European Journal of Clinical Investigation (2000) 30, 905±914 Paper 727

Virological and immunological effects of antioxidant

treatment in patients with HIV infection

F. MuÈller*, A. M. Svardal², I. Nordùy*, R. K. Berge², P. Aukrust* and S. S. Frùland*

*
University of Oslo, The National Hospital, Rikshospitalet, Oslo, ²University of Bergen, Haukeland Hospital, Bergen,
Norway

See Commentary on page 841 and paper on page 915.

Abstract Background Intracellular oxidative stress in CD4‡ lymphocytes due to disturbed glu-
tathione homeostasis may lead to impaired lymphocyte functions and enhanced HIV
replication in patients with HIV infection, especially in those with advanced immuno-
de®ciency. The aim of the present study was to assess whether short-term, high-dose
antioxidant treatment might have effects on immunological and virological parameters in
patients with HIV infection.
Materials and methods In this pilot study, we examined virological and immunological
effects of antioxidant combination treatment for 6 days with high doses of N-acetylcysteine
(NAC) and vitamin C in 8 patients with HIV infection. The following were assayed before,
during and after antioxidant treatment: HIV RNA plasma levels; numbers of CD4‡, CD8‡,
and CD14‡ leukocytes in blood; plasma thiols; intracellular glutathione redox status in
CD4‡ lymphocytes and CD14‡ monocytes; lymphocyte proliferation; lymphocyte apop-
tosis and plasma levels of tumour necrosis factor (TNF)a; soluble TNF receptors and
neopterin in plasma.
Results No signi®cant changes in HIV RNA plasma levels or CD4‡ lymphocyte counts in
blood were noted during antioxidant treatment in the patient group. However, in the 5
patients with the most advanced immunode®ciency (CD4‡ lymphocyte counts
< 200 ´ 106 L 1), a signi®cant rise in CD4‡ lymphocyte count, a reduction in HIV RNA
plasma level of 0´8 log, an enhanced lymphocyte proliferation and an increased level of
intracellular glutathione in CD4‡ lymphocytes were found. No change in lymphocyte
apoptosis was noted.
Conclusions Short-term, high-dose combination treatment with NAC and vitamin C in
patients with HIV infection and advanced immunode®ciency lead to immunological and
virological effects that might be of therapeutic value.
Keywords Glutathione, HIV, N-acetylcysteine, oxidative stress, vitamin C
Eur J Clin Invest 2000; 30 (10): 905±914

Introduction

Infection with human immunode®ciency virus type 1

Section of Clinical Immunology and Infectious Diseases,
Medical Department and Research Institute for Internal
Medicine, University of Oslo, The National Hospital,
Rikshospitalet, Oslo (F. MuÈller, I. Nordùy, P. Aukrust, S.
Frùland) Department of Pharmacology (A. M. Svardal) and
Department of Clinical Biochemistry (R. Berge) University of
Bergen, Haukeland Hospital, Bergen, Norway
Correspondence: Fredrik MuÈller, Institute of Medical
Microbiology Rikshospitalet, N-0027 Oslo. Tel.: ‡47 23071141;
fax: ‡47 23071110; e-mail: fredrik.muller@labmed.uio.no
Received 15 December 1999; accepted 31 March 2000
(hereafter referred to as HIV) results in a progressive
impairment of immune function, ultimately leading to
clinically manifested disease including opportunistic infec-
tions and malignancies characterising the acquired immu-
node®ciency syndrome (AIDS). A fundamental
immunologic abnormality in HIV infection is the progres-
sive decrease and functional impairment of CD4‡ lym-
phocytes. The functional capacity of lymphocytes is
critically dependent on the intracellular redox balance,
and oxidative stress, i.e. an inadequate ability of antiox-
idants to neutralize the formation of reactive oxygen species
(ROS), has been shown to impair lymphocyte functions
[1±3].

