Free Radical Biology and Medicine 69 (2014) 136–144

Contents lists available at ScienceDirect

Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Immunoneuropathogenesis of HIV-1 clades B and C: Role of redox

expression and thiol modification
Thangavel Samikkannu, Kurapati V.K. Rao, Sudhessh Pilakka Kanthikeel,
Venkata Subba Rao Atluri, Marisela Agudelo, Upal Roy, Madhavan P.N. Nair n
Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL
33199, USA

art ic l e i nf o a b s t r a c t

Article history: Previous studies have shown that, during infection, HIV-1 clade B and clade C differentially contribute to
Received 18 July 2013 the neuropathogenesis and development of HIV-associated neurocognitive disorders (HANDs). The low-
Received in revised form molecular-weight tripeptide glutathione (GSH) alters the redox balance and leads to the generation of
19 December 2013
reactive oxygen species, which play a significant role in the neuropathogenesis of HANDs. We
Accepted 22 December 2013
Available online 27 January 2014
hypothesized that the HIV-1 clade B and clade C viruses and their respective Tat proteins exert
differential effects on monocyte-derived immature dendritic cells (IDCs) and neuroblastoma cells
Keywords: (SK-N-MC) by redox activation, which leads to immunoneuropathogenesis. The GSH/GSSG ratio and
HIV-1 clade B mRNA expression levels and protein modification of glutathione synthetase (GSS), glutathione perox-
HIV-1 clade C
idase 1 (GPx1), superoxide dismutase 1 (SOD1), and catalase (CAT) were analyzed in IDCs infected with
Dendritic cells
HIV-1 clade B or clade C as well as in cells treated with the respective Tat proteins. The results indicated
Neuron
Oxidative stress that HIV-1 clade B virus and its Tat protein significantly increased the production of reactive oxygen
Glutathione species and reduced the GSH/GSSG ratio and subsequent downregulation of gene expression and protein
Free radicals modification of GSS, GPx1, SOD1, and CAT compared to infection with the clade C virus or treatment with
the clade C Tat protein. Thus, our studies demonstrate that HIV-1 clades B and C exert differential effects
of redox expression and thiol modification. HIV-1 clade B potentially induces oxidative stress, leading to
more immunoneuropathogenesis than infection with HIV-1 clade C.
& 2014 Elsevier Inc. All rights reserved.

Human immunodeficiency virus type-1 (HIV-1) displays extra- leading to immune dysfunction [9–11]. These immune dysfunctions
ordinary genetic variation in its global distribution. It is classified and pathogenic mechanisms, tentatively imbued with the ability to
into four groups (M, N, O, and P) and nine different subtypes or enter the CNS, can then induce neuropathogenesis. Studies have
clades (A–K) based on phylogenetic analysis [1]. A large majority shown that HIV-1 directly and indirectly affects the CNS, causing
(4 86%) of circulating HIV-1 are B and C clade, and clade C is neurological impairments that are manifested by cognitive, beha-
responsible for more than 56% of HIV-1 infections. The subgroups vioral, and motor abnormalities due to the massive death of neurons
M, N, O, and P differ in amino acids by 30–40% and subtypes or in all regions of the brain [12]. The HIV Tat protein is known to cause
clades (A–K) by 5–20% [2]. The predominant subtype of HIV-1 cellular oxidative stress and progressively affects the CNS. It is also
clade B infections occur in North America, Western Europe, essential for viral replication and disease progression-induced neu-
and Australia. Conversely, HIV-1 clade C is found in Africa, Latin ronal impairments [13,14] and stimulates nitric oxide synthase [15]
America, and Asia [3–5]. as well as other toxic factors. Previous studies have shown that Tat is
HIV-1 directly affects the immune function causing a deficiency released extracellularly by HIV-1-infected lymphocytes and micro-
in antigen presentation. This is manifested by a dysregulation of glial cells [16]. Interestingly, Tat mRNA expression is increased during
inflammatory cytokines, chemokines, and other factors such as HIV brain dementia [17]. In astrocytes, Tat induces oxidative stress,
oxidative stress-induced reactive oxygen species and reactive nitro- affecting mitochondrial function and leading to cell death [18]. Tat
gen species [6–8]. In vitro and in vivo evidence shows that HIV may also interact with cell surface receptors, leading to the activation
infection affects peripheral cells such as monocytes (MCs) and of intracellular signaling pathways [19]. Previous studies have
dendritic cells (DCs) as well as the central nervous system (CNS), suggested that the biological properties of HIV clades influence
disease progression, transmission efficiency, and dissemination
[20]. The amino acid divergence of Tat among HIV-1 clades may
n
Corresponding author. Fax: þ1 305 348 1109. influence its binding and transactivation functions [21] and is also
E-mail address: nairm@fiu.edu (M.P.N. Nair). associated with the varying degrees of associated neurological

0891-5849/$ - see front matter & 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.025
 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144 137

