ASSOCIATION.html
ORIGINAL ARTICLE
Association of -308 TNF-alpha promoter
polymorphism with viral load and CD4 T-helper cell apoptosis in HIV-1
infected black South Africans
Shivona Gounden, MMedSci
Devapregasan Moodley, PhD
Anil A Chuturgoon, PhD
Department of Medical Biochemistry, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban
Leshern Karamchand, MMedSci
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA
Halima Dawood, MB ChB, FCP
Department of Medicine, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban
Corresponding author: A Chuturgoon (chutur@ukzn.ac.za)
Objective.
To determine whether the -308 TNF-α promoter polymorphism is associated
with markers of HIV progression in the South African population.
Methods. Polymerase
chain reaction-restriction fragment length polymorphism was used to
detect the -308 TNF-α polymorphism in 75 patients and 76 healthy
controls. Serum TNF-α concentrations were measured using ELISA in each
cohort. CD4+
T cell apoptosis and HIV-1 RNA viral load were determined using
Annexin-V-FITC assay and Nuclisens Easy Q HIV-1 assay respectively. CD4
+ T cell counts were measured flow cytometrically.
Results. The
frequency of -308 G allele was similar in the HIV-1 and control
cohorts. The -308GG genotype was associated with lower TNF-α
concentrations and markers of increased HIV progression indicated by
higher TH lymphocyte apoptosis, lower TH lymphocyte count and higher plasma viral load, irrespective of treatment.
Conclusion. The
presence of the TNF-α -308 G allele in HIV-1 patients may be associated
with increased risk of HIV-1 progression. Further research is required
to investigate the nature of this association.
S Afr J HIV Med 2012;13(2):72-77.
Patients infected with human immunodeficiency virus (HIV) show a decline in CD4+ T-helper (TH)
lymphocyte levels and an increase in viral load that ultimately results
in compromised immune function and increased susceptibility to various
opportunistic infections.1
In early stages of infection, HIV-1 has the ability to manipulate the
immune response to ensure its own replication and survival.2
Consequently, there has been much controversy as to whether eliciting a
robust immune response towards the virus early in infection will be
beneficial or detrimental for the patient.2
The differential rate of HIV progression and chronic inflammatory disorders3 may be induced by viral, environmental and host genetic factors. Dean et al.
observed a 32 base pair deletion in the chemokine receptor 5 (CCR5)
that showed better protection against HIV and slower progression to
AIDS.6
Another study investigated a chemokine receptor 2 (CCR2) polymorphism
with a G→A transition at position 190, that also resulted in
slower progression to AIDS.7 Crawley et al.
found that a polymorphism associated with IL-10 at the -592 position
resulted in decreased production of IL-10, inhibition of macrophage
growth and decreased proliferation of HIV-1 in infected individuals.8
,
9
The molecular mechanisms of most polymorphisms have not been fully
elucidated. There is a need to explore more the role of host genetics
in understanding HIV disease.
In vitro and in vivo
studies have shown that HIV-1 infection can induce the secretion of
pro-inflammatory cytokines such as tumour necrosis factor alpha
(TNF-α).10
TNF-α is the central mediator of the inflammatory response, and
high concentrations of TNF-α may influence HIV-1 replication via
clonal expansion of infected T lymphocytes.13
In addition, TNF-α is also a potent inducer of apoptosis, which
is a function dependent on the death receptor configuration of immune
cells.1
,
14 HIV-1 induces immune suppression by rapid apoptosis of bystander TH lymphocytes.
TNF-α production is tightly controlled but genotypic differences may influence transcriptional regulation.15
,
16 Reports have shown that promoter polymorphisms affect TNF-α gene expression.17 A common polymorphism occurs at the -308 locus in the promoter region that results in a guanine (G) to adenine (A) transition.20
The -308 A allele has been associated with higher transcriptional
activation and, therefore, increased TNF-α expression in
different populations.4
,
17
,
19
This association has also been linked to pathogenesis of various
inflammatory disorders and, consequently, poorer disease prognosis.4
,
17
The presence of various allelotypes, especially in promoter regions of
cytokines, may severely affect immune responses to infection, given
that they exert a large degree of transcriptional control over cytokine
production. These effects, however, have not been comprehensively
investigated in the context of infection. The precise mechanisms of
genotypic influences on transcriptional regulation are currently
unknown. However, it is thought that the G to A transition at the -308
locus is associated with conformational changes that increase binding
affinity of transcription factors such as nuclear factor-kappa B
(NF-κB).15
Considering the influence of the -308 TNF-α promoter polymorphism on TNF-α concentration, CD4 TH
lymphocyte apoptosis and HIV-1 replication, genotype may severely
influence clinical outcomes in HIV-1 infected patients. The influence
of the -308 TNF-α promoter polymorphism on HIV-1 infected black
South Africans has not been studied. This is important as South Africa
has the highest burden of HIV-1 infected individuals, and polymorphic
variation may not only affect disease progression, but also response to
treatment.