Q 2000 Blackwell Science Ltd

906 F. MuÈller et al.

Glutathione, a cysteine-containing tripeptide, is the
dominant intracellular antioxidant [4,5]. Several reports
have suggested that decreased antioxidant defence due to
disturbed glutathione homeostasis plays a role in the
immunopathogenesis of HIV infection [6±10]. ROS have
also been shown to enhance HIV replication through
activation of NF-kB [11,12]. Antioxidants such as N-
acetyl cysteine (NAC), glutathione, glutathione-esters
and vitamin C have been demonstrated to inhibit HIV
replication in vitro [11,13±21]. Several antioxidants appear
to have co-operative interactions. For example, glutathione
has been found to be involved in the recycling of vitamin C
and vice versa [22,23]. Notably, it has been shown that
vitamin C and NAC act synergistically in inhibition of HIV
replication in vitro [15]. Based on these data, several
investigators have suggested that clinical trials with anti-
oxidants, in particular with glutathione replenishing drugs,
should be carried out [15,24±27].
In this pilot study, we have examined the effects of short-
term antioxidant treatment on a number of immunological
and virological parameters in patients with HIV infection.
Based on in vitro data regarding interactions between NAC
and vitamin C [15,22,23], these compounds were given as
combination treatment.
Methods
Patients and controls
Eight HIV-seropositive men were included in the study
(median age 34 years; range 22±56 years). They were clini-
cally classi®ed according to the revised criteria from Centers
for Disease Control and Prevention (CDC) [28] as shown in
Table 1. None of the patients had suffered from any clinical
events in the last six months before blood sampling. Six
patients received antiretroviral therapy as shown in Table 1.
None of them had initiated therapy or changed dosage
regimen during the last 3 months. The study was approved
by the Regional Ethical Committee. Signed informed con-
sent was obtained from all patients. For comparison of
baseline levels of plasma and cellular measurements in the
HIV patient group, a control group consisting of 10 HIV-
seronegative blood donors was included.
Study design
All patients received N-acetylcysteine (NAC) and vitamin
C for 6 days. NAC was administered as continuous intra-
venous infusion for 72 h (300 mg kg 1 24 h 1 in glucose
50 mg mL 1; 1000 mL 24 h 1). Thereafter, the drug was
given perorally (5 g q6 h) for three days. Vitamin C was
given perorally (3 g q6 h) for six days. Clinical evaluation
and blood samples were performed at baseline, 3, 6 and
13 days after treatment was started.
Sampling of plasma
Blood was drawn into sterile pyrogen-free vacuum blood
collection tubes (Becton Dickinson, San Jose, CA) using
EDTA as an anticoagulant. Tubes were immediately
immersed in melting ice and centrifuged within 10 min
(600 g and 4 8C for 10 min). Plasma was stored at 80 8C in
multiple aliquots until analysis. Samples were thawed only
once.
For determination of various thiol components in plasma,
blood was collected into three evacuated tubes placed in
melting ice containing heparin as anticoagulant and isotonic
solutions containing either monobromobimane (mBrB,
Molecular Probes, Eugene, OR, USA), N-ethylmaleimide
(NEM, Sigma Chemical Co., St. Louis, MO, USA) as thiol-
derivatizing reagents or no addition [29].
Plasma thiol redox status
Assays of reduced, free oxidized and total forms of

Table 1 Clinical and laboratory parameters in the HIV-seropositive patients participating in the study

CDC Antiretroviral HIV RNA CD4‡ lymphocytes CD8‡ lymphocytes

classi®cation treatment Age copies ml plasma ´106 L 1 ´106 L 1

1 C2 ZDV 41 1 267 650 98 1310

2 B ddI 21 4 018 95 218
3 A ZDV 32 4 649 275 375
4 A ZDV 35 64 086 108 280
5 A ddI 26 21 219 15 200
6 A ± 55 66 551 160 1510
7 A ZDV ‡ 3TC 24 29 120 305 570
8 A ± 40 64 761 250 920
All patients 34 46 603 134 473
(21±55)* (12 934±65 656) (97±263) (249±1115)

Data are given as medians and 25±75% percentiles (*range).

CDC, Centers for Disease Control and Prevention; ZDV, zidovudine; ddI, didanosine; 3TC, lamivudine.

Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914

homocysteine, cysteine and cysteinylglycine have been
described in detail elsewhere [29,30]. Brie¯y, the amounts
of reduced thiols were obtained from the mBrB-treated
plasma samples, free oxidized forms were quanti®ed in
NEM-treated plasma, while the total thiol were obtained
from untreated plasma.
Processed and derivatized samples were stored at 80 8C
(median storage time 8 weeks; range 4±16 weeks) until
chromatographic analysis and were thawed only once.
Isolation of cells
Peripheral blood mononuclear cells (PBMC) were obtained
from heparinized blood by Isopaque-Ficoll (Lymphoprep,
Nycomed Pharma AS, Oslo, Norway) gradient centrifuga-
tion within 1 h after blood sampling as previously described
[31]. PBMC were resuspended in RPMI 1640 with 2 mM L-
glutamine and 25 mM HEPES buffer (RPMI, Gibco BRL,
Paisley, UK) supplemented with 10% heat inactivated
pooled human AB‡ serum (culture medium) at a concen-
tration of 2 ´ 106 cells mL 1. The fraction of CD4‡ lym-
phocytes and CD14‡ monocytes in the isolated PBMC was
determined by immunomagnetic quanti®cation [32].
Positive selection of CD14‡ monocytes and CD4‡
lymphocytes was performed as described elsewhere [33].
Puri®ed CD14‡ and CD4‡ cells were immediately trans-
ferred to liquid nitrogen and stored. The purity of the cell
populations was > 98% [33].
Intracellular glutathione in CD4‡ lymphocytes and
CD14‡ monocytes
Glutathione analysis was performed as described elsewhere
[10,29,34]. Brie¯y, ice-cold 5% sulphosalicylic acid
(Merck AG; Darmstadt, Germany) containing 50 mM
dithioerythritol (Sigma, St. Louis, MO, USA) was added
to the frozen cell pellets to prevent in vitro oxidation of thiol
groups. After thawing, precipated proteins and immuno-
magnetic beads were immediately removed by centrifuga-
tion. Storing of cells with immunomagnetic beads did not
in¯uence intracellular glutathione levels (data not shown).
Total free glutathione (reduced glutathione ‡ glutathione
disulphide ‡ soluble glutathione mixed disulphide; for
simplicity referred to as total glutathione in the text) and
reduced glutathione were determined in the acid extract
according to a modi®cation [29] of a chromatographic
procedure described previously [34]. Measurements were
performed on blind samples in duplicates.
Lymphocyte proliferation
PBMC were stimulated with phytohemagglutinin (PHA;
Murex Diagnostics Ltd, Dartford, UK; ®nal concentration
1 : 100), inactivated in¯uenza virus A/Singapore/6/86
(INF, formalin-inactivated; a gift from The National
Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914
Antioxidant treatment in HIV infection 907
Institute of Public Health, Oslo, Norway; ®nal concentra-
tion 1 : 1000) or left unstimulated. Cells were cultured
(0´2 ´ 106 cells well 1) in ¯at-bottomed 96-well microtitre
plates (Costar, Cambridge, MA, USA). The endotoxin
level in culture medium was less than 10 pg mL 1 (Quan-
titative chromogenic limulus amebocyte lysate test,
BioWhittaker Inc., Walkerswille, MD, USA). Cells were
pulsed with 1 mCi of 3H-thymidine (Amersham Interna-
tional plc., Little Chalfont, UK) after 48 h of culture, and
then harvested 16 h later onto glass ®lter strips, using an
automated multi-sample harvester (Skatron, Lier,
Norway). 3H-thymidine incorporation was determined by
liquid scintillation as counts per minute (cpm). The
stimulatory ratio was determined as cpm (stimulated
cells)/cpm (unstimulated cells).
Lymphocyte apoptosis
Lymphocyte apoptosis was quanti®ed as described else-
where with some modi®cations [35]. Brie¯y, PBMC
(4 ´ 106, 2 ´ 106 cells mL 1) were seeded in 24-well plates
(Costar; 1 mL well 1) in culture medium for 24 h. Non-
adherent cells were washed once in PBS, ®xed in 1%
paraformaldehyde in PBS at ‡ 4 8C for 10 min, washed
once in PBS and resuspended in 100% methanol at 20 8C
over night. Cells were then washed once in PBS and
incubated in 50 mL buffer consisting of: 10 mM biotin-16-
dUTP (Boehringer Mannheim, Mannheim, Germany);
0´25 mg mL 1 BSA (Calbiochem, San Diego, CA, USA);
0´1 mM dithiothreitol (Sigma); 2 mM cobalt chloride
(Sigma); 0´2 M sodium cacodylate (Sigma); 25 mM Tris-
HCl (Sigma) and 5 U terminal deoxynucleotidyl transfer-
ase (TdT; Boehringer Mannheim) for 30 min at 37 8C.
Control cells were incubated in the same buffer, but with-
out TdT. After one wash in 0´1% (v/v) Triton X-100
(Sigma) and 5% (w/v) BSA in PBS (washing buffer), cells
were incubated in FITC-streptavidin (1/50; Amersham),
0´1% (v/v) Triton X-100 and 3% (w/v) nonfat-dried milk
(Sigma) in PBS for 30 min at 20 8C. After a ®nal wash in
washing buffer, cells were resuspended in PBS and ana-
lysed by ¯ow cytometry within 2 h using a FACScan
(Becton-Dickinson, San Jose, CA, USA) with CellQuest
software (Becton-Dickinson). List mode ®les were col-
lected for 10,000 cells from each sample. Calibration and
adjustment of compensation were done with ¯uorescence
beads (Calibrite, Becton-Dickinson) and FacsComp soft-
ware (Becton-Dickinson). The gate for apoptotic cells was
set so that < 1% of cells were positive in the control samples
(without TdT).
HIV-RNA in plasma
HIV-RNA levels were measured in plasma by quantitative
reverse polymerase reaction (Amplicor Monitor, Roche
Diagnostic Systems, Branchburg, NY, USA). The limit of
detection was 400 copies mL 1 plasma.
908 F. MuÈller et al.