problems [12]. Recently, Mishra et al. [22] reported that alterations in HIV clade B and C virus infection of IDC
the dicysteine motif at position C30C31 in the clade C Tat protein
probably alter its functional properties. IDCs (1  106 cells/ml) were treated with Polybrene (4 mg/ml)
Recent studies have suggested that HIV dementia is associated for 5 h and then infected with HIV-1 clade B or clade C at 20 ng/
with increased oxidative stress and altered lipid peroxidation in 106 or TCID50 (the dose that produces approximately the same
the brain [23]. The oxidative stress-induced free radicals H2O2 and levels of p24 antigen in culture) at 37 1C. After 18 h, the cells were
O2 cause cellular damage in many diseases, including HIV/AIDS washed with PBS, and the HIV infections were maintained for 15
[24,25]. Glutathione (GSH) is one of the main players in intracel- days. Every third day, half of the medium was replaced with fresh
lular antioxidant defense mechanisms, and low levels of GSH have medium, and the supernatant obtained from the removed medium
been associated with impaired immune responses and neuronal was used for the p24 antigen estimation using an enzyme-linked
dysfunction [26]. The reduced level of GSH in HIV-positive immunosorbent assay (ELISA) kit. Uninfected control cells were
individuals results in an increased production of H2O2 and O2 included in all of the experiments.
[27], leading to AIDS dementia complex [28,29]. It has been shown
that sequence variations in HIV-1 viral proteins lead to differential HIV-1 clade B and C Tat protein treatment of IDCs and SK-N-MC cells
expression of dementia and neurocognitive disorders [20,12].
However, the underlying mechanisms of how immune dysfunction IDCs and SK-N-MC cells (1  106 cells/ml) were treated with
in immature dendritic cells (IDCs) leads to neuronal cell loss and either HIV clade B Tat or HIV clade C Tat protein using an
ultimately HIV-associated neurocognitive disorders (HANDs) are optimized concentration of 50 ng/ml for 24 h treatment. The
not well understood. Therefore, we investigated the mechanism of untreated cells served as the control.
differential induction of oxidative stress by HIV-1 clades B and C
by assessing alterations in redox expression and thiol modification ELISA p24 measurements
in IDCs and SK-N-MC cells.
We demonstrated that HIV-1 clade B virus and the clade B Tat A commercially available ELISA kit (Zeptometrix, Buffalo, NY, USA)
protein induced oxidative stress and potentiated redox-induced was used to quantitate the amount of p24 in the culture supernatants.
gene expression and protein modification of glutathione synthe-
tase (GSS), glutathione peroxidase 1 (GPx1), superoxide dismutase Determination of reactive oxygen species (ROS) production
1 (SOD1), and catalase (CAT) in IDCs and neuronal cells; the effects and intracellular thiol content
were significantly different from those associated with clade C
infection or clade C Tat protein. The HIV-1-induced ROS production and intracellular thiol levels
were analyzed by flow cytometry in a FACSCalibur (BD Bioscience,
San Jose, CA, USA). Briefly, IDCs (5  105) were infected with 20 ng
HIV-1 clade B or clade C for 6 days. At the end of that time, 10 mM
Materials and methods either 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) or
Amplex red and 50 mM monobromobimane (mBrB; Invitrogen,
HIV-1 clade B and C viruses and Tat recombinant proteins Carlsbad, CA, USA) was added directly to the medium and the cells
were incubated at 37 1C for 30 min. The cells were then washed with
HIV-1 clade B (Bal strain) and clade C (CN54 strain) viruses and the PBS, and ROS production and intracellular thiol levels were analyzed.
HIV-1 clade B Tat protein were obtained from the NIH AIDS Research
and Reference Reagent Program (Cat. Nos. 510, 4164, and 2222, Effect of HIV-1 clades B and C on DC-specific intercellular adhesion
respectively). The HIV-1 clade C Tat was obtained from Diatheva (Fano molecule-3-grabbing nonintegrin (DC-SIGN) expression in IDCs
(PU), Italy). The purity and functional properties of the recombinant
Tat proteins were confirmed by a transactivation assay. The HIV-1-induced expression of DC-SIGN was analyzed by
flow cytometry in a FACSCalibur (BD Bioscience). Briefly, IDCs
(5  105) were infected with HIV-1 clade B or C (20 ng/106 cells)
Isolation of monocyte-derived IDCs for 6 days. Then, the cells were washed, and surface staining was
performed using FITC-conjugated anti-DC-SIGN antibody (Invitro-
IDCs were prepared from peripheral blood mononuclear cells gen). The cells were then washed with PBS, and DC-SIGN expres-
(PBMCs) as described by Nair et al. [30]. Briefly, PBMCs were sion levels were analyzed.
separated on a density gradient and adhered to plastic culture
plates containing medium plus serum. Nonadherent cells were RNA extraction and real-time quantitative PCR (qPCR)
removed after incubation for 2 h at 37 1C, and adherent MCs were
harvested and washed with phosphate-buffered saline (PBS)/1% Total RNA from 1  106 IDCs and cultured SK-N-MC cells was
fetal calf serum. MCs were grown for 5–7 days in RPMI culture extracted using a Qiagen kit (Invitrogen) following the manufacturer’s
medium containing 500 U/ml GM-CSF and 500 U/ml IL-4 to instructions. The total RNA (5 mg) was used for the synthesis of the
generate IDCs. Subsequently, the IDCs were stained with the first strand of cDNA. The amplification of cDNA was performed using
surface markers CD80, CD86, CD40, and CD11c and analyzed by primers specific for the LTR-R/U5 domain (Sigma), GSS (Assay ID
flow cytometry. Hs00609286), GSR (Hs00167317), SOD1 (Hs00166575), catalase
(Hs00156308), and β-actin (Hs99999903) (Applied Biosystems, Foster
City, CA, USA). β-Actin served as an internal control. The relative
Neuronal cell culture abundance of each mRNA species was assessed using the Brilliant
Q-PCR master mix from Stratagene using the Mx3000P instrument,
The neuroblastoma cell line SK-N-MC was purchased from the which detects and plots the increase in fluorescence versus PCR cycle
ATCC (Manassas, VA, USA), and the cells were cultured in Eagle’s number to produce a continuous measure of PCR amplification. The
minimum essential medium supplemented with fetal bovine relative mRNA species expression was quantitated, and the mean fold
serum to a final concentration of 10% and 1% antibiotic/antimycotic change in expression of the target gene was calculated using the
ΔΔ
solution (Sigma–Aldrich, St. Louis, MO, USA). comparative CT method (transcript accumulation index, TAI¼2  CT).
138 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144

