# Aflatoxin-Related Immune Dysfunction in Health and in  Human Immunodeficiency Virus Disease

Authors: Yi Jiang; Pauline E. Jolly; Peter Preko; Jia-Sheng Wang; William O. Ellis; Timothy D. Phillips; Jonathan H. Williams
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Both aflatoxin and the human immunodeficiency virus (HIV) cause immune suppression and millions of HIV-infected people in developing countries are chronically exposed to aflatoxin in their diets. We investigated the possible interaction of aflatoxin and HIV on immune suppression by comparing immune parameters in 116 HIV positive and 80 aged-matched HIV negative Ghanaians with high (≥0.91 pmol/mg albumin) and low (<0.91 pmol/mg albumin) aflatoxin B1 albumin adduct (AF-ALB) levels. AF-ALB levels and HIV viral load were measured in plasma and the percentages of leukocyte immunophenotypes and cytokine expression were determined using flow cytometry. The cross-sectional comparisons found that (1) among both HIV positive and negative participants, high AF-ALB was associated with lower perforin expression on CD8+ T-cells (P=.012); (2) HIV positive participants with high AF-ALB had significantly lower percentages of CD4+ T regulatory cells (Tregs; P=.009) and naive CD4+ T cells (P=.029) compared to HIV positive participants with low AF-ALB; and (3) HIV positive participants with high AF-ALB had a significantly reduced percentage of B-cells (P=.03) compared to those with low AF-ALB. High AF-ALB appeared to accentuate some HIV associated changes in T-cell phenotypes and in B-cells in HIV positive  participants.


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## Body

## 1. Introduction

Numerous studies
conducted in animals and in animal and human cell cultures have shown that
aflatoxin exposure can suppress immune function especially cell-mediated immune
responses [1, 2]. More specifically, these studies on the immunotoxic effect of aflatoxin have
shown that exposure to aflatoxin decreased T or B lymphocyte activity [3, 4], impaired
macrophage/neutrophil effector functions [5–8], modified
synthesis of inflammatory cytokines [8, 9], suppressed NK cell-mediated cytolysis
[10], decreased resistance to infectious diseases [2, 11–15], induced
reactivation of chronic infection [16, 17], decreased immunity to vaccination
[18, 19], and impaired immune function in developing animals [7, 20].However, only two studies have been conducted on the immune
effects of aflatoxin in humans exposed to low levels of aflatoxin in
contaminated foods. One study conducted in
Gambian children reported that secretory immunoglobulin A in saliva may be
reduced by dietary levels of aflatoxin [21]. We previously reported that the
percentages of CD8+ T-cells that expressed perforin, or both perforin and granzyme
A were significantly lower in participants with high AFB1-albumin adduct (AF-ALB) levels in
plasma compared to those with low AF-ALB [22]. We also found that low levels of
CD3+CD69+ and CD19+CD69+ cells were significantly associated with high AF-ALB
levels. These alterations in immunological parameters in participants with high
AF-ALB levels could result in impairments in cellular immunity in these
individuals that could decrease their resistance to infections.Hendrickse et al. [23] investigated the
reasons for the rapid progression of human immunodeficiency virus (HIV) and
acquired immune deficiency syndrome (AIDS) in heroin addicts in the Netherlands
and Scotland. They found that street heroin was often contaminated with
aflatoxin, and that aflatoxin derivatives were commonly found in the body
fluids of the addicts. They speculated that the accelerated rate of HIV
progression was due to aflatoxin-related immune suppression, but did not
undertake studies to examine this. This suggestion of synergy between aflatoxin
and HIV progression is also supported by the broad correlation between
estimated aflatoxin exposure and the commonly perceived faster rate of HIV
progression in Africa than in developed countries in Europe or the United
States of America [24, 25]. The HIV
pandemic is critical enough for this possibility to be investigated as a matter
of urgency.HIV
infection results in impaired immune function that can be measured by changes
in immunphenotypically
defined lymphocyte subsets and other in vitro functional
assays. The altered expression of lymphocyte surface antigens reflects the
dynamic interaction between the immune system and HIV. Investigation of the
effect of aflatoxin on the immune system in HIV positive individuals is
urgently needed since both aflatoxin and HIV are immune suppressive and  millions of people who  are chronically exposed to aflatoxin in their
diet in developing countries are also HIV positive. In this study, we examined
the potential immune suppressive interaction of aflatoxin and HIV by measuring
a broad array of immune indices in HIV-infected individuals with high and low
AF-ALB levels. HIV negative participants were included as a control group.


