnoted is unclear. The studies actually contradict several earlier studies (1992, 1993) by another team, which found that NAC posed no problem for macrophages [8].

Marcel Blanc [private communication] has suggested that it is possible to reconcile the contradictory results of the two sets of studies. His analysis follows.

"I have found a clue that may help resolve the contradiction between Ho et al and Nottet et al on NAC. Nottet et al (1994 and 1997) monitored the process of viral transcription as assessed by the p24 production (that is the production of a protein capsid). In other words, they monitored the amount of infectious viral particles released from macrophages. And they found it was increased by NAC (through stimulation of NF-kB binding). On the other hand, Ho et al. (1992 and 1993) monitored the process of viral retrotranscription (leading to proviral DNA) after the viral entry into macrophages. And they found that the process of retrotranscription is inhibited by NAC by indirect inhibition, that is by inhibition of the viral entry in cells, through changes induced in the membrane resulting in limited virus adsorption on the cell membrane : "NAC and GSH suppression of HIV- 1 in cells which were pretreated suggests that intracellular GSH was increased enough possibly limiting adsorption of the virus through changes on the cell membrane." (Ho et al., 1993, p. 497. Ho et al. allows also that decrease of viral reproduction in some circumstances (that is infection, then NAC treatment 2 week after infection) may also result from inhibition of viral transcription through inhibition of NF-KB binding. But it is only an indirect speculation, since they really assess the decrease of viral replication by the amount of retrotranscription. It is more likely, on my view, that the decrease of viral replication induced by NAC in the aforementioned circumstances is also due to the decrease of viral penetration in macrophages.

“To sum up, even if NAC increases the release of infectious viral particles by macrophages (as shown by Nottet el al.), NAC-induced membrane changes forbid the increase of pro-virally infected macrophages (as implied by Ho et al. results). That is, the surnumerary released viral particles induced by enhanced transcription are simply lost : they never gain entry in new macrophages."

This analysis does not entirely settle the issue, but may well have merit. Further complicating the issue is the fact that, as already noted, NF-kB, the host transcriptional factor which Nottet et al found to be stimulated by NAC, is felt to be less important to FIV replication than to HIV replication. All HIV-1 provirus (viral genes embedded in human DNA) have a binding site for NF-kB. Some FIV provirus do not, although all have binding sites for several other host transcriptional factors. So NAC may have a different effect in infected feline macrophages than in infected human macrophages.

There is also information (http://www.jimmunol.org/cgi/content/full/171/10/5107) describing NAC as a promoter of the proinflammatory cytokine IL-4 and as an enhancer of AP-1 binding activity. (AP-1 is a host transcriptional factor like NF-kB for which there appear to be binding sites on all FIV strains). In the in vitro study, these effects did not occur in naive T cells alone, but by some unexplained mechanism, in the presence of antigen-presenting macrophages it did [9]. A previous study working with T cells only did not find this phenomenon.

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References:

[1] Wikipedia, "oxidative stress," "reactive oxygen species."

[2] Allard, J, Aghdassi E, Chau, J; Tam C, Kovacs CM, Salit IE, Walmsley S. Effects of vitamin E and C supplementation on oxidative stress and viral load in HIV-infected subjects. AIDS: Volume 12(13)10 September 1998p 1653-1659.

http://www.aidsonline.com/pt/re/aids/fulltext.00002030-199813000-00013.htm; jsessionid=FsvS1rnDksyv9wJTz30GQHJB0QTdmlj07ZkXQ2lLwkhRJNK8KLTm! -910938601!-949856144!8091!-1

[3]Mortola E, Okuda M, Ohno K, Watari Y, Tsujimoto H, Hasgawa A. Inhibition of apoptosis and Viral Replication in Feline Immunodeficiency Virus-Infected Cells by N-Acetylcysteine and Ascorbic Acid. J Vet Med Sci 60 (11): 1187-1193,1998.

http://www.jstage.jst.go.jp/article/jvms/60/11/1187/_pdf

[4] Müller F, Svardal AM, Nordoy I, Berge RK, Aukrust P, Frøland SS. Virological and immunological effects of antioxidant treatment in patients with HIV infection. Eur J Clin Invest 2000 Oct;30(10):905-14. http://www.ncbi.nlm.nih.gov/pubmed/11029606?

[5] TreitingerA, Spada C, Masokawa IY, Verdi JCV, Silveira M, Chaves M, Reis M, FerreiraV S, AbdallaVI D. Effect of N-acetyl-L-cysteine on lymphocyte apoptosis, lymphocyte viability, TNF-a and IL-8 in HIV-infected patients undergoing anti-retroviral treatment. Braz J Infect Dis 8 (5) Oct. 2004, no pp. provided.

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702004000500005&lng=en&nrm=iso&tlng=en

[6] Phillips TR, Talbott RL,Muir LS, Lovelace K, Elder JH. Comparison of Two Host Cell Range Variants of Feline Immunodeficiency Virus, Journal of Virology Oct. 1990, p. 4605-4613, Vol. 64, No. 10. http://jvi.asm.org/cgi/reprint/64/10/4605.pdf

[7] Nottet H, Asbeck S, Graaf L, Vos NM, Visser MR, Verhoef J. Role for Oxygen radicals in self-sustained HIV-1 replication in monocyte-derived macrophages: enhanced HIV-1 replication by N-acetyl-L-cysteine. J Leukoc Biol 56: 702-707; 1994.

http://www.jleukbio.org/cgi/reprint/56/6/702.pdf;

[8] Ho WZ, Douglas SD. Glutathione and N-acetylcysteine suppression of human immunodeficiency virus replication in human monocyte/macrophages in vitro. AIDS Res Hum Retroviruses. 1992 Jul;8(7):1249-53. http://www.ncbi.nlm.nih.gov/pubmed/1520537?

Lioy J, Ho WZ, Cutilli JR, Polin RA, Douglas SD. Thiol suppression of human immunodeficiency virus type 1 replication in primary cord blood monocyte-derived macrophages in vitro. J Clin Invest 1993 Feb;91(2):495-8.

http://www.ncbi.nlm.nih.gov/pubmed/7679409?

[9] Monick MM, Samavati L, Butler NS, Mohning M, Powers LS, Yarovinsky T, Spitz DR, and Hunninghake GW. Intracellular Thiols Contribute to Th2 Function via a Positive Role in IL-4 Production. The Journal of Immunology, 2003, 171: 5107-5115.

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