All of the data were normalized for the quantity of RNA input by Tat or clade C Tat for 24 h, and the cells were washed with PBS and
performing measurements on an endogenous reference gene, β-actin. then resuspended in 1 ml of a homogenization buffer containing
In addition, the results obtained with RNA from treated samples were Hepes buffer (pH 7.4) with 137 mM NaCl, 4.6 mM KCl, 1.1 mM
normalized to the results obtained with RNA from the control, KH2PO4, and 0.6 mM MgSO4 and homogenized with 10 strokes
untreated sample [31]. using a chilled Dounce homogenizer. The homogenate was cen-
trifuged at 14,000 rpm for 10 min at 4 1C, and the supernatant was
Western blot collected. The GSH/GSSG ratio was determined according to the
manufacturer’s instructions and expressed as GSH equivalents
To assess protein expression, IDCs and SK-N-MC cells (1  106) using a standard curve.
were treated with HIV-1 clade B or clade C Tat proteins (50 ng/ml)
for 24 h at 37 1C. After incubation, the cells were washed with PBS Statistical analysis
and lysed using lysis buffer (Pierce, Rockford, IL, USA) with a 1 
complete cocktail of protease inhibitors. The extracts were cen- The results presented in this study are representative of three
trifuged at 14,000 g for 5 min at 4 1C, and the total amount of or more independent experiments performed in triplicate. Statis-
cellular protein was determined by a Bio-Rad protein determina- tical significance was determined by ANOVA, Student’s t test, or
tion assay (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins unpaired observations using GraphPad Prism software(GraphPad,
were resolved by 4–15% polyacrylamide gel electrophoresis and La Jolla, CA, USA). The values are presented as the mean 7SD, and
transferred to a nitrocellulose membrane. After being blocked, the a p value of r0.05 was considered significant.
membrane was probed with primary rabbit polyclonal GSS and
GPx1 and mouse monoclonal SOD1 antibody (Chemicon Interna- Results
tional, Temecula, CA, USA), followed by secondary goat anti-rabbit
IgG antibody and goat anti-mouse IgG antibody (Santa Cruz HIV-1 clade B and C infection induced oxidative stress and redox-
Biotechnology, Paso Robles, CA, USA). The immunoreactive bands dependent gene expression in IDCs
were visualized using a chemiluminescence Western blotting
system according to the manufacturer’s instructions (Amersham, It is known that HIV infection and Tat protein induce ROS
Piscataway, NJ, USA). production and alter redox regulation, leading to apoptosis and
peroxidative damage [17,24,25,32]. However, the effects of specific
Determination of GSH/GSSG HIV clades on redox-dependent gene expression have not yet been
studied. Therefore, we examined whether HIV clade B and C
The cellular GSH/GSSG ratio was determined by a colorimetric viruses induce similar or distinct mechanisms of redox-
assay (Cat. No. GT 35, Oxford Biomedical Research, Oxford, MI, dependent expression in IDCs. The results demonstrated that after
USA). Briefly, IDCs (1  106) were treated with 50 ng HIV-1 clade B 6 days of either clade B or clade C virus infection, oxidative stress

Fig. 1. The effects of HIV-1 clade B and C viruses on IDCs. IDCs (1  106 cells/ml) were infected with HIV-1 clade B (Bal strain) or HIV-1 clade C (CN54 strain) at (A) 20 ng/106
cells or (B) the TCID50 for 18 h. The supernatants were collected at (A) various time points and (B) 6 h and used to estimate the expression of p24 antigen by ELISA. Total RNA
was extracted from the infected IDCs and then reverse transcribed and subjected to quantitative real-time PCR using gene-specific primers to measure the expression of
(C) LTR, (D) GSS, and the housekeeping gene β-actin. The data are expressed as the mean 7SD of the transcript accumulation index values of three independent experiments.
 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144 139

was significantly increased and redox-dependent expression was (Fig. 1C) and GSS (Fig. 1D) gene expression. These results demon-
subsequently inhibited in IDCs. Fig. 1 shows the effects of clade B strate that compared with HIV clade C infection, HIV clade B
and C viruses on the level of p24 antigen using 20 ng virus (Fig. 1A) infection results in a significant increase in p24 antigen secretion
or TCID50 (Fig. 1B), as well as the effects of the viruses on LTR-R/U5 and LTR-R/U5 expression (po 0.01) and a subsequent inhibition of

Fig. 2. The effects of HIV clade B and C viruses on ROS production and intracellular thiol levels. IDCs (1  106/ml) were seeded in six-well plates and infected with HIV-1
clade B (Bal) or C (CN 54) (20 ng/ml) for 18 h. Next, the cells were washed and maintained for 6 days. The IDC positive cells were stained for (A) CD80 and ROS production by
DCFH-DA, (B) H2O2 by Amplex red, and (C) intracellular thiol content by mBrB and analyzed by flow cytometry. The data are expressed as the mean7 SD of three
independent experiments.

Fig. 3. The effects of HIV clade B and C viruses on DC-SIGN expression in IDCs. IDCs (5  105 cells/ml) were cultured and infected with HIV-1 clade B (Bal) or C (CN 54) (20 ng/ml)
for 18 h. The cells were then washed to remove unbound virus and cultured for 6 days. At the end of the 6th day, the cells were stained for DC-SIGN, and expression was analyzed
by flow cytometry. The data are expressed as the mean7SD of three independent experiments.
140 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144

GSS gene expression (po 0.004). Fig. 2 shows that infection with the capacity of HIV-1 clade B and clade C Tat to influence this ratio
HIV clade B virus induced oxidative stress in IDCs, leading to in IDCs. Twenty-four hours after treatment, a significantly lower
increased ROS production by DCFH-DA (Fig. 2A; p o0.02) and GSH/GSSG ratio was detected in clade B Tat-treated IDCs (po0.01)
Amplex red (Fig. 2B; p o0.02) and subsequently depleting intra- compared to control. The ratio in clade C Tat-treated cells was also
cellular thiol (Fig. 2C; po0.004) compared to clade C infection. lower but not significantly different compared to the control (Fig. 5A),
These results suggest that HIV-1 clade B and clade C differentially indicating that clade B Tat has a more pronounced effect on the GSH/
regulate thiol protein and redox-dependent gene expression; clade GSSG ratio than clade C Tat. We also analyzed the level of intracellular
B has a more potent effect than clade C. thiols in IDCs treated with clade B Tat or clade C Tat. Fig. 5B shows that
IDCs treated with clade B Tat showed significantly reduced thiol levels
HIV clade B and C infection induced DC-SIGN expression in IDCs (po0.02) compared to IDCs treated with clade C Tat or control. These
results are consistent with the observed effects of HIV-1 clades B and C
DC-SIGN, also referred to as CD209, plays a key role in the on ROS production and GSS gene expression (Figs. 1 and 2).
dissemination of HIV infection [33,34]. However, the ability of
specific HIV clades to induce DC-SIGN expression has not yet been Effects of clade B and C Tat on redox-induced protein modification
studied. Therefore, we determined the effects of HIV clade B and C
viruses on DC-SIGN expression in IDCs. Fig. 3 demonstrates that We also examined whether clade B and clade C Tat differen-
HIV clade B infection showed a significant increase in DC-SIGN tially modulate thiol oxidation and redox enzymes leading to
expression at 6 days compared with HIV clade C infection. These protein modification. Fig. 6 demonstrates that clade B Tat-treated
observations confirmed that clade B infection potentiates and IDCs showed a significant decrease in GSS (Fig. 6A), GPx1 (Fig. 6B),
activates DC-SIGN-mediated pathogenesis compared to clade C and SOD1 (Fig. 6C) protein modification at 24 h of treatment
infection. compared with untreated cells, whereas no changes were detected
in clade C Tat-treated IDCs. Taken together, these findings suggest
HIV-1 clade B and clade C Tat induced redox-dependent gene an important role for redox expression by HIV-1 clade B and clade
expression in IDCs C infection leading to distinct mechanisms of immune function.