## 2. Materials and Methods

### 2.1. Study Design and Study Participants

This was a cross-sectional analysis of
116 HIV positive individuals and 80 HIV negative control participants. The HIV positive patients and HIV
negative controls of comparable age were recruited from the St. Markus Hospital
and surrounding communities in Kumasi, Ghana. The protocol for the study was
approved by the Institutional Review Board of the University of Alabama at
Birmingham (UAB) and the Medical School Ethics Committee of the Kwame Nkrumah
University of Science and Technology (KNUST) and participants gave informed consent.
The participants were asked to complete a survey on socio-demographic
characteristics, and a 20 mL blood sample was collected from each in EDTA vacutainer tubes. Plasma was separated and peripheral blood mononuclear cells
(PBMCs) were prepared using ficoll-hypaque density gradients as previously done
[26]. PBMCs were stored frozen in liquid nitrogen and shipped to UAB for
analysis.


### 2.2. Determination of AF-ALB Levels in Plasma by Radioimmunoassay

AF-ALB levels in plasma of study
participants were determined by radioimmunoassay (RIA)
as published previously [27]. Briefly, human plasma samples were concentrated
by high-speed centrifugal filtration using Microcon-50 microconcentrator with a
50000 mol. wt. filter cutoff. The concentrated protein was resuspended in
100–150 μL PBS and the amount of human plasma albumin determined in each sample
using a bromocresol purple dye binding method. In addition, the amount of total
protein was determined by the procedure of Bradford (Pierce Biotechnology Inc.,
Rockford, IL). Total protein was then digested with Pronase (Calbiochem, La
Jolla, Calif, USA; 70000 proteolytic units/g dry weight was dissolved in PBS at
10 mg/mL) at a ratio of 4:1 (Protein:pronase) in a shaking water bath (50 strokes/min) at 37C° for 16–18 hours. Digestion was stopped by cooling on ice.
Two volumes ice-cold acetone were added and the sample mixed and allowed to
remain at 4C° for 1 hour. The suspension was then centrifuged at 11000rpm (9800 g) for 15 minutes. The resulting supernatant containing the bound
aflatoxin was decanted and dried in vacuo using
a savant speed-vac concentrator. The RIA procedure was used to quantify
aflatoxin B1-albumin adducts in duplicate human plasma protein digests each
containing 2 mg protein. Nonspecific inhibition in the assay was determined by
processing pooled normal human plasma standards obtained from Sigma Aldrich
(St. Louis, MO, USA) and the average value of the background was subtracted
from the values of test samples in calculating AFB1-albumin adduct levels. The standard
curve for the RIA was determined using a nonlinear regression method [28] and
values were expressed as the amount of AFB1 per
mg albumin [27]. The detection limit of the assay was 0.01 pmol/mg albumin.