The aim of this study was to investigate genotypic frequencies of
the -308 TNF-α promoter polymorphism in a cohort of HIV-1
infected black South African patients and determine whether genotype at
this locus influenced serum TNF-α concentrations. In addition,
the influence of this promoter polymorphism on CD4 TH lymphocyte apoptosis and HIV-1 burden was investigated.
Materials and methods
Patient recruitment
This cross-sectional study was approved by the
University of KwaZulu-Natal, Biomedical Research Ethics Administration
(H129/04). Patients (N=75)
were recruited by purposeful sampling from an antiretroviral (ARV)
rollout clinic at King Edward VII Hospital, Durban, after obtaining
informed consent. All patients had confirmed HIV-1 infection.
Twenty-five patients were on NRTI-based HAART (NRTI: nucleoside reverse
transcriptase inhibitor; HAART: highly active anti-retroviral therapy);
50 patients were HAART-naive. Healthy controls (N
=76) were sourced from the South African National Blood Service. There
was no follow-up of patients to assess changes in measures or outcomes
over time.
Peripheral lymphocyte preparation
Buffy coats containing peripheral blood lymphocytes (PL) were extracted as previously described by our laboratory.21 Cell density was adjusted to 1×106 cells/ml with the trypan blue exclusion test.
DNA extraction
Genomic DNA was extracted from PLs for each
patient. Cells were transferred to 500 μl lysis buffer containing
0.5% SDS, 150 mM NaCl, 10 mM EDTA, and 10 mM Tris-HCl (pH 8.0). To
this, RNase A (100 μg/ml, DNase-free) was added, and the solution
was incubated at 37°C for 1 hour. Following the RNase A step,
proteinase K (200 μg/ml) was added to the solution and thereafter
incubated for 3 hours at 50°C. Protein contaminants were then
precipitated by addition of 0.1 volume 5 mM potassium acetate and
centrifuging (5 000 ×g, 15 minutes). Supernatants containing
genomic DNA were transferred to fresh tubes and extracted with 100%
isopropanol on ice and then washed with 70% ethanol. DNA samples were
then dissolved in 10 mM Tris and 0.1 mM EDTA (pH 7.4) at 4°C
overnight. To verify DNA extraction, equal amounts of DNA (300 ng) were
electrophoresed (150 V, 50 min.) on a 1.8% agarose gel containing 0.5
mg/ml ethidium bromide. DNA bands were visualised by UV light and
digitally photographed using a gel documentation system (Chemi-Doc XRS,
Bio-Rad) and Quantity One Image Analysis software (Bio-Rad). The
concentration of each sample was determined spectrophotometrically.
Genotyping for the -308 TNF-α promoter polymorphism
Polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) was used to determine the -308
TNF-α promoter polymorphism. A 107bp PCR product was amplified
using 20 pmol of forward and reverse primer in a 25 μl reaction
containing 0.2 mM of each dNTP, 1.5 mM MgCl2,
1X Green GoTa0071 Flexi buffer (Promega), 1 U GoTaq DNA polymerase
(Promega) and 100 ng genomic DNA template. The forward and reverse
primers were those according to Wilson et al.20 (5’AGGCAATAGGTTTTGAGGGCCAT 3’; 5’ TCCTCCCTGCTCCGATTCCG 3’).
DNA was amplified for 35 cycles
with denaturation at 94°C for 3 minutes, annealing at 60°C for
1 minute, extension at 72°C for 1 minute and a final extension at
72°C for 5 minutes. The PCR product was then digested with the
restriction enzyme NcoI
for 12 hours at 37°C. Digestion of the PCR product confirmed 2
alleles viz. -308 G allele which resulted in 2 fragments (87 bp and 20
bp), and –308 A allele which resulted in a single 107 bp fragment
(Fig. 1).20
TNF-α enzyme-linked immunosorbent assay (ELISA)
Plasma was collected by centrifuging whole blood.
Plasma TNF-α concentration was measured using the human
TNF-α Max Standard ELISA kit (Biolegend). A high-affinity
microtitre plate was coated with TNF-α capture antibody (100
µl/well, 18 hours at 4oC).
Plates were washed and treated with 200 µl assay diluent.
Thereafter, 100 µl standards and samples were added. Biotinylated
anti-human TNF-α detection antibody and avidin-horseradish
peroxidase were then added, followed by the TMB substrate and the stop
solution. Absorbance was measured at 450 nm (570 nm reference) (Bio-Tek
µQuant ELISA plate reader). Plasma concentrations of TNF-α
were calculated by extrapolation from the standard curve.
CD4 TH cell apoptosis, CD4 TH cell counts and viral loads
CD4 TH lymphocyte apoptosis, CD4 TH cell counts and viral loads were determined as described previously.21
Statistical analysis
Genotype and allelic frequencies of the TNF-α
-308 polymorphism for the control and HIV-1 cohort were compared by
direct counting. Hardy-Weinberg statistics were used to determine
whether our study cohort was representative of the larger population.
Statistical analyses and correlations were done using Graphpad Prism
Software (version 5).