Next, we tested whether exposure to HIV-1 clade B or C Tat HIV-1 clade B and clade C induced thiol modification and redox
proteins induces oxidative stress and redox-dependent gene expression
expression similar to HIV viral infection. IDCs treated with clade
B Tat for 24 h showed a greater inhibition of GSS (p o0.01; Fig. 4A), HIV infection affects immune function and ultimately has an
GPx1 (p o0.009; Fig. 4C), SOD1 (p o0.03; Fig. 4B), and CAT impact on CNS functions [35,36]. DCs are one of the major
(p o0.003; Fig. 4D) gene expression compared with IDCs exposed reservoirs of connection between immune and CNS function.
to clade C Tat. These observations confirmed that clades B and C Therefore, we examined the effect of redox expression by clade B
differentially regulate redox-dependent gene expression. and clade C Tat on SK-N-MC neuroblastoma cells. However,
structural differences in the Tat sequence change the motif and
Effects of clade B and C Tat on GSH/GSSG ratio and intracellular thiol alter the functional properties [22], which may result in distinct
functional effects. Fig. 7 shows a comparison of the clade B and
The GSH/GSSG ratio plays an important role in oxidative stress clade C Tat primary structural sequences (Fig. 7A) and the
mechanisms and the maintenance of redox balance. We investigated increased production of ROS (Fig. 7B) and subsequent reduction

Fig. 4. Differential effects of redox gene expression by HIV-1 clade B and C Tat protein. IDCs (1  106/ml) were treated with HIV-1 clade B or clade C Tat protein (50 ng/ml) for
24 h. RNA was extracted and reverse transcribed followed by RT-qPCR for (A) GSS, (B) SOD1, (C) GPx1, (D) catalase, and the housekeeping gene β-actin. The data are expressed
as the mean7 SD of the transcript accumulation index values of three independent experiments.
 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144 141

Fig. 5. The effects of HIV-1 clade B and C Tat proteins on the GSH/GSSG ratio and intracellular thiol content. IDCs (1  106 cells/ ml) were seeded in six-well plates and treated
with HIV-1 clade B or clade C Tat protein (50 ng/ml) for 24 h. (A) The GSH/GSSG ratio was estimated spectrophotometrically, and (B) the intracellular expression of thiol was
determined by the percentage of mBrB-positive cells. The data are expressed as the mean 7SD of three independent experiments.

Fig. 6. Differential effects of redox proteins by HIV-1 clade B and C Tat protein. IDCs were treated with HIV-1 clade B or clade C Tat protein (50 ng/ml) for 24 h and analyzed
by Western blot to detect (A) GSS, (B) GPx1, and (C) SOD1. The data are expressed as the mean 7 SD of three independent experiments.

in GSS gene expression (Fig. 7C) and protein modifications potentiate more immune-neuropathogenic dysfunction compared
(Fig. 7D) in SK-N-MC cells treated with clade B Tat compared with with clade C virus and Tat protein.
cells treated with clade C Tat. This study suggests that immune Previous studies have shown that HIV-1 Tat protein is capable
dysfunction subsequently leads to impaired neuronal function, of facilitating HIV infection [37], viral replication, and disease
and this effect might be mediated differentially by HIV-1 clade B progression [38]. HIV Tat treatment of various CNS cells induces
and C infections. oxidative stress and alters the mitochondrial membrane function,
possibly leading to brain dementia and neuronal cell death [39,23].
It has been reported that oxidative stress is coupled with different
Discussion genetic or environmental insults to affect different CNS regions
[40] and trigger astrocytes and microglia [41,42]. Studies have
This study provides new insights into the differential impacts of shown that HIV-1 Tat-induced oxidative stress leads to decreased
HIV-1 clade B and C infection on oxidative stress-induced redox GSH levels in DCs in vitro and in vivo, which could affect antigen-
expression and thiol modification in IDCs and SK-N-MC cells. Here, presenting cells [43] as well as CNS function. In this regard,
we demonstrated how the clade-specific viruses and Tat proteins concomitant low levels of SOD could lead to elevated ROS
modulate the efficacy of HIV-1 infection through redox balance production and lipid peroxidase levels [44,45]. Previous studies
and antioxidant status. This is the first report to demonstrate that have demonstrated that HIV-1 clade B significantly potentiates the
clade B and clade C infections differentially induce redox expres- inhibition of antigen processing and presentation in IDCs [46].
sion and thiol modification. In addition, our results suggest that However, DC-SIGN is the major player in DC function and directly
the HIV-1 clade B virus- and Tat protein-induced redox regulation or indirectly affects the signaling machinery via ROS. HIV infection
and thiol modification significantly decrease GSH levels and is known to cause DC-mediated immune function [30]. Recently, Li
142 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144

Fig. 7. HIV clade B and clade C Tat sequence alignment and effects on SK-N-MC neuronal cells. (A) Differences in the primary structure of clade B and clade C Tat sequences.
SK-N-MC cells were cultured in six-well plates and separately treated with HIV-1 clade B Tat or clade C Tat (50 ng/ml) for 24 h. (B) ROS production was analyzed by flow
cytometry; (C) RNA was extracted, reverse transcribed, and then subjected to quantitative real-time PCR using GSS-specific primers to measure GSS gene expression; and
(D) the cell lysates were resolved by Western blot using GSS antibody. The data are expressed as the mean 7 SD of three independent experiments.