### 2.3. Determination of Percentages of Leukocyte
Immunophenotypes Using Flow Cytometry

We determined the percentages of
leukocyte immunophe-notypes in PBMCs from the study participants. The
percentages of T-cells (CD3+), subsets of T-cells (CD4+ and CD8+), B-cells
(CD19+), and NK-cells (CD3-CD56+) were measured by flow cytometry. Naive CD4
cells were defined as CD45RA+CD62L+ and memory cells as CD45RO+CD45RA-. The
expression of the costimulatory molecule CD28, activation marker HLA-DR, and
CD38 on CD4
and CD8 T-cells were also measured. CD8+ T-cell subset classification has been
proven useful in monitoring the immune system in several clinical situations
[29]. Therefore, we classified CD8+ T-cell subsets into naive (CD8+CD27+CD45RA+), memory (CD8+CD27+CD45RA-),
and effector (CD8+CD27-CD45RA+) CD8+ T-cells by flow cytometry.
Subtypes of NK-cells, CD3-CD56+CD16-, and CD3-CD56+CD16+ were determined. The
percentages of activated (marker CD69) CD3+ T-cells and CD19+ B-cells (CD3+CD69+
and CD19+CD69+), and CD4+CD25+, CD4+CD25+ CD45RO+ T regulatory cell were also
measured.PBMCs were incubated with combinations
of fluorescein FITC-, PE-, PerCP-labeled monoclonal antibodies (MAbs) against
CD3, CD4, CD8, CD16, CD19, CD25, CD27, CD38, CD45RA,
CD45RO, CD56, CD62, CD69, and HLA-DR (BD PharMingen, San Diego, Calif) for 30 minutes
at 4C°. Isotype-matched irrelevant FITC-,
PE-, and PerCP-labeld MAbs (BD PharMingen,
San Diego, Calif) were used as controls in the experiments. After washing the cells
three times in PBS, cell fluorescence for each phenotype was analyzed using
Becton Dickinson
(San Diego, Calif, USA), FACS, and CELLQuest software.


### 2.4. Determination of Cytokine Expressing CD8+ and
CD3-CD56+ Cells

CD8+ T-cell cytokine expression
(perforin and granzyme A) was measured by intracellular cytokine staining and
multiparameter flow cytometry. Also, the
cytotoxicity potential of NK-cells was examined by detecting perforin
expression in phenotypically defined NK-cells (CD3-CD56+).For intracellular cytokine staining,
PBMCs(1×106) were collected in dPBS and washed once with cold dPBS
containing 1% BSA. Cells were then resuspended in 100 μL of staining buffer
(PBS supplemented with 0.1% sodium azide and 1% FBS pH 7.4) and the phenotypic
MAb (CD3, CD8, and CD56) and incubated at 4C° for 30 minutes. After
staining, the cells were washed with PBS and resuspended in 1 mL of fix/perm
buffer (BD PharMingen, San
Diego, Calif). The cells were then fixed for 30 minutes at 4C°, washed,
resuspended in 3 mL perm staining buffer and incubated with cytokine antibodies
(antiperforin, antigranzyme A) (BD
PharMingen, San Diego, Calif) in the presence of 50 uL of permeabilization buffer for 30 minutes
at 4C°. The cells were then washed with perm buffer and
resuspended in 300 μL of fixative buffer (BD PharMingen) for flow cytometric
analysis on a Becton Dicknson FACS using CELLQuest software.


### 2.5. Quantitative Viral Load Assay

HIV RNA was measured using a
quantitative reverse transcriptase polymerase chain reaction assay (Amplicor Monitor,
Roche Diagnostic System, Brandersburg,
NJ, USA). Virus from 0.2 mL of plasma was
lysed using the kit lysis buffer and the HIV RNA was precipitated
using isopropanol and pelleted by
centrifugation. After washing with ethanol, the RNA was resuspended
using the kit dilution buffer.
Extracted RNA was amplified and detected according to the manufacturer's instructions, and
results were reported as HIV
RNA copies/mL. All undetectable values (below 400 copies) were assigned a value
of 399.