Results
-308 TNF-α promoter polymorphism
Genotypic distribution did not deviate from those predicted by the Hardy-Weinberg equilibrium (HIV-1: p=0.331, chi-square statistic=0.946; controls: p=0.194,
chi-square statistic=1.688). There were no significant differences in
genotypic distribution between the HIV-1 and control cohorts
respectively (GG 60% and 65.8%; GA 37.3% and 27.6%; and AA 2.7% and
6.6%). However, when allelic distribution was investigated, we found
that the -308 G allele was more frequent in the control population
(79.6% v. 78.7%) but this difference did not reach statistical
significance (chi square test p=0.888, odds ratio=1.06, 95% CI (confidence interval) 0.607 - 1.84; see Table 1).
Plasma TNF-α concentration
Mean plasma TNF-α concentration was
determined in patients and controls by ELISA. The HIV-1 infected
subjects showed significantly higher TNF-α concentration than
controls (10.87 pg/ml and 3.57 pg/ml, p<0.0001, 95% CI: HIV-1 infected patients 9.39 - 12.36 pg/ml, controls 0.74 - 6.41 pg/ml; see Table 2).
We then investigated whether
genotypic variation at the -308 locus influenced plasma TNF-α
concentration in the HIV-1 infected cohort. Mean TNF-α
concentrations were determined after grouping patients according to
genotype. Higher plasma TNF-α concentrations were recorded in the
-308GA genotype than in the -308GG genotype (15.52 pg/ml v. 15.01
pg/ml). This difference did not reach statistical significance
(Mann-Whitney test, p=0.404,
95% CI: GA 13.35 - 17.70 pg/ml, GG 12.19 - 17.83 pg/ml; see Table 2).
The mean TNF-α concentration in patients with the -308AA genotype
was 19.35 pg/ml.
Genotype and clinical parameters
Since genotypic differences in TNF-α
concentration were noted, we investigated whether genotype influenced
viral load and CD4 TH cell counts. Lower mean plasma viral load and lower mean CD4 TH
cell counts were observed in the -308GG genotype than in the -308GA
genotype (3.69 log copies/ml v. 3.92 log copies/ml and 256.10
cells/μl v. 288.60 cells/μl respectively), with no significant
difference (Mann-Whitney, p=0.970, 95% CI: GG 3.00 - 4.38 log copies/ml, GA 3.25 - 4.58 log copies/ml and p=0.242, 95% CI: GG 204.80 - 307.40 cells/μl, GA 245.30 - 331.90 cells/μl; Table 2).
Mean plasma viral load and CD4 TH cell counts in patients with the -308AA genotype were 3.59 log copies/ml and 197.00 cells/μl respectively.
Genotype and HAART
Following the observation of genotypic differences
in the clinical markers of infection, we investigated whether genotype
influenced patient response to treatment. Patients were grouped into
HAART-naive and HAART-treated cohorts, and these groups further
stratified according to genotype. Mean plasma viral load and CD4 TH cell counts were analysed according to genotype and treatment.
In the HAART-naive cohort, higher plasma viral loads and lower CD4 TH
cell counts were observed in the -308GG genotype than in the -308GA
genotype (4.92 log copies/ml v. 4.54 log copies/ml and 244.30
cells/µl v. 283.80 cells/µl) but there were no significant
differences (Mann-Whitney test, p=0.101, 95% CI: GG 4.68 - 5.16 log copies/ml, GA 4.17 - 4.90 log copies/ml and p=0.250, 95% CI: GG 179.70 - 308.80 cells/µl, GA 233.80 - 333.80 cells/µl; see Table 4).
Higher CD4 TH
cell counts and statistically significant lower plasma viral loads were
recorded in the HAART-treated cohort than in the HAART-naive cohort
(288.64 cells/µl v. 264.80 cells/µl and 1.19 log copies/ml
v. 4.72 log copies/ml) (Mann-Whitney test, p=0.451, 95% CI: HAART-naive 226.80 - 302.80 cells/µl, HAART-treated 216.70 - 360.60 cells/µl and p<0.0001,
95% CI: HAART-naive 4.51 - 4.93 log copies/ml, HAART-treated 0.940 -
1.44 log copies/ml; Table 3). This result was expected as HAART is
associated with lower plasma viral loads and higher CD4 TH
cell counts. Interestingly, we noticed genotypic differences in the
HAART-treated cohort in the -308GG genotype. The -308GG genotype showed
higher plasma viral loads and lower CD4 TH
cell counts than in the -308GA genotype (1.22 log copies/ml v. 1.13 log
copies/ml and 278 cells/µl v. 314.0 cells/µl); however, the
differences did not reach statistical significance (Mann-Whitney, p=0.251, 95% CI: GG 0.855 - 1.58 log copies/ml, GA 1.02 - 1.23 log copies/ml and p=0.374, 95% CI: GG 177.70 - 379.30 cells/µl, GA 185.40 - 442.60 cells/µl; see Table 4).