et al. [47] suggested the potential role of DC-SIGN as a receptor for Campbell et al. [59] demonstrated that when clade B was bound,
DNA and protein and proposed that it induces tolerogenicity in DC it induced significantly more proinflammatory cytokine TNF-α and
through DC-SIGN-mediated negative signals. Studies have shown CCL2 receptors in monocytes. TNF-α is elevated during HIV
that changes in cellular redox status induce oxidative stress in progression and is negatively correlated with GSH and also serves
neuronal cells, which results in the depletion of GSH [48] and an as a second messenger in oxidative stress [25,60]. Also, studies
increase in oxidized protein carbonyls [49] and reactive oxygen have demonstrated that depleted GSH in HIV-positive individuals
intermediates in the cerebral spinal fluid of patients with severe [61] plays a major role in the apoptosis of CD4 þ T cells [62]. GSH
dementia [50]. This suggests that redox state imbalance and GSH levels in DCs maintain redox homeostasis and protect DCs from
activity are associated with neuronal dysfunction. However, there oxidative stress. DCs are highly specialized in their ability to
are no reports on the cellular and molecular mechanism of redox- process and present exogenous antigen to CD4 þ T helper cells
induced gene expression initiated by HIV-1 clades B and C. and endogenous antigen to CD8 þ T cells and accordingly influence
In this study, we have shown for the first time that clade B the immune response by Th1 cytokine secretion [63–65]. The
infection increases ROS production and decreases intracellular present observations indicate that redox gene expression modu-
thiol and GSS gene expression compared with clade C infection lates immune cells as well as the neuronal response during clade B
(Figs. 1 and 2). HIV-1 clade B and clade C differentially induce infection.
DC-SIGN expression (Fig. 3). Furthermore, our results demonstrate Increases in the expression of various proinflammatory genes
that both clades induce oxidative damage and antioxidant enzyme have been shown to be dependent on the cellular GSH level [66],
activity; however, clade B Tat is significantly stronger than clade C and in turn, GSH levels are regulated by oxidative stress and redox
Tat at inhibiting GSS, GPx1, SOD1 and catalase expression (Fig. 4). balance and are known to promote neuropathogenesis [67]. These
These results indicate that HIV subtypes may affect redox-induced findings suggest that GSH plays a role in the development of
mRNA expression levels by different mechanisms in IDCs. HADs/HANDs. Our recent studies showed that HIV-1 clade B Tat
Furthermore, the GSH/GSSG ratio plays a major role in main- induced significantly higher levels of neurotoxin kynurenine in
taining cellular redox homeostasis. In IDCs treated with clade B astrocytes and dendritic cells than clade C Tat [46,56]. Another
Tat, the GSH/GSSG ratio was markedly decreased compared with study reported that oxidative stress induces quinolinic acid [68],
cells treated with clade C Tat (Fig. 5). Previous reports have which then inhibits antioxidant enzymes and redox expression
indicated that HIV infection and HIV Tat protein downregulate (Fig. 7). Therefore, it is possible that the secretion of immuno-
glutathione synthesis as evidenced by γ-GCS [51,52] and pathogenic molecules during HIV infection leads to GSH dysregu-
decreased GPx mRNA [32,53], Mn-SOD [44,54], and catalase levels lation and ROS production. Further studies are needed to fully
[55]. Our results demonstrated a significant increase in clade define the mechanism of clade B and C Tat-induced redox gene
B-induced oxidative stress, which inhibited transcription of redox expression and enzyme activation. Overall, the data provide
genes and protein modification (Fig. 6). Interestingly, there was no evidence that there is a connection between redox gene and
significant change in redox gene expression in response to clade C protein expression enhancing the level of ROS production and
Tat. Recent studies have shown that clade B Tat induces contin- inhibiting intracellular signaling mechanisms in clade B and clade
uous synthesis and secretion of cytokine, chemokine, and neuro- C Tat-treated cells. The downregulation of redox gene expression
toxic factors, which may be involved in the observed effects by the Tat protein may lead to increased oxidative damage and
[56,57]. However, the structural and functional aspects of ultimately cell death. This study supports the idea that oxidative
sequence alterations in the dicysteine motif at position C30C31 stress and redox-dependent gene expression are associated with
in the clade C Tat protein probably decrease chemotactic function immune dysfunction, particularly with the reduction of GSH [69].
compared with clade B Tat [22]. Another study demonstrated that In conclusion, infection with HIV-1 clade B and C or treatment
clade B Tat binds CCR2, whereas clade C Tat does not [58]. with the respective clade-specific Tat proteins modulated the
 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144 143