Lymphocyte subsets in peripheral blood
The numbers of CD4‡ and CD8‡ lymphocytes were
determined by immunomagnetic quanti®cation [36].
Neopterin, interleukin-10, TNFa, and TNF
receptors in plasma
calculated by the Spearman rank test. The calculations
were performed using the STATISTICA (StatSoft, Tulsa,
OK, USA) software package. Data are given as medians
and 25th to 75th percentiles if not otherwise stated. P-
values are two sided and considered signi®cant when
P < 0´05.

Neopterin was quanti®ed by radioimmunoassay (IMMUt- Results

est Neopterin; Henning Berlin GMBH, Berlin, Germany).
Enzyme immunoassay was used for quanti®cation of IL-10
and sTNFR-I and sTNFR-II (R & D Systems, Minneapo-
lis, MN, USA) and TNFa (Medgenix, Fleurus, Belgium).
Statistical analysis
Adverse reactions
Two patients experienced moderate abdominal discomfort
during the ®rst three days of antioxidant treatment, while
all patients reported distaste of the perorally administered
NAC.

For each parameter, patients and controls were compared Cysteine, cysteinylglycine, homocysteine and
by the Mann±Whitney U-test, while pre- and post-treat- glutathione in plasma
ment values in the patients were compared by the Wilcoxon
matched pairs test. Coef®cients of correlation (r) were While total cysteinylglycine and homocysteine levels were

Table 2 Antioxidant levels in CD4 ‡ lymphocytes, CD14 ‡ monocytes and in plasma from 10 blood donor controls and from 8
patients with HIV infection before, during and after treatment with N-acetylcysteine and vitamin C

Baseline Treatment with NAC and vitamin C

Controls 0 3 days 6 days 13 days

CD4‡ lymphocytes
Total glutathione 1´0 1´0 1´3 1´8 1´4
(nmol/106 cells) (0´8±1´1) (0´7±1´7) (0´7±1´7) (1´0±2´2) (0´8±2´9)
Reduced glutathione 0´9 0´9 1´0 1´5 1´2
(nmol/106 cells) (0´8±1´0) (0´6±1´3) (0´6±1´5) (0´9±1´9) (0´7±2´4)
Reduced:total 0´91 0´82 0´80 0´85 0´83
glutathione ratio (0´89±0´96) (0´77±0´88) (0´77±0´96) (0´83±0´88) (0´80±0´86)
CD14‡ monocytes
Total glutathione 1´5 2´3* 3´1 2´1 2´2
(nmol/106 cells) (1´4±1´7) (1´9±3´5) (2´2±4´3) (1´4±2´5) (1´8±3´5)
Reduced glutathione 1´3 2´1 2´7 1´8 2´1
(nmol/106 cells) (1´2±1´5) (1´6±3´0) (1´9±3´4) (1´3±2´2) (1´6±2´9)
Reduced:total 0´87 0´86 0´86 0´86 0´88
glutathione ratio (0´85±0´87) (0´81±0´91) (0´83±0´87) (0´83±0´90) (0´85±0´97)
Plasma
Total glutathione 6´7 6´7 6´8 5´6 5´0
(mmol L 1) (6´1±9´2) (5´7±8´9) (4´0±8´8) (4´8±6´1) (4´7±6´5)
Total cysteine 169´2 222´4* 117´1** 195´2** 233´6
(mmol L 1) (161´6±209´4) (212´0±252´0) (101´1±136´2) (171´7±205´7) (174´3±273´6)
Reduced cysteine 4´0 6´1 14´0** 6´3 6´1
(mmol L 1) (3´2±5´4) (3´2±8´0) (12´7±16´9) (5´3±8´4) (4´4±8´1)
Reduced:total 0´02 0´03 0´12** 0´04 0´03
cysteine ratio (0´01±0´03) (0´01±0´04) (0´10±0´20) (0´03±0´05) (0´02±0´04)
Total cysteinylglycine 18´5 19´1 11´1** 17´2** 18´4
(mmol L 1) (17´6±25) (18´5±19´4) (8´8±12´9) (14´0±17´8) (14´2±21´2)
Reduced cysteinylglycine 0´6 0´7 2´0** 0´9** 0´8
(mmol L 1) (0±0´7) (0´3±0´8) (1´6±2´6) (0´7±1´3) (0´5±1´1)
Reduced/total 0´03 0´03 0´17** 0´05 0´05
cysteinylglycine ratio (0±0´03) (0´01±0´04) (0´14±0´29) (0´04±0´09) (0´03±0´07)
Total homocysteine 3´4 4´1 0** 1´6** 4´2
(mmol L 1) (3´22±4´32) (3´2±5´3) (0±0) (0´9±2´6) (3´7±5´3)