### 2.6. Statistical Analysis

Data were entered and analyzed using
Windows SPSS version 11.5 (SPSS Inc., Clay, NC,
USA). Data are expressed as the means ± SD and the median. For analysis, HIV
negative and HIV positive participants were divided into high and low AF-ALB
subgroups based on the median AF-ALB level for the group. Groups were compared
by intragroup comparisons (high versus low, among either HIV positive or HIV
negative participants) and by intergroup comparisons (HIV positive versus HIV
negative participants, among either high or low AF-ALB), using Mann-Whitney
U-tests. Possible correlates of AF-ALB-associated accelerated HIV disease
progression included those indices that were similarly and significantly
associated with AF-ALB, as determined by intragroup
comparisons among both HIV positive and HIV negative participants, and with HIV
infection, as determined by at least 1 intergroup comparison among high and low
AF-ALB participants. A probability value ofP<.05 was considered
statistically significant. Correlations using nonparametric methods were also conducted
to examine the association between AF-ALB and the immune parameters for the
entire group and for HIV positive and HIV negative groups separately.


## 2.1. Study Design and Study Participants

This was a cross-sectional analysis of
116 HIV positive individuals and 80 HIV negative control participants. The HIV positive patients and HIV
negative controls of comparable age were recruited from the St. Markus Hospital
and surrounding communities in Kumasi, Ghana. The protocol for the study was
approved by the Institutional Review Board of the University of Alabama at
Birmingham (UAB) and the Medical School Ethics Committee of the Kwame Nkrumah
University of Science and Technology (KNUST) and participants gave informed consent.
The participants were asked to complete a survey on socio-demographic
characteristics, and a 20 mL blood sample was collected from each in EDTA vacutainer tubes. Plasma was separated and peripheral blood mononuclear cells
(PBMCs) were prepared using ficoll-hypaque density gradients as previously done
[26]. PBMCs were stored frozen in liquid nitrogen and shipped to UAB for
analysis.


## 2.2. Determination of AF-ALB Levels in Plasma by Radioimmunoassay

AF-ALB levels in plasma of study
participants were determined by radioimmunoassay (RIA)
as published previously [27]. Briefly, human plasma samples were concentrated
by high-speed centrifugal filtration using Microcon-50 microconcentrator with a
50000 mol. wt. filter cutoff. The concentrated protein was resuspended in
100–150 μL PBS and the amount of human plasma albumin determined in each sample
using a bromocresol purple dye binding method. In addition, the amount of total
protein was determined by the procedure of Bradford (Pierce Biotechnology Inc.,
Rockford, IL). Total protein was then digested with Pronase (Calbiochem, La
Jolla, Calif, USA; 70000 proteolytic units/g dry weight was dissolved in PBS at
10 mg/mL) at a ratio of 4:1 (Protein:pronase) in a shaking water bath (50 strokes/min) at 37C° for 16–18 hours. Digestion was stopped by cooling on ice.
Two volumes ice-cold acetone were added and the sample mixed and allowed to
remain at 4C° for 1 hour. The suspension was then centrifuged at 11000rpm (9800 g) for 15 minutes. The resulting supernatant containing the bound
aflatoxin was decanted and dried in vacuo using
a savant speed-vac concentrator. The RIA procedure was used to quantify
aflatoxin B1-albumin adducts in duplicate human plasma protein digests each
containing 2 mg protein. Nonspecific inhibition in the assay was determined by
processing pooled normal human plasma standards obtained from Sigma Aldrich
(St. Louis, MO, USA) and the average value of the background was subtracted
from the values of test samples in calculating AFB1-albumin adduct levels. The standard
curve for the RIA was determined using a nonlinear regression method [28] and
values were expressed as the amount of AFB1 per
mg albumin [27]. The detection limit of the assay was 0.01 pmol/mg albumin.