GSH/GSSG ratio and downregulated GSS, GPx1, SOD1, and catalase [18] Chauhan, A.; Turchan, J.; Pocernich, C.; Bruce-Keller, A.; Roth, S.; Butterfield, D. A.;
in IDCs and neuronal cells. Interestingly, we also demonstrated Major, E. O.; Nath, A. Intracellular human immunodeficiency virus Tat expression in
astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant
that clade B and C infection and Tat treatment differentially sites via axonal transport. J. Biol. Chem. 278:13512–13519; 2003.
inhibited redox expression, leading to immune cellular dysfunc- [19] Faller, E. M.; Sugden, S. M.; McVey, M. J.; Kakal, J. A.; MacPherson, P. A. Soluble
tion and perhaps potentiation of neuronal disease progression. To HIV Tat protein removes the IL-7 receptor alpha-chain from the surface of
resting CD8 T cells and targets it for degradation. J. Immunol. 185:2854–2866;
the best of our knowledge, this is the first report that HIV-1 clade B 2010.
has a greater impact on oxidative stress than clade C. Taken [20] Kaleebu, P.; Ross, A.; Morgan, D.; Yirrell, D.; Oram, J.; Rutebemberwa, A.;
together, these results suggest that clade-specific mechanisms in Lyagoba, F.; Hamilton, L.; Biryahwaho, B.; Whitworth, J. Relationship between
HIV-1 Env subtypes A and D and disease progression in a rural Ugandan
cellular oxidative stress might play a critical role in the immuno-
cohort. AIDS 15:293–299; 2001.
neuropathogenesis of HANDs. [21] Kandathil, A. J.; Kannangai, R.; Abraham, O. C.; Pulimood, S. A.; Sridharan, G.
Amino acid sequence divergence of Tat protein (exon1) of subtype B and C
HIV-1 strains: does it have implications for vaccine development? Bioinforma-
tion 4:237–241; 2009.
Conflict of interest
[22] Mishra, M.; Vetrivel, S.; Siddappa, N. B.; Ranga, U.; Seth, P. Clade-specific
differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat
The authors declare that they have no conflicts of interest. of human neurons: significance of dicysteine C30C31 motif. Ann. Neurol.
63:366–376; 2008.
[23] Pocernich, C. B.; Sultana, R.; Mohmmad-Abdul, H.; Nath, A.; Butterfield, D. A.
HIV-dementia, Tat-induced oxidative stress, and antioxidant therapeutic
Funding Statements considerations. Brain Res. Brain Res. Rev. 50:14–26; 2005.
[24] Turchan, J.; Pocernich, C. B.; Gairola, C.; Chauhan, A.; Schifitto, G.; Butterfield,
The present study was supported by grants from National D. A.; Buch, S.; Narayan, O.; Sinai, A.; Geiger, J.; Berger, J. R.; Elford, H.; Nath, A.
Oxidative stress in HIV demented patients and protection ex vivo with novel
Institute of Health (NIH); MH085259 and MH096640. antioxidants. Neurology 60:307–314; 2003.
[25] Aukrust, P.; Müller, F.; Svardal, A. M.; Ueland, T.; Berge, R. K.; Frøland, S. S.
Disturbed glutathione metabolism and decreased antioxidant levels in human
References immunodeficiency virus-infected patients during highly active antiretroviral
therapy—potential immunomodulatory effects of antioxidants. J. Infect. Dis.
[1] Robertson, D. L.; Anderson, J. P.; Bradac, J. A.; Carr, J. K.; Foley, B.; Funkhouser, R. K.; 188:232–238; 2003.
Gao, F.; Hahn, B. H.; Kalish, M. L.; Kuiken, C.; Learn, G. H.; Leitner, T.; McCutchan, F.; [26] Droge, W.; Schulze-Osthoff, K.; Mihm, S.; Galter, D.; Schenk, H.; Eck, H. P.;
Osmanov, S.; Peeters, M.; Pieniazek, D.; Salminen, M.; Sharp, P. M.; Wolinsky, S.; Roth, S.; Gmünder, H. Functions of glutathione and glutathione disulfide in
Korber, B. HIV-1 nomenclature proposal. Science 288:55–56; 2000. immunology and immunopathology. FASEB J. 8:1131–1138; 1994.
[2] Osmanov, S.; Pattou, C.; Walker, N.; Schwardländer, B.; Esparza, J. Estimated [27] Piette, J.; Poels-legarand, S. HIV-1 reactivation after an oxidative stress mediated
global distribution and regional spread of HIV-1 genetic subtypes in the year by different reactive oxygen species. Chem.-Biol. Interact 91:79–89; 1994.
2000. J. Acquired Immune Defic. Syndr 29:184–190; 2000. [28] Purohit, V.; Rapaka, R.; Shurtleff, D. Drugs of abuse, dopamine, and HIV-
[3] Korber, B.; Kuiken, C.; Foley, B.; Hanh, B.; McCutchan, F.; Mellors, J.; Sodroski, J. associated neurocognitive disorders/HIV-associated dementia. Mol. Neurobiol.
Human retroviruses and AIDS: a compilation and analysis of nucleic acid and 44:102–110; 2011.
amino acid sequences. Los Alamos, NM: Theoretical Biology and Biophysics [29] Steiner, J.; Haughey, N.; Li, W.; Venkatesan, A.; Anderson, C.; Reid, R.; Malpica, T.;
Group, Los Alamos National Laboratory; 1998. Pocernich, C.; Butterfield, D. A.; Nath, A. Oxidative stress and therapeutic
[4] Rambaut, A.; Robertson, D. L.; Pybus, O. G.; Peeters, M.; Holmes, E. C. Human approaches in HIV dementia. Antioxid. Redox Signaling 8:2089–2100; 2006.
immunodeficiency virus: phylogeny and the origin of HIV-1. Nature [30] Nair, M. P.; Mahajan, S. D.; Schwartz, S. A.; Reynolds, J.; Whitney, R.; Bernstein, Z.;
410:1047–1048; 2001. Chawda, R. P.; Sykes, D.; Hewitt, R.; Hsiao, C. B. Cocaine modulates dendritic cell-
[5] Myers, G.; MacInnes, K.; Korber, B. The emergence of simian/human immu- specific C type intercellular adhesion molecule-3-grabbing non-integrin expres-
nodeficiency viruses. AIDS Res. Hum. Retroviruses 8:373–386; 1992. sion by dendritic cells in HIV-1 patients. J. Immunol. 174:6617–6626; 2005.