* P < 0´05 vs. controls; ** P < 0´05 vs. patient baseline levels.

Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914

Figure 1 Effect of antioxidant combination treatment with
NAC and vitamin C in 5 HIV-infected patients with CD4‡
lymphocyte counts in blood < 200 ´ 106 L 1 on: (a) reduced:total
glutathione ratio in CD4‡ lymphocytes; (b) total glutathione
level in CD4‡ lymphocytes; (c) reduced:total glutathione ratio
in CD14‡ monocytes; (d) total glutathione level in CD14‡
similar in controls and patients before antioxidant treat-
ment, cysteine levels were signi®cantly higher in the patient
group (Table 2). During antioxidant treatment, a pro-
nounced reduction of total levels of all plasma thiols
except glutathione was noted, reaching the lowest levels
after 3 days as shown in Table 2. Concomitantly, the
reduced forms of cysteine and cysteinylglycine increased
markedly, resulting in a marked increase in the reduced:-
total ratio for these thiols during therapy (Table 2).
Intracellular glutathione in CD4‡ lymphocytes and
CD14‡ monocytes
While total intracellular glutathione levels in CD4‡ lympho-
cytes were similar in the control and patient groups (Table 2),
the reduced:total glutathione ratios tended to be lower in the
patient group (P ˆ 0´07 vs. controls). When patients with
CD4‡ lymphocyte counts in blood < 200 ´ 106 L 1 were
analysed separately, they had a signi®cantly lower reduced:-
total glutathione ratio compared to controls (Fig. 1a).
During antioxidant treatment, total glutathione levels in
Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914
Antioxidant treatment in HIV infection 909
monocytes. Corresponding levels for blood donor controls
(n ˆ 10) are given for comparison. Data are given as medians
and 25±75 percentiles. The horizontal bar below the x-axis
shows the period of antioxidant treatment. *P < 0´05 vs. patient
baseline levels; **P < 0´05 vs. controls.
CD4‡ lymphocytes tended to increase (P ˆ 0´09; Table 2).
However, in the subgroup of patients with CD4‡ lympho-
cyte counts in blood < 200 ´ 106 L 1, the intracellular total
glutathione level rose signi®cantly from 0´75 (0´6 to 1´74)
nmol/106 cells to 2´23 (2´01±2´24) nmol/106 cells before
and after 6 days of treatment, respectively (P ˆ 0´04; Fig.
1b). No signi®cant change in the reduced/total glutathione
ratio in CD4‡ lymphocytes was observed during antiox-
idant treatment (Fig. 1a).
In monocytes, intracellular glutathione levels were sig-
ni®cantly higher in the patient group before treatment
compared to controls (Fig. 1d). No apparent changes in
glutathione levels (total, reduced or reduced:total ratios)
were noted during antioxidant treatment either in the
patient group as a whole or in those with CD4‡
lymphocyte counts < 200 ´ 106 L 1 (Table 2, Fig. 1c,d).
HIV RNA in plasma
A trend to reduction in plasma HIV RNA levels was noted
during 6 days of antioxidant treatment, also persisting after
910 F. MuÈller et al.