Genotype and apoptosis
Since genotypic differences were observed in TNF-α concentration, we investigated whether genotype influenced CD4 TH
cell apoptosis. Significantly higher mean apoptosis levels were
observed in HIV-1 infected patients than in controls (25.98% v. 8.52%;
Mann-Whitney test, p<0.0001,
95% CI: control 6.71 - 10.32%, HIV-1 infected 22.35 - 29.61%; see Table
2). In the HIV-1 cohort, higher apoptosis levels were observed in the
-308GG genotype (28.04%); however, there was no statistical difference
between genotypes (Mann-Whitney, p=0.223, 95% CI: GG 22.87 - 33.21%, GA 17.56 - 27.58%; see Table 2).
We investigated mean apoptosis
levels in patients on treatment, and observed higher apoptosis levels
in the HAART-naive cohort than in the HAART-treated HIV-1 infected
cohorts; however, the differences did not reach statistical
significance (27.13% v. 23.68%, Mann-Whitney test, p=0.482,
95% CI: HAART-naive 22.14 - 32.13%, HAART treated 18.99 - 28.38%; see
Table 3). The -308GG genotype showed higher apoptosis levels in both
the HAART-naive and HAART-treated HIV-1 infected cohorts than in the
-308GA genotype (32.12% v. 29.58% and 23.77% v. 21.57%); however,
differences in both cohorts were not statistically significant
(Mann-Whitney test, p=0.404, 95% CI: GG 25.17 -39.07%, GA 22.79 - 36.37% and p
=0.786, 95% CI: GG 18.19 - 29.35%, GA 4.82 - 38.32%; see Table 4). The
mean apoptosis level in the patients with the -308AA genotype was
27.77%.
Discussion
TNF-α is an immune regulatory cytokine that is released in response to viral antigens to combat infection.10 However, chronically high concentrations of TNF-α may facilitate progression of HIV-1 and apoptosis of bystander T cells.22
TNF-α indirectly induces viral replication by activating NF-κB23 which binds to the long terminal repeat (LTR) of HIV.23
,
24
This may lead to production of viral proteins such as Tat and Nef which
further induce TNF-α production via the inflammatory response.23
,
24 The -308 TNF-α promoter polymorphism has been associated with altered TNF-α concentration.15
,
16
Genotypic variation may induce conformational changes in the promoter
region that increase binding affinity of transcription factors, such as
NF-κB.15
,
16
,
23
Ours is the first report on the -308 TNF-α promoter
polymorphism in HIV-1 infected black South Africans. It is probable
that elevated levels of TNF-α may alter clinical outcomes in the
patient.4
,
17 A previous study showed lower CD4 TH cell apoptosis and plasma viral load in a cohort of HIV-1 infected patients on HAART.21
The current study aimed to investigate whether the -308 TNF-α
promoter polymorphism influenced TNF-α concentration, CD4 TH cell count, CD4 TH cell apoptosis and plasma viral load in HIV-1 infected black South Africans.
It is well established that TNF-α concentration is elevated early in infection.10
,
11
However, during HIV-1 infection, consistently high levels of
TNF-α may be attributed to constant antigenic stimulation from
viral proteins such as Tat and Nef.23
,
24
Our study shows that the -308 G allele was similar in both the
HIV-1 infected and control cohorts. This finding is consistent with
other studies that reported similar allelic frequencies in different
demographic groups.4
,
25
The -308 G allele in the HIV-1 infected cohort was associated with
significantly high levels of TNF-α, which may be due to increased
binding affinity of transcription factors.
In addition to high TNF-α
concentration, this study showed a cross-sectional association between
allelic frequency and markers of HIV disease progression, which was
indicated by high bystander TH
cell apoptosis and viral replication. High TNF-α concentration is
involved in HIV-1 replication via clonal expansion of infected TH cells.13
,
23 It is also involved in rapid apoptosis of bystander TH
cells, which may account for the high viral titres and high levels of
apoptosis observed in this study. During HIV-1 infection, TNF-α
may act as a molecular rheostat that switches between clonal expansion
and bystander TH cell apoptosis, depending on membrane receptor profile.1
,
14 Genotypic differences in the TNF-α
promoter
that influence a cell’s inherent ability to produce the cytokine
may exacerbate these functions during HIV-1 infection. In response to
rapid apoptosis, the immune system may compensate by increasing bone
marrow turnover of mononuclear cells. These may, however, not reach
complete maturation and lead to impaired TH cell recovery, ultimately contributing to HIV-1 progression.1
,
22
,
28
This study differs from previous
studies which have associated the -308 A allele with high TNF-α
concentration and disease.4
,
17
,
19 The -308AA genotype has been widely associated with poorer clinical outcomes and disease progression in Leishmaniasis, cerebral malaria and insulin-dependent diabetes mellitus.29 Interestingly, some reports showed no association between this genotype and disease severity.25
,
32
,
33
In studies that showed the association between the -308AA genotype and
disease severity, frequencies of the -308 A allele were low, which may
have conferred low statistical power and, as such, these conclusions
warrant confirmation in other populations.4
,
34
Furthermore, the bulk of these studies were performed in populations of
white ancestry. No studies to date have investigated the influence of
the -308 TNF-α promoter polymorphism in infectious diseases in a
black African population.