[6] Torre, D.; Ferrario, G. Immunological aspects of nitric oxide in HIV-1 infection. [31] Shively, C. A.; Mirkes, S. J.; Lu, N. Z.; Henderson, J. A.; Bethea, C. L. Soy and
Med. Hypotheses 47:405–407; 1996. social stress affect serotonin neurotransmission in primates. Pharmacoge-
[7] Granelli-Piperno, A.; Moser, B.; Pope, M.; Chen, D.; Wei, Y.; Isdell, F.; nomics J. 3:114–121; 2006.
O'Doherty, U.; Paxton, W.; Koup, R.; Mojsov, S.; Bhardwaj, N.; Clark-Lewis, I.; [32] Gil, L.; Martínez, G.; González, I.; Tarinas, A.; Alvarez, A.; Giuliani, A.; Molina,
Baggiolini, M.; Steinman, R. M. Efficient interaction of HIV-1 with purified R.; Tápanes, R.; Pérez, J.; León, O. S. Contribution to characterization of
dendritic cells via multiple chemokine coreceptors. J. Exp. Med. 184: oxidative stress in HIV/AIDS patients. Pharmacol. Res. 47:217–224; 2003.
2433–2438; 1996. [33] Geijtenbeek, T. L.; Torensma, R. Sequence and expression of a membrane-
[8] Kadiu, I.; Wang, T.; Schlautman, J. D.; Dubrovsky, L.; Ciborowski, P.; Bukrinsky, M.; associated C-type lectin that exhibits CD4-independent binding of human
Gendelman, H. E. HIV-1 transforms the monocyte plasma membrane proteome. immunodeficiency virus envelope glycoprotein gp120. Proc. Natl. Acad. Sci.
Cell. Immunol. 258:44–58; 2009. USA 89:8356–8360; 2001.
[9] Zheng, L.; Yang, Y.; Guocai, L.; Pauza, C. D.; Salvato, M. S. HIV Tat protein [34] Soilleux, E. J. DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin) and
increases Bcl-2 expression in monocytes which inhibits monocyte apoptosis DC-SIGN-related (DC-SIGNR): friend or foe? Clin. Sci. (London) 104:437–446;
induced by tumor necrosis factor-alpha-related apoptosis-induced ligand. 2003.
Intervirology 50:224–228; 2007. [35] Venkataramana, A.; Pardo, C. A.; McArthur, J. C.; Kerr, D. A.; Irani, D. N.;
[10] Wu, L.; KewalRamani, V. N. Dendritic–cell interactions with HIV: infection and Griffin, J. W.; Burger, P.; Reich, D. S.; Calabresi, P. A.; Nath, A. Immune reconstitution
viral dissemination. Nat. Rev. Immunol. 6:859–868; 2006. inflammatory syndrome in the CNS of HIV-infected patients. Neurology 67:
[11] Canque, B.; Rosenzwajg, M.; Camus, S.; Yagello, M.; Bonnet, M. L.; Guigon, M.; 383–388; 2006.
Gluckman, J. C. The effect of in vitro human immunodeficiency virus infection [36] Ancuta, P.; Kamat, A.; Kunstman, K. J.; Kim, E. Y.; Autissier, P.; Wurcel, A.;
on dendritic-cell differentiation and function. Blood 88:4215–4228; 1996. Zaman, T.; Stone, D.; Mefford, M.; Morgello, S.; Singer, E. J.; Wolinsky, S. M.;
[12] Satishchandr, P.; Nalini, A.; Gourie-Devi, M.; Khanna, N.; Santosh, V.; Ravi, V.; Gabuzda, D. Microbial translocation is associated with increased monocyte
Desai, A.; Chandramuki, A.; Jayakumar, P. N.; Shankar, S. K. Profile of activation and dementia in AIDS patients. PLoS One 3:e2516; 2008.
neurologic disorders associated with HIV/AIDS from Bangalore, south India [37] Izmailova, E.; Bertley, F. M.; Huang, Q.; Makori, N.; Miller, C. J.; Young, R. A.;
(1989–96). Indian J. Med. Res. 111:14–23; 2000. Aldovini, A. HIV-1 Tat reprograms immature dendritic cells to express
[13] Bansal, A. K.; Mactutus, C. F.; Nath, A.; Maragos, W.; Hauser, K. F.; Booze, R. M. chemoattractants for activated T cells and macrophages. Nat. Med 9:191–197;
Neurotoxicity of HIV-1 proteins gp120 and Tat in the rat striatum. Brain Res. 2003.
879:42–49; 2000. [38] Frankel, A. D. Activation of HIV transcription by Tat. Curr. Opin. Genet. Dev
[14] Toborek, M.; Lee, Y. W.; Pu, H.; Malecki, A.; Flora, G.; Garrido, R.; Hennig, B.; 2:293–298; 1992.
Bauer, H. C.; Nath, A. HIV-Tat protein induces oxidative and inflammatory [39] Shor-Posner, G.; Lecusay, R.; Morales, G.; Campa, A.; Miguez-Burbano, M. J.
pathways in brain endothelium. J. Neurochem. 84:169–179; 2003. Neuroprotection in HIV-positive drug users: implications for antioxidant
[15] Polazzi, E.; Levi, G.; Minghetti, L. Human immunodeficiency virus type 1 Tat therapy. J. Acquired Immune Defic. Syndr 31(Suppl. 2):S84–88; 2002.
protein stimulates inducible nitric oxide synthase expression and nitric oxide [40] Moosmann, B.; Behl, C. Antioxidants as treatment for neurodegenerative
production in microglial cultures. J. Neuropathol. Exp. Neurol 58:825–831; disorders. Expert Opin. Invest. Drugs 11:1407–1435; 2002.
2003. [41] Manning, P.; Cookson, M. R.; McNeil, C. J.; Figlewicz, D.; Shaw, P. J. Superoxide-
[16] Ensoli, B.; Buonaguro, L.; Barillari, G.; Fiorelli, V.; Gendelman, R.; Morgan, R. A.; induced nitric oxide release from cultured glial cells. Brain Res. 911:203–210;
Wingfield, P.; Gallo, R. C. Release, uptake, and effects of extracellular human 2001.
immunodeficiency virus type 1 Tat protein on cell growth and viral transacti- [42] Turchan-Cholewo, J.; Dimayuga, F. O.; Gupta, S.; Keller, J. N.; Knapp, P. E.;
vation. J. Virol. 67:277–287; 1993. Hauser, K. F.; Bruce-Keller, A. J. Morphine and HIV-Tat increase microglial-free
[17] Nath, A. Human immunodeficiency virus (HIV) proteins in neuropathogenesis radical production and oxidative stress: possible role in cytokine regulation.
of HIV dementia. J. Infect. Dis 186(Suppl. 2):S193–198; 2002. J. Neurochem. 108:202–215; 2009.
144 T. Samikkannu et al. / Free Radical Biology and Medicine 69 (2014) 136–144