## 2.3. Determination of Percentages of Leukocyte
Immunophenotypes Using Flow Cytometry

We determined the percentages of
leukocyte immunophe-notypes in PBMCs from the study participants. The
percentages of T-cells (CD3+), subsets of T-cells (CD4+ and CD8+), B-cells
(CD19+), and NK-cells (CD3-CD56+) were measured by flow cytometry. Naive CD4
cells were defined as CD45RA+CD62L+ and memory cells as CD45RO+CD45RA-. The
expression of the costimulatory molecule CD28, activation marker HLA-DR, and
CD38 on CD4
and CD8 T-cells were also measured. CD8+ T-cell subset classification has been
proven useful in monitoring the immune system in several clinical situations
[29]. Therefore, we classified CD8+ T-cell subsets into naive (CD8+CD27+CD45RA+), memory (CD8+CD27+CD45RA-),
and effector (CD8+CD27-CD45RA+) CD8+ T-cells by flow cytometry.
Subtypes of NK-cells, CD3-CD56+CD16-, and CD3-CD56+CD16+ were determined. The
percentages of activated (marker CD69) CD3+ T-cells and CD19+ B-cells (CD3+CD69+
and CD19+CD69+), and CD4+CD25+, CD4+CD25+ CD45RO+ T regulatory cell were also
measured.PBMCs were incubated with combinations
of fluorescein FITC-, PE-, PerCP-labeled monoclonal antibodies (MAbs) against
CD3, CD4, CD8, CD16, CD19, CD25, CD27, CD38, CD45RA,
CD45RO, CD56, CD62, CD69, and HLA-DR (BD PharMingen, San Diego, Calif) for 30 minutes
at 4C°. Isotype-matched irrelevant FITC-,
PE-, and PerCP-labeld MAbs (BD PharMingen,
San Diego, Calif) were used as controls in the experiments. After washing the cells
three times in PBS, cell fluorescence for each phenotype was analyzed using
Becton Dickinson
(San Diego, Calif, USA), FACS, and CELLQuest software.


## 2.4. Determination of Cytokine Expressing CD8+ and
CD3-CD56+ Cells

CD8+ T-cell cytokine expression
(perforin and granzyme A) was measured by intracellular cytokine staining and
multiparameter flow cytometry. Also, the
cytotoxicity potential of NK-cells was examined by detecting perforin
expression in phenotypically defined NK-cells (CD3-CD56+).For intracellular cytokine staining,
PBMCs(1×106) were collected in dPBS and washed once with cold dPBS
containing 1% BSA. Cells were then resuspended in 100 μL of staining buffer
(PBS supplemented with 0.1% sodium azide and 1% FBS pH 7.4) and the phenotypic
MAb (CD3, CD8, and CD56) and incubated at 4C° for 30 minutes. After
staining, the cells were washed with PBS and resuspended in 1 mL of fix/perm
buffer (BD PharMingen, San
Diego, Calif). The cells were then fixed for 30 minutes at 4C°, washed,
resuspended in 3 mL perm staining buffer and incubated with cytokine antibodies
(antiperforin, antigranzyme A) (BD
PharMingen, San Diego, Calif) in the presence of 50 uL of permeabilization buffer for 30 minutes
at 4C°. The cells were then washed with perm buffer and
resuspended in 300 μL of fixative buffer (BD PharMingen) for flow cytometric
analysis on a Becton Dicknson FACS using CELLQuest software.


## 2.5. Quantitative Viral Load Assay

HIV RNA was measured using a
quantitative reverse transcriptase polymerase chain reaction assay (Amplicor Monitor,
Roche Diagnostic System, Brandersburg,
NJ, USA). Virus from 0.2 mL of plasma was
lysed using the kit lysis buffer and the HIV RNA was precipitated
using isopropanol and pelleted by
centrifugation. After washing with ethanol, the RNA was resuspended
using the kit dilution buffer.
Extracted RNA was amplified and detected according to the manufacturer's instructions, and
results were reported as HIV
RNA copies/mL. All undetectable values (below 400 copies) were assigned a value
of 399.