Conclusion
In contrast with other studies, our study
reports for the first time that the -308 G allele may contribute to
mechanisms that lead to poorer response to HAART therapy in Black South
Africans infected with HIV-1. Similarly, we found the -308AA genotype
to be least frequent (N=2),
which may preclude disease association studies until adequate sample
sizes are collected. Comparable clinical outcomes were observed in
heterozygote individuals, providing further evidence that the presence
of the -308 G allele may be associated with markers of HIV-1
progression in this study.
Single nucleotide polymorphisms that affect regulation of cytokines
may affect host response to HIV-1 infection. This effect may influence
disease progression and clinical outcomes. To provide holistic
management of patients infected with HIV-1 and develop individual
treatment strategies, it is imperative to study genotypic differences
between individuals. Such approaches may curb the advent of adverse
drug reactions, minimise therapeutic failures and also address not only
the medical, but also the economic burdens of this disease.
Acknowledgements. The authors thank LIFElab for funding.
REFERENCES
1. Badley AD, Pilon AA, Landay A, Lynch DH. Mechanisms of HIV-associated lymphocyte apoptosis. Blood 2000;96(9):2951-2964.
1. Badley AD, Pilon AA, Landay A, Lynch DH. Mechanisms of HIV-associated lymphocyte apoptosis. Blood 2000;96(9):2951-2964.
2. Furler RL, Uittenbogaart CH. Signaling through the P38 and ERK
pathways: a common link between HIV replication and the immune
response. Immunol Res 2010;48(1-3):99-109.
2. Furler RL, Uittenbogaart CH. Signaling through the P38 and ERK
pathways: a common link between HIV replication and the immune
response. Immunol Res 2010;48(1-3):99-109.
3. Fernandez-Real JM, Gutierrez C, Ricart W, et al. The TNF-alpha gene
Nco I polymorphism influences the relationship among insulin
resistance, percent body fat, and increased serum leptin levels.
Diabetes 1997;46(9):1468-1472.
3. Fernandez-Real JM, Gutierrez C, Ricart W, et al. The TNF-alpha gene
Nco I polymorphism influences the relationship among insulin
resistance, percent body fat, and increased serum leptin levels.
Diabetes 1997;46(9):1468-1472.
4. Rodriguez-Carreon AA, Zuniga J, Hernandez-Pacheco G, et al. Tumor
necrosis factor-alpha -308 promoter polymorphism contributes
independently to HLA alleles in the severity of rheumatoid arthritis in
Mexicans. J Autoimmun 2005;24(1):63-68.
4. Rodriguez-Carreon AA, Zuniga J, Hernandez-Pacheco G, et al. Tumor
necrosis factor-alpha -308 promoter polymorphism contributes
independently to HLA alleles in the severity of rheumatoid arthritis in
Mexicans. J Autoimmun 2005;24(1):63-68.
5. Dalziel B, Gosby AK, Richman RM, Bryson JM, Caterson ID. Association
of the TNF-alpha -308 G/A promoter polymorphism with insulin resistance
in obesity. Obes Res 2002;10(5):401-407.
5. Dalziel B, Gosby AK, Richman RM, Bryson JM, Caterson ID. Association
of the TNF-alpha -308 G/A promoter polymorphism with insulin resistance
in obesity. Obes Res 2002;10(5):401-407.
6. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1
infection and progression to AIDS by a deletion allele of the CKR5
structural gene. Science 1996;273(5283):1856-1862.
6. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1
infection and progression to AIDS by a deletion allele of the CKR5
structural gene. Science 1996;273(5283):1856-1862.
7. Smith MW, Dean M, Carrington M, et al. Contrasting genetic influence
of CCR2 and CCR5 variants on HIV-1 infection and disease progression.
Science 1997;277(5328):959-965.
7. Smith MW, Dean M, Carrington M, et al. Contrasting genetic influence
of CCR2 and CCR5 variants on HIV-1 infection and disease progression.
Science 1997;277(5328):959-965.
8. Crawley E, Kay R, Sillibourne J,
et al. Polymorphic haplotypes of the interleukin-10 5’ flanking
region determine variable interleukin-10 transcription and are
associated with particular phenotypes of juvenile rheumatoid arthritis.
Arthritis Rheum 1999;42(6):1101-1108.
8. Crawley E, Kay R, Sillibourne J,
et al. Polymorphic haplotypes of the interleukin-10 5’ flanking
region determine variable interleukin-10 transcription and are
associated with particular phenotypes of juvenile rheumatoid arthritis.
Arthritis Rheum 1999;42(6):1101-1108.
9. Winkler C, Modi W, Smith MW, et al. Genetic restriction of AIDS
pathogenesis by an SDF-1 chemokine gene variant. Science
1998;279(5349):389-393.