[43] Zocchi, M. R.; Poggi, A.; Rubartelli, A. The RGD-containing domain of 3-dioxygenase (IDO) by HIV-1 clade B and C Tat protein. AIDS Res. Hum.
exogenous HIV-1 Tat inhibits the engulfment of apoptotic bodies by dendritic Retroviruses 25:329–335; 2009.
cells. AIDS 11:1227–1235; 1997. [58] Albini, A.; Ferrini, S.; Benelli, R.; Sforzini, S.; Giunciuglio, D.; Aluigi, M. G.;
[44] Flores, S. C.; Marecki, J. C.; Harper, K. P.; Bose, S. K.; Nelson, S. K.; McCord, J. M. Proudfoot, A. E.; Alouani, S.; Wells, T. N.; Mariani, G.; Rabin, R. L.; Farber, J. M.;
Tat protein of human immunodeficiency virus type 1 represses expression of Noonan, D. M. HIV-1 Tat protein mimicry of chemokines. Proc. Natl. Acad. Sci.
manganese superoxide dismutase in HeLa cells. Proc. Natl. Acad. Sci. USA USA 95:13153–13158; 1998.
90:7632–7636; 1993. [59] Campbell, G. R.; Watkins, J. D.; Singh, K. K.; Loret, E. P.; Spector, S. A. Human
[45] Sönnerborg, A.; Carlin, G.; Akerlund, B.; Jarstrand, C. Increased production immunodeficiency virus type 1 subtype C Tat fails to induce intracellular
of malondialdehyde in patients with HIV infection. Scand. J. Infect. Dis. calcium flux and induces reduced tumor necrosis factor production from
20:287–290; 1988. monocytes. J. Virol. 81:5919–5928; 2007.
[46] Samikkannu, T.; Rao, K. V.; Gandhi, N.; Saxena, S. K.; Nair, M. P. Human [60] Aukrust, P.; Svardal, A. M.; Muller, F.; Lunden, B.; Berge, R. K.; Ueland, P. M.;
immunodeficiency virus type 1 clade B and C Tat differentially induce Frøland, S. S. Increased levels of oxidized glutathione in CD4 þ lymphocytes
indoleamine 2,3-dioxygenase and serotonin in immature dendritic cells: associated with disturbed intracellular redox balance in human immunode-
implications for neuroAIDS. J. Neurovirol. 16:255–263; 2010. ficiency virus type 1 infection. Blood 86:258–267; 1995.
[47] Li, J.; Geng, S.; Liu, X.; Liu, H.; Jin, H.; Liu, C. G.; Wang, B. DNA and protein [61] Buhl, R.; Jaffe, H. A.; Holroyd, K. J.; Wells, F. B.; Mastrangeli, A. Systemic
co-administration induces tolerogenic dendritic cells through DC-SIGN glutathione deficiency in symptom-free HIV seropositive individuals. Lancet
mediated negative signals. Hum. Vaccines Immunother 9:2038–2040; 2013. 2:1294–1298; 1989.
[48] Agrawal, L.; Louboutin, J. P.; Strayer, D. S. Preventing HIV-1 Tat-induced [62] Guerra, C.; Morris, D.; Sipin, A.; Kung, S.; Franklin, M.; Gray, D.; Tanzil, M.;
neuronal apoptosis using antioxidant enzymes: mechanistic and therapeutic Guilford, F.; Khasawneh, F. T.; Venketaraman, V. Glutathione and adaptive
implications. Virology 363:462–472; 2007. immune responses against Mycobacterium tuberculosis infection in healthy
[49] Stadtman, E. R. Protein oxidation and aging. Science 257:1220–1224; 1992.
and HIV infected individuals. PLoS One 6; 2011e28378 6; 2011.
[50] Boven, L. A.; Middel, J.; Portegies, P.; Verhoef, J.; Jansen, G. H.; Nottet, H. S.
[63] Suthanthiran, M.; Anderson, M. E.; Sharma, V. K.; Meister, A. Glutathione
Overexpression of nerve growth factor and basic fibroblast growth factor in
regulates activation-dependent DNA synthesis in highly purified normal
AIDS dementia complex. J. Neuroimmunol. 97:154–162; 1999.
human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. Natl.
[51] Choi, J.; Liu, R. M.; Kundu, R. K.; Sangiorgi, F.; Wu, W.; Maxson, R.; Forman, H. J.
Acad. Sci. USA 87:3343–3347; 1990.
Molecular mechanism of decreased glutathione content in human immuno-
[64] D'Angelo, J. A.; Dehlink, E.; Platzer, B.; Dwyer, P.; Circu, M. L.; Garay, J.; Aw, T.
deficiency virus type 1 Tat-transgenic mice. J. Biol. Chem. 275:3693–3698;
Y.; Fiebiger, E.; Dickinson, B. L. The cystine/glutamate antiporter regulates
2000.
dendritic cell differentiation and antigen presentation. J. Immunol. 185:
[52] Price, T. O.; Ercal, N.; Nakaoke, R.; Banks, W. A. HIV-1 viral proteins gp120 and
Tat induce oxidative stress in brain endothelial cells. Brain Res. 1045:57–63; 3217–3226; 2010.
2005. [65] Hirsch, C. S.; Hussain, R.; Toossi, Z.; Dawood, G.; Shahid, F.; Ellner, J. J. Cross-
[53] Richard, M. J.; Guiraud, P.; Didier, C.; Seve, M.; Flores, S. C.; Favier, A. Human modulation by transforming growth factor beta in human tuberculosis:
immunodeficiency virus type 1 Tat protein impairs selenoglutathione perox- suppression of antigen-driven blastogenesis and interferon gamma produc-
idase expression and activity by a mechanism independent of cellular tion. Proc. Natl. Acad. Sci. USA 93:3193–3198; 1996.
selenium uptake: consequences on cellular resistance to UV-A radiation. Arch. [66] Rahman, I.; MacNee, W. Regulation of redox glutathione levels and gene
Biochem. Biophys 386:213–220; 2001. transcription in lung inflammation: therapeutic approaches. Free Radic. Biol.
[54] Ngondi, J. L.; Oben, J.; Forkah, D. M.; Etame, L. H.; Mbanya, D. The effect of Med. 28:1405–1420; 2000.
different combination therapies on oxidative stress markers in HIV infected [67] Calabrese, V.; Ragusa, N.; Antico, A.; Mangiameli, S.; Rizza, V. Cysteine-
patients in Cameroon. AIDS Res. Ther 3:19; 2006. induced enhancement of lipid peroxidation in substantia nigra: comparative
[55] Jaruga, P.; Jaruga, B.; Gackowski, D.; Olczak, A.; Halota, W.; Pawlowska, M.; effect with exogenous administration of reduced glutathione. Drugs Exp. Clin.
Olinski, R. Supplementation with antioxidant vitamins prevents oxidative Res. 23:25–31; 1997.
modification of DNA in lymphocytes of HIV-infected patients. Free Radic. Biol. [68] Behan, W. M.; McDonald, M.; Darlington, L. G.; Stone, T. W. Oxidative stress as
Med. 32:414–420; 2002. a mechanism for quinolinic acid-induced hippocampal damage: protection by
[56] Gandhi, N.; Saiyed, Z.; Samikkannu, T.; Rodriguez, J. W.; Rao, K. V. K.; Nair, M. melatonin and deprenyl. Br. J. Pharmacol. 128:1754–1760; 1999.
N. Differential effects of HIV-1 clade B and clade C Tat protein on expression of [69] Ghezzi, P.; Romines, B.; Fratelli, M.; Eberini, I.; Gianazza, E.; Casagrande, S.;
pro- and anti-inflammatory cytokines by primary monocytes. AIDS Res. Hum. Laragione, T.; Mengozzi, M.; Herzenberg, L. A.; Herzenberg, L. A. Protein
Retroviruses 25:691–699; 2009. glutathionylation: coupling and uncoupling of glutathione to protein thiol
[57] Samikkannu, T.; Saiyed, Z. M.; Rao, K. V.; Babu, D. K.; Rodriguez, J. W.; groups in lymphocytes under oxidative stress and HIV infection. Mol. Immunol.
Papuashvili, M. N.; Nair, M. P. Differential regulation of indoleamine-2, 38:773–780; 2002.