Table 3 Immunological and virological parameters in 10 blood donor controls and in 8 patients with HIV infection before (baseline),
during (3 and 6 days) and after (13 days) treatment with N-acetylcysteine and vitamin C

Baseline Treatment with NAC and vitamin C

Controls 0 3 days 6 days 13 days

HIV-RNA ± 46 603 28 010 13 438 17 097

(copies mL 1 plasma) (12 934±65 656) (12 007±70 057) (8500±81 115) (3787±75 009)
CD4 ‡ lymphocytes 630 134* 187 178 164
(´106 L 1) (500±930) (97±263) (101±258) (115±200) (113±237)
CD8 ‡ lymphocytes 420 473 445 515 590
(´106 L 1) (310±580) (249±1115) (360±1110) (280±1300) (360±1115)
CD14 ‡ monocytes 430 335 400 370 360
(´106 L 1) (320±530) (272±400) (305±555) (325±550) (285±403)
CD19 ‡ lymphocytes 150 123 170 100 125
(´106 L 1) (120±180) (83±183) (120±215) (70±140) (98±148)
Unstimulated lymphocyte 371 603* 464 523 422
proliferation (cpm) (308±586) (479±718) (402±495) (369±711) (378±585)
In¯uenza-antigen stimulated 5070 984* 767 1307 1217
lymphocyte proliferation (cpm) (2648±7437) (499±1738) (467±1649) (684±2296) (705±1736)
PHA stimulated lymphocyte 125 333 58 918* 72 850 59 785 67 550
proliferation (cpm) (103 450±157 739) (36 226±89 378) (33 410±79 481) (36 981±90 576) (32 302±74 047)
Lymphocyte apoptosis in vitro 3´3 12´5* 14´9 14´9 11´3
(% apoptotic cells) (2´3±5´1) (5´4±19´8) (7´5±19´3) (7´4±18´8) (8´4±15´2)
Neopterin in plasma 9´0 31´3* 27´5** 31´5 29´9
(nmol L 1) (7´3±10´4) (29´7±39´4) (22´7±33´4) (20´3±33´8) (22´1±38´4)
TNFa in plasma 3´8 46´4* 53´5 51´3 50´8
(pg mL 1) (3´7±6´2) (40´6±55´3) (36´8±71´3) (37´7±58´4) (38´1±60´6)
Soluble TNFR-I 602 916* 889 912 887
(pg mL 1) (581±606) (705±1081) (707±1034) (868±1008) (832±1012)
Soluble TNFR-II 2142 6514* 3489** 5877** 6504
(pg mL 1) (2057±2452) (5622±6998) (2863±4009) (4512±6090) (5629±7662)
IL-10 in plasma 1´0 3´3* 3´2 2´8 2´5**
(pg mL 1) (0´8±1´4) (2´6±4´2) (2´5±4´5) (2´2±3´3) (2´1±3´1)

PHA, phytohemagglutinin.
* P < 0´05 vs. controls; **P < 0´05 vs. patient baseline levels.

13 days, but these changes did not reach statistical signi®-
cance (Table 3). However, in the 5 patients with CD4‡
lymphocyte counts in blood < 200 ´ 106 L 1, a signi®cant
reduction of 0´8 log was found after 6 days of treatment
(P ˆ 0´04; Fig. 2a).
Lymphocyte subsets in peripheral blood
No signi®cant changes in CD4‡ , CD8‡ , or CD19‡
lymphocytes or in CD14‡ monocytes (Table 3) were
found during antioxidant treatment. However, CD4‡
lymphocyte counts tended to rise after 6 days of treatment.
When patients with CD4‡ lymphocyte counts in blood
< 200 ´ 106 L 1 were analysed separately, a signi®cant
median increase from 102 to 130 CD4‡ lymphocytes was
observed after 6 days of treatment (P ˆ 0´04), and this
increase persisted after 13 days (P ˆ 0´04 compared to
base line; Fig. 2b).
antigen (in¯uenza) or the nonspeci®c mitogen (PHA), or
stimulatory ratios were noted during antioxidant treatment,
although there was a trend towards increased stimulated
responses (Table 3). Again, in patients with CD4‡ lympho-
cyte counts in blood < 200 ´ 106 l 1, a signi®cant increase in
the PHA stimulatory ratio from 88 (57 to 94) to 123 (71±
173) was observed after 3 days of treatment (P ˆ 0´04),
re¯ecting both decreased spontaneous and increased PHA
stimulated responses (data not shown).
After 24 h in culture without any stimulants, the percen-
tage of apoptotic lymphocytes at base-line from the patients
was signi®cantly higher than in lymphocytes from the
controls (Table 3). Antioxidant treatment did not lead to
any signi®cant alteration in the percentage of apoptotic
lymphocytes either in the patient group as a whole (Table 3)
or in those with CD4‡ lymphocyte counts in blood
< 200 ´ 106 L 1 (data not shown).