## 2.6. Statistical Analysis

Data were entered and analyzed using
Windows SPSS version 11.5 (SPSS Inc., Clay, NC,
USA). Data are expressed as the means ± SD and the median. For analysis, HIV
negative and HIV positive participants were divided into high and low AF-ALB
subgroups based on the median AF-ALB level for the group. Groups were compared
by intragroup comparisons (high versus low, among either HIV positive or HIV
negative participants) and by intergroup comparisons (HIV positive versus HIV
negative participants, among either high or low AF-ALB), using Mann-Whitney
U-tests. Possible correlates of AF-ALB-associated accelerated HIV disease
progression included those indices that were similarly and significantly
associated with AF-ALB, as determined by intragroup
comparisons among both HIV positive and HIV negative participants, and with HIV
infection, as determined by at least 1 intergroup comparison among high and low
AF-ALB participants. A probability value ofP<.05 was considered
statistically significant. Correlations using nonparametric methods were also conducted
to examine the association between AF-ALB and the immune parameters for the
entire group and for HIV positive and HIV negative groups separately.


## 3. Results

### 3.1. Demographic and Selected Clinical
Characteristics of Participants

Demographic and clinical
characteristics for the 116 HIV positive study participants and 80 HIV negative
controls are summarized in Table1. The mean age for the HIV positive group was
38.25±9.44 years and for the HIV negative
group was 40.77±17.52 years. Approximately 65% of HIV positive participants
were females compared to 44% females in the HIV negative group. AF-ALB levels
for the 196 study participants ranged from 0–3.48 pmoL/mg albumin with a mean
of 1.01±0.53 and median of 0.91 pmoL/mg albumin. The mean AF-ALB was 1.01±0.61 with median of 0.91 pmoL/mg albumin for the HIV positive group and 1.01±0.41 with median of 0.91 pmoL/mg albumin for the HIV negative control group.
Both groups of participants were divided into high AF-ALB (≥0.91)and low
AF-ALB (<0.91) subgroups based on the median AF-ALB levels. For HIV positive
participants, the mean virus load was higher among the high AF-ALB group
compared to the low AF-ALB group (85,049 versus 70,260 copies/mL), but this
difference was not statistically significant (P=.709). The mean CD4+ T-cell
count in the HIV positive participants was 307.46±248.37 cells/μL with a
range of 37–1505 cells/μL. The mean CD4+ T-cell count in the HIV negative
participants was 1099.04±454.91 cells/μL (range 472–2099 cells/μL). Based on
the CDC classification system [30], 17 HIV positive patients were placed in
category A, 52 in category B, and 47 in category C.Table 1
Demographic and clinical
characteristics of HIV positive and HIV negative study participants.
CharacteristicHIV positiveHIV negativeHigh AF-ALBLow AF-ALBHigh AF-ALBLow AF-ALBTotal number of participants58584040Age (mean ± SD years)38.28±9.6538.22±9.3039.50±18.2542.08±16.86Gender number (%)Male24 (41.4)17 (29.3)23 (57.5)22 (55)Female34 (58.6)41 (70.7)17 (42.5)18 (45)Viral load (mean ± SD copies/ml)85049±23069670260±19316300CD4 count (mean ± SD cells/μL)316±266298±2301001±2091197±614CDC classification number (%)A10 (17.3)7 (12.1)B25 (43.1)27 (46.5)C23 (39.7)24 (41.4)AF-ALB (mean ± SD pmol/mg albumin)1.44±0.560.59±0.241.31±0.360.70±0.14Aflatoxin B1
albumin adducts (AF-ALB)—Mean ± SD =1.01±0.53 pmoL/mg albumin; median = 0.91 pmoL/mg albumin;
range = 0–3.48 pmoL/mg albumin.“High AF-ALB”
participants were≥0.91 pmoL/mg
albumin; “low AF-ALB” participants were <0.91 pmoL/mg albumin.SD = standard deviation.