9. Winkler C, Modi W, Smith MW, et al. Genetic restriction of AIDS
pathogenesis by an SDF-1 chemokine gene variant. Science
1998;279(5349):389-393.
10. Bergamini A, Faggioli E, Bolacchi F, et al. Enhanced production of
tumor necrosis factor-alpha and interleukin-6 due to prolonged response
to lipopolysaccharide in human macrophages infected in vitro with human
immunodeficiency virus type 1. J Infect Dis 1999;179(4):832-842.
10. Bergamini A, Faggioli E, Bolacchi F, et al. Enhanced production of
tumor necrosis factor-alpha and interleukin-6 due to prolonged response
to lipopolysaccharide in human macrophages infected in vitro with human
immunodeficiency virus type 1. J Infect Dis 1999;179(4):832-842.
11. Molina JM, Scadden DT, Byrn R, Dinarello CA, Groopman JE.
Production of tumor necrosis factor alpha and interleukin 1 beta by
monocytic cells infected with human immunodeficiency virus. J Clin
Invest 1989;84(3):733-737.
11. Molina JM, Scadden DT, Byrn R, Dinarello CA, Groopman JE.
Production of tumor necrosis factor alpha and interleukin 1 beta by
monocytic cells infected with human immunodeficiency virus. J Clin
Invest 1989;84(3):733-737.
12. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor alpha
functions in an autocrine manner in the induction of human
immunodeficiency virus expression. Proc Natl Acad Sci USA
1990;87(2):782-785.
12. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor alpha
functions in an autocrine manner in the induction of human
immunodeficiency virus expression. Proc Natl Acad Sci USA
1990;87(2):782-785.
13. Folks TM, Clouse KA, Justement J, et al. Tumor necrosis factor
alpha induces expression of human immunodeficiency virus in a
chronically infected T-cell clone. Proc Natl Acad Sci USA
1989;86(7):2365-2368.
13. Folks TM, Clouse KA, Justement J, et al. Tumor necrosis factor
alpha induces expression of human immunodeficiency virus in a
chronically infected T-cell clone. Proc Natl Acad Sci USA
1989;86(7):2365-2368.
14. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADD-TRAF2 and TRADD-FADD
interactions define two distinct TNF receptor 1 signal transduction
pathways. Cell 1996;84(2):299-308.
14. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADD-TRAF2 and TRADD-FADD
interactions define two distinct TNF receptor 1 signal transduction
pathways. Cell 1996;84(2):299-308.
15. Baseggio L, Bartholin L, Chantome A, et
al. Allele-specific binding to the -308 single nucleotide polymorphism
site in the tumour necrosis factor-alpha promoter. Eur J Immunogenet
2004;31(1):15-19.
15. Baseggio L, Bartholin L, Chantome A, et
al. Allele-specific binding to the -308 single nucleotide polymorphism
site in the tumour necrosis factor-alpha promoter. Eur J Immunogenet
2004;31(1):15-19.
16. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis
factor-alpha promoter polymorphism effects transcription. Mol Immunol
1997;34(5):391-399.
16. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis
factor-alpha promoter polymorphism effects transcription. Mol Immunol
1997;34(5):391-399.
17. Abraham LJ, Kroeger KM. Impact of the -308 TNF promoter
polymorphism on the transcriptional regulation of the TNF gene:
relevance to disease. J Leukoc Biol 1999;66(4):562-566.
17. Abraham LJ, Kroeger KM. Impact of the -308 TNF promoter
polymorphism on the transcriptional regulation of the TNF gene:
relevance to disease. J Leukoc Biol 1999;66(4):562-566.
18. Louis E, Franchimont D, Piron A, et al. Tumour necrosis factor
(TNF) gene polymorphism influences TNF-alpha production in
lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy
humans. Clin Exp Immunol 1998;113(3):401-406.
18. Louis E, Franchimont D, Piron A, et al. Tumour necrosis factor
(TNF) gene polymorphism influences TNF-alpha production in
lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy
humans. Clin Exp Immunol 1998;113(3):401-406.
19. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of
a polymorphism in the human tumor necrosis factor alpha promoter on
transcriptional activation. Proc Natl Acad Sci USA 1997;94(7):3195-3199.
19. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of
a polymorphism in the human tumor necrosis factor alpha promoter on
transcriptional activation. Proc Natl Acad Sci USA 1997;94(7):3195-3199.
20. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base
polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene
detectable by NcoI restriction of PCR product. Hum Mol
Genet1992;1(5):353.
20. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base
polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene
detectable by NcoI restriction of PCR product. Hum Mol
Genet1992;1(5):353.
21. Karamchand L, Dawood H, Chuturgoon AA. Lymphocyte mitochondrial
depolarization and apoptosis in HIV-1-infected HAART patients. J Acquir
Immune Defic Syndr 2008;48(4):381-388.
21. Karamchand L, Dawood H, Chuturgoon AA. Lymphocyte mitochondrial
depolarization and apoptosis in HIV-1-infected HAART patients. J Acquir
Immune Defic Syndr 2008;48(4):381-388.