Neopterin, interleukin-10, TNFa and soluble TNF

Lymphocyte proliferation and apoptosis receptors in plasma

No signi®cant changes in unstimulated lymphocyte Patients had elevated levels of neopterin, IL-10, TNFa,
proliferation, proliferative responses of lymphocytes to sTNFR-I and sTNFR-II at baseline compared to

Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914

Figure 2 Effect of antioxidant combination treatment with
NAC and vitamin C in 5 HIV-infected patients with CD4‡
lymphocyte counts in blood < 200 ´ 106 L 1 on: (a) number of
HIV RNA copies in plasma; (b) CD4‡ lymphocyte counts in
blood; (c) CD8‡ lymphocyte counts in blood. Data are given as
medians and 25±75 percentiles. The horizontal bar below the
x-axis shows the period of antioxidant treatment. *P < 0´05 vs.
patient baseline levels.
Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914
Antioxidant treatment in HIV infection 911
controls (Table 3), re¯ecting the immune activation seen
in patients with HIV infection. During antioxidant treat-
ment, signi®cant reduction in the levels of neopterin
(approximately 12%) and sTNFR-II (approximately
46%) was observed after 3 days, while IL-10 levels
showed a declining trend with a signi®cant reduction
13 days after initiation of treatment (Table 3). No sig-
ni®cant changes were noted for TNFa or sTNFR-I
neither in the patient group (Table 3) nor in those with
with CD4‡ lymphocyte counts in blood < 200 ´ 106 L 1
(data not shown).
Discussion
Our data indicate that high dose combination treatment
with N-acetylcysteine and vitamin C for 6 days may have
immunological and virological short-term effects in
patients with HIV infection and advanced immuno-
de®ciency which might be of therapeutic value. In fact, in
patients with CD4‡ lymphocyte counts in blood
< 200 ´ 106 L 1 there was a signi®cant decrease of 0´8 log
in viral load, and signi®cant increases in CD4‡ lymphocyte
counts in peripheral blood and in lymphocyte proliferative
responses. At the same time a down-regulation of certain
parameters of immune activation was observed. Notably,
all these changes were associated with a three-fold increase
in intracellular levels of glutathione in CD4‡ lymphocytes.
These ®ndings therefore underscore the potential utility of
antioxidant therapy in HIV-infected patients, at least in
those with advanced immunode®ciency as re¯ected in low
CD4‡ lymphocyte counts.
This study was designed as an uncontrolled, small pilot
study, and the results should be interpreted with caution.
On the other hand, the study design permitted an in-depth
analysis of a number of parameters of relevance to the
pathogenesis of HIV infection.
The effect of NAC administration to patients with HIV
infection has been addressed in other studies. De Quay et
al. [37] noted a moderate increase in glutathione level in
PBMC from eight HIV-infected patients in response to a
single dose of NAC. In another study, six patients were
given NAC for two weeks, but no increase in glutathione
level in PBMC or plasma was noted [38]. In a double-blind
placebo-controlled trial, Akerlund et al. [39] treated 45
HIV seropositive patients with CD4‡ lymphocyte counts
> 200 ´ 106 L 1 for 4 months with orally administered NAC
(800 mg daily) or placebo and found that the decline of the
CD4‡ lymphocyte count was less steep in the NAC group
compared to the placebo group. Herzenberg et al. [40]
administered NAC for 8 weeks to 27 HIV-seropositive
patients and found a signi®cant increase in whole blood
GSH as well as increased survival compared to placebo.
Finally, Look et al. [41] administered NAC and sodium
selenite for 24 weeks to asymptomatic HIV-infected
patients and found increased CD4:CD8 ratios, a trend
towards an increase in the percentage of CD4‡
lymphocytes, but no alteration in viral load.
912 F. MuÈller et al.
Compared to several of these studies, the patients with
advanced immunode®ciency in our study showed more
extensive effects of antioxidant treatment in terms of rise
in CD4‡ lymhocyte counts and reduction of viral load.
These discrepancies may have several explanations. Firstly,
it may be due to the more advanced level of immunode®-
ciency in our patients compared to the patients in most of
the other studies. In fact, we have previously found that
severe glutathione disturbances in CD4‡ lymphocytes are
con®ned to patients with advanced clinical and immuno-
logical disease [10]. Secondly, orally administered NAC, as
used in all the other studies mentioned, has a low bioavail-