### 3.2. T, B, and NK Cell Phenotypes of Intergroup
and Intragroup Comparisons

Nominally,
significant inter- and intragroup differences are summarized in Figure1 and
Table 2. When intergroup comparisons were made, HIV associated differences were
mostly similar among both high AF-ALB and low AF-ALB groups. Relative and
absolute CD4+ T-cells were significantly decreased in HIV positive participants
compared to HIV negative controls (Figure 1(a)) and the proportions of CD8+ T-cells
were higher in HIV positive participants than in the negative controls (P=.000, Figure 1(b)). Differences in
lymphocyte antigen expression that were evident in the HIV positive group were
the CD28+ and HLA-DR+CD38+ percentages within both the CD4+ and CD8+ lymphocyte
populations (all P≤.001, Table 2, Figures 1(c), 1(d), 1(i),
and 1(j)). There were fewer CD4+CD25+
and CD4+CD25+CD45RO+ regulatory T-cells in HIV positive participants than HIV
negative controls among both high and low AF-ALB groups (Table 2, Figure 1(k). The difference was statistically
significant among the high AF-ALB group (P=.000) and was not statistically
significant among the low AF-ALB group (P=.061).Table 2
Summary
of the immune indices in which significant differences (P value) were
identified by intra- and intergroup comparisons among high and low aflatoxin B1 albumin adduct (AF-ALB) and HIV positive and HIV negative participants.
Cell subset (percentage)Intergroup comparisonIntragroup comparisonHigh AF-ALBLow AF-ALBHIV positiveHIV negativeHIV positive versus negativeHIV positive versus negativeHigh versus Low AF-ALBHigh versus Low AF-ALBLymphocytes (count)↓ 0.003↓ 0.045CD4+ cells↓ 0.000↓ 0.000CD4+ cells (count)↓ 0.000↓ 0.000CD4+CD28+↓ 0.000↓ 0.000CD45RA+CD62L+CD4+↓ 0.029CD45RO+CD45RA-CD4+CD4+HLA-DR+CD38+↑ 0.000↑ 0.000CD4+CD25+↓ 0.001↓ 0.016CD4+CD25+CD45RO+↓ 0.000↓ 0.061↓ 0.009CD4+CD25-CD45RO-↑ 0.000↑ 0.000CD4+CD25-CD45RO+↑ 0.000↑ 0.000CD8↑ 0.000↑ 0.000CD8+CD28+↓ 0.000↓ 0.000↑ 0.022CD45RA+CD62L+CD8+↓ 0.017↓ 0.003CD45RO+CD45RA-CD8+↑ 0.006↑ 0.002CD8+HLA-DR+CD38+↑ 0.000↑ 0.000CD8+CD27-CD45RA+↑ 0.038CD8+CD27+CD45RA+↓ 0.03CD8+CD27-CD45RA-CD8+Perforin+↑ 0.000↑ 0.000↓ 0.012↓ 0.008CD8+Granzyme A+↑ 0.000↑ 0.003CD8+Granzyme A+Perforin+↑ 0.000↑ 0.002↓ 0.010CD3-CD19+↓ 0.03CD19+CD69+↓ 0.000↓ 0.027CD3+CD3+CD69+↓ 0.024CD3-CD56+CD3-CD16+CD56+↑ 0.052CD3+CD56+PerforinPercentages of T-, B-, and NK-cells in PBMCs in relation to aflatoxin B1 albumin adduct (AF-ALB) levels and HIV
infection. The
percentages are shown for HIV positive high AF-ALB(n=58) and low AF-ALB (n=58) groups, and HIV negative high AF-ALB (n=40) and
low AF-ALB (n=40) groups.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)HIV infection was associated with less naive
CD8+ (CD45RA+CD62L+CD8+) T-cells (Table2, Figure 1(f)), and more memory CD8+ (CD45RO+CD45RA-CD8+)
T-cells (Table 2, Figure 1(h)) among both high AF-ALB (P=.017,P=.006) and low AF-ALB
groups (P=.003,P=.002). There were more CD8+CD27-CD45RA+ cells
in HIV positive participants than HIV negative controls (Table 2, Figure 1(l)) among
the low AF-ALB group (P=.038) and less CD8+CD27+CD45RA+ cells among the
high AF-ALB group (P=.03, Table 2). When CD8+ cells were analyzed for
the presence of intracellular perforin and granzyme A without stimulation, we
found that the percentages of CD8+ T-cells containing both perforin and
granzyme A were statistically significantly higher in HIV positive participants
with high AF-ALB (P=.000) and low AF-ALB (P≤.003) compared with HIV negative
controls (Table 2, Figures 1(m) and 1(n)).There was lower CD69 expression on
CD19+ B-cells in HIV positive participants among both high and low AF-ALB group