22. Khoo SH, Pepper L, Snowden N, et al. Tumour necrosis factor c2
microsatellite allele is associated with the rate of HIV disease
progression. AIDS 1997;11(4):423-428.
22. Khoo SH, Pepper L, Snowden N, et al. Tumour necrosis factor c2
microsatellite allele is associated with the rate of HIV disease
progression. AIDS 1997;11(4):423-428.
23. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB. Tumor necrosis
factor alpha activates human immunodeficiency virus type 1 through
induction of nuclear factor binding to the NF-kappa B sites in the long
terminal repeat. Proc Natl Acad Sci USA 1989;86(15):5974-5978.
23. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB. Tumor necrosis
factor alpha activates human immunodeficiency virus type 1 through
induction of nuclear factor binding to the NF-kappa B sites in the long
terminal repeat. Proc Natl Acad Sci USA 1989;86(15):5974-5978.
24. Munoz-Fernandez MA, Navarro J, Garcia A, et al. Replication of
human immunodeficiency virus-1 in primary human T cells is dependent on
the autocrine secretion of tumor necrosis factor through the control of
nuclear factor-kappa B activation. J Allergy Clin Immunol 1997;100(6 Pt
1):838-845.
24. Munoz-Fernandez MA, Navarro J, Garcia A, et al. Replication of
human immunodeficiency virus-1 in primary human T cells is dependent on
the autocrine secretion of tumor necrosis factor through the control of
nuclear factor-kappa B activation. J Allergy Clin Immunol 1997;100(6 Pt
1):838-845.
25. Azmy IA, Balasubramanian SP, Wilson AG, et
al. Role of tumour necrosis factor gene polymorphisms (-308 and -238)
in breast cancer susceptibility and severity. Breast Cancer Res
2004;6(4):R395-400.
25. Azmy IA, Balasubramanian SP, Wilson AG, et
al. Role of tumour necrosis factor gene polymorphisms (-308 and -238)
in breast cancer susceptibility and severity. Breast Cancer Res
2004;6(4):R395-400.
26. Cuenca J, Cuchacovich M, Perez C, et al. The -308 polymorphism in
the tumour necrosis factor (TNF) gene promoter region and ex vivo
lipopolysaccharide-induced TNF expression and cytotoxic activity in
Chilean patients with rheumatoid arthritis. Rheumatology (Oxford)
2003;42(2):308-313.
26. Cuenca J, Cuchacovich M, Perez C, et al. The -308 polymorphism in
the tumour necrosis factor (TNF) gene promoter region and ex vivo
lipopolysaccharide-induced TNF expression and cytotoxic activity in
Chilean patients with rheumatoid arthritis. Rheumatology (Oxford)
2003;42(2):308-313.
27. Maher B, Alfirevic A, Vilar FJ, et al. TNF-alpha promoter region
gene polymorphisms in HIV-positive patients with lipodystrophy. AIDS
2002;16(15):2013-2018.
27. Maher B, Alfirevic A, Vilar FJ, et al. TNF-alpha promoter region
gene polymorphisms in HIV-positive patients with lipodystrophy. AIDS
2002;16(15):2013-2018.
28. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+
lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med
1997;126(12):946-954.
28. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+
lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med
1997;126(12):946-954.
29. Cabrera M, Shaw MA, Sharples C, et al. Polymorphism in tumor
necrosis factor genes associated with mucocutaneous leishmaniasis. J
Exp Med 1995;182(5):1259-1264.
29. Cabrera M, Shaw MA, Sharples C, et al. Polymorphism in tumor
necrosis factor genes associated with mucocutaneous leishmaniasis. J
Exp Med 1995;182(5):1259-1264.
30. McGuire W, Hill AV, Allsopp CE, Greenwood BM, Kwiatkowski D.
Variation in the TNF-alpha promoter region associated with
susceptibility to cerebral malaria. Nature 1994;371(6497):508-510.
30. McGuire W, Hill AV, Allsopp CE, Greenwood BM, Kwiatkowski D.
Variation in the TNF-alpha promoter region associated with
susceptibility to cerebral malaria. Nature 1994;371(6497):508-510.
31. Pociot F, Briant L, Jongeneel CV, et al. Association of tumor
necrosis factor (TNF) and class II major histocompatibility complex
alleles with the secretion of TNF-alpha and TNF-beta by human
mononuclear cells: a possible link to insulin-dependent diabetes
mellitus. Eur J Immunol 1993;23(1):224-231.
31. Pociot F, Briant L, Jongeneel CV, et al. Association of tumor
necrosis factor (TNF) and class II major histocompatibility complex
alleles with the secretion of TNF-alpha and TNF-beta by human
mononuclear cells: a possible link to insulin-dependent diabetes
mellitus. Eur J Immunol 1993;23(1):224-231.