ability of < 10% [42]. In our study, the initial intravenous
administration of NAC might thus lead to more potent
antioxidant effects. An indication of the ef®ciency of the
treatment is the three-fold increase in intracellular glu-
tathione level in CD4‡ lymphocytes. Thirdly, the combi-
nation of NAC with vitamin C in our study might be of
bene®t as it has been shown in vitro that vitamin C
potentiates the antioxidant effects of NAC [22,23] and
that the two compounds act synergistically in inhibition of
HIV replication in vitro [15]. However, in vitro data [11,43]
suggest that relatively high concentrations of NAC are
needed for inhibition of HIV replication. Thus, even the
high NAC dosage employed in our study may be insuf®-
cient to reach ef®cient inhibition of HIV replication in vivo.
In addition to immunode®ciency, HIV-infected patients
are characterized by persistent immune activation, as also
reported by ourselves [44]. Thus, in the present study the
patients had raised plasma levels of neopterin, TNFa and
sTNFRs as well as elevated spontaneous lymphocyte pro-
liferation, suggesting a sustained and inappropriate
immune activation in these patients. Notably, several para-
meters re¯ecting immune activation decreased during anti-
oxidant treatment. While there was no decline in TNFa
levels, sTNFRs, and particularly sTNFR-II, are believed to
be better and more stable circulating parameters of activa-
tion of the TNF system than TNFa itself [45]. Indeed,
there was a signi®cant decline in sTNFR-II during anti-
oxidant therapy. We and others have previously suggested
that enhanced oxidative stress during HIV infection might
be related to enhanced immune activation, particularly in
the TNF system, which also enhances oxidative stress,
resulting in a vicious circle (reviewed in [5]). Thus the
effect of antioxidant therapy on the persistent immune
activation in HIV-infected patients may be important,
breaking the vicious circle as described above.
During antioxidant therapy there was a marked
decrease, rather than increase, in total levels of the thiols
cysteine, cysteinylglycine and homocysteine. This is in
agreement with a report from Wiklund et al. [46] where
high doses of NAC were administered to patients with
hyperlipidemia. Signi®cantly reduced plasma levels of the
same thiol compounds were noted and the authors sug-
gested that the reduction might be due to displacement of
the thiols by NAC from binding proteins leading to
increased elimination of the low molecular nonprotein
bound thiol forms. Concomitant with the reduction in
total thiol levels in our study, the concentrations of the
Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 905±914
reduced thiol forms increased signi®cantly, thus increasing
the plasma antioxidant capacity. The mechanisms under-
lying this rise in reduced thiol forms are unclear.
The patients in this study were included during the
beginning of the HAART (highly active antiretroviral ther-
apy) era. At present, HAART, with combinations of differ-
ent classes of reverse transcriptase inhibitors and protease
inhibitors [47,48], is the cornerstone in treatment of HIV-
infected patients. Although the results of HAART have
been impressive, there are also several problems related to
this treatment, such as the emergence of drug resistant HIV
variants, long-term side-effects, dif®culties with strict
adherence to treatment regimens, and frequent lack of
complete immunological reconstitution [47±51]. These
dif®culties have led to a renewed interest in other treatment
modalities in addition to antiviral therapy, including immu-
nomodulating agents such as glutathione replenishing
drugs.
In conclusion, a number of immunological and virolo-
gical short-term effects were found in patients with HIV
infection and advanced immunode®ciency during short-
term high dose combination treatment with NAC and
vitamin C, which may be therapeutically useful. Larger,
controlled studies are needed to further assess the effects of
antioxidant treatment in patients with HIV infection, pre-
ferably with other glutathione replenishing drugs with
better bioavailability than NAC. In future trials, such
agents must be given in combination with state of the art
HAART regimens.
Acknowledgements
This work was supported by the Norwegian Research
Council, the Norwegian Cancer Society, Medinnova Foun-
dation, Anders Jahre's Foundation and Odd KaÊre Rabben's
Memorial Fund for AIDS research.
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