32. Gander ML, Fischer JE, Maly FE, von Kanel R. Effect of the G-308A
polymorphism of the tumor necrosis factor (TNF)-alpha gene promoter
site on plasma levels of TNF-alpha and C-reactive protein in smokers: a
cross-sectional study. BMC Cardiovasc Disord 2004;4:17.
32. Gander ML, Fischer JE, Maly FE, von Kanel R. Effect of the G-308A
polymorphism of the tumor necrosis factor (TNF)-alpha gene promoter
site on plasma levels of TNF-alpha and C-reactive protein in smokers: a
cross-sectional study. BMC Cardiovasc Disord 2004;4:17.
33. Veloso S, Olona M, Garcia F, et al. Effect of TNF-alpha genetic
variants and CCR5 Delta 32 on the vulnerability to HIV-1 infection and
disease progression in Caucasian Spaniards. BMC Med Genet 2010;11:63.
33. Veloso S, Olona M, Garcia F, et al. Effect of TNF-alpha genetic
variants and CCR5 Delta 32 on the vulnerability to HIV-1 infection and
disease progression in Caucasian Spaniards. BMC Med Genet 2010;11:63.
34. Corbett EL, Mozzato-Chamay N, Butterworth AE, et al. Polymorphisms
in the tumor necrosis factor-alpha gene promoter may predispose to
severe silicosis in black South African miners. Am J Respir Crit Care
Med 2002;165(5):690-693.
34. Corbett EL, Mozzato-Chamay N, Butterworth AE, et al. Polymorphisms
in the tumor necrosis factor-alpha gene promoter may predispose to
severe silicosis in black South African miners. Am J Respir Crit Care
Med 2002;165(5):690-693.
Fig. 1.
Restriction
fragment length polymorphism (RFLP) showing alleles of the -308
TNF-α promoter polymorphism. The -308 G allele gave rise to a 87
bp and 20 bp fragment, and the -308 A allele to a 107 bp fragment.
Table 1. Genotypic and allelic frequencies of the -308
TNF-α promoter region polymorphism in both HIV-positive and
control populations
HIV
(
N
=75)
p
value
Controls
(
N
=76)
p
value
Genotype frequency
G/G
60%
0.331*
65.8%
0.194*
G/A
37.3%
27.6%
A/A
2.7%
6.6%
Allelic frequency
A
21.3%
20.4%
0.888
G
78.7%
79.6%
*Hardy-Weinberg equilibrium (HIV-1: chi-square statistic=0.946; controls: chi-square statistic=1.688).
Table
2. Mean TNF-α concentration and markers of HIV-1 progression in the
HIV-positive and control cohorts. Markers of HIV-1 progression within
the -308 GG and -308 GA genotypes of the HIV-positive cohort
HIV-positive patients
Controls
p
value
TNF-α concentration (pg/ml)
10.87±0.73
(14.40)
3.57±1.36
(0.00)
p<0.0001
% apoptosis of CD4+ T cells
25.98±1.82
(24.30)
8.52±0.90
(6.94)
p<0.0001
HIV-positive patients
GG
GA
TNF-α concentration (pg/ml)
15.01±1.40
(14.04)
15.52±1.05
(15.39)
p=0.403
Plasma viral load (log copies/ml)
3.69±0.337
(4.66)
3.92±0.321
(4.36)
p=0.970
CD4+ T cell count (cells/µl)
256.10±25.04
(243.00)
288.60±20.97
(275.00)
p=0.242
% apoptosis of CD4+ T cells
28.04±2.57
(24.52)
22.57±2.45
(23.30)
p=0.223
All values reported as mean±SEM (median).
Table 3. Markers of HIV progression in HAART-naive and HAART-treated groups in HIV-positive patients
HAART-naive
HAART-treated
p
value
Plasma viral load (log copies/ml)
4.72±0.105
(4.85)
1.19±0.115
(1.06)
p<0.0001
CD4+ T cell count (cells/µl)
264.80±18.80
(256.00)
288.64±33.31
(293.00)
p=0.451
% apoptosis of CD4+ T cells
27.13±2.49
(24.77)
23.68±2.27
(22.86)
p=0.482
All values reported as mean±SEM (median).
Table 4. Markers of HIV progression in the -308 GG and -308 GA genotypes in the HAART-naive and HAART-treated groups
GG
GA
p
value
HAART-naive
Plasma viral load (log copies/ml)
4.92±0.115 (4.91)
4.54±0.173 (4.57)
p=0.101
CD4+ T cell count (cells/µl)
244.30±30.72 (188.00)
283.80±23.97 (273.00)
p=0.250
% apoptosis of CD4+ T cells
32.12±3.40 (26.49)
29.58±3.34 (24.54)
p=0.404
HAART-treated
Plasma viral load (log copies/ml)
1.22±0.16 (1.06)
1.13±0.03 (1.15)
p=0.251
CD4+ T cell count (cells/µl)
278.50±44.57 (273.50)
314.00±40.42 (314.00)
p=0.374
% apoptosis of CD4+ T cells
23.77±2.67 (22.36)
21.57±5.26 (22.48)
p=0.786
All values reported as mean±SEM (median).