(Table2, Figure 1(p)), but this difference was only statistically significant
in the low AF-ALB group (P=.000).
No significant HIV associated difference in CD3-CD56+ and perforin expressing
NK-cells were apparent by intergroup comparison among either the high AF-ALB or
low AB-ALB groups. With the exception of CD19+CD69+,
CD8+CD27-CD45RA+, and CD8+CD27+CD45RA+ cells, many of the significant T-cell
perturbations that were associated with HIV infection in high AF-ALB HIV
positive participants were present in their low AF-ALB counterparts.When intragroup comparisons were made,
aflatoxin associated differences depended in part on the HIV serostatus of the
participants. Among both the HIV
positive and HIV negative participants, higher AF-ALB was associated with lower
expression of perforin on CD8+
T-cells (Table2, Figure 1(m)). Lower perforin and granzyme A expressing CD8+ T-cells also were seen in both groups (Table 2, Figure 1(n)), but the difference
was only statistically significant for the HIV negative groups (P=.01).
Additional aflatoxin associated differences among HIV positive participants
included lower percentage of
CD4+CD25+CD45RO+ regulatory T-cells (P=.009), which was associated with HIV
infection in both high and low AF-ALB
HIV positive participants (Table 2, Figure 1(k)), and lower percentage
of naive CD4+ (CD4+CD45RA+CD62L+) T-cells (Table 2, Figure 1(e)) in the high
AF-ALB group.AF-ALB
associated reduction in the B-cells was apparent in HIV positive participants (P=.03)
but not in HIV negative participants (Table 2, Figure 1(o)). High AF-ALB associated
differences among HIV negative controls included less CD69 expression on both
CD3+ T-cells (P=.024) and CD19+ B-cells (P=.027) (Table 2, Figure 1(p)).No significant aflatoxin-related differences in NK (CD3-CD56+
or CD3-CD56+CD16+) cells and perforin expressing NK (CD3-CD56+Perforin) cells were
apparent by intragroup comparisons among either the HIV positive or the HIV
negative participants (Table2, Figures 1(q) and 1(r)).When we conducted correlation analyses
between AF-ALB and the immune parameters among HIV positive participants, we
found significant correlations between AF-ALB and perforin-expressing CD8+ T-cells(r=−0.170;P=.045), T-regulatory cells (r=−0.395;P=.002),
and B-cells (r=−0.212;P=.012). These findings are similar to our
findings above. Also our results were consistent with those presented above for
the entire study group and for HIV negative participants.


## 3.1. Demographic and Selected Clinical
Characteristics of Participants

Demographic and clinical
characteristics for the 116 HIV positive study participants and 80 HIV negative
controls are summarized in Table1. The mean age for the HIV positive group was
38.25±9.44 years and for the HIV negative
group was 40.77±17.52 years. Approximately 65% of HIV positive participants
were females compared to 44% females in the HIV negative group. AF-ALB levels
for the 196 study participants ranged from 0–3.48 pmoL/mg albumin with a mean
of 1.01±0.53 and median of 0.91 pmoL/mg albumin. The mean AF-ALB was 1.01±0.61 with median of 0.91 pmoL/mg albumin for the HIV positive group and 1.01±0.41 with median of 0.91 pmoL/mg albumin for the HIV negative control group.
Both groups of participants were divided into high AF-ALB (≥0.91)and low
AF-ALB (<0.91) subgroups based on the median AF-ALB levels. For HIV positive
participants, the mean virus load was higher among the high AF-ALB group
compared to the low AF-ALB group (85,049 versus 70,260 copies/mL), but this
difference was not statistically significant (P=.709). The mean CD4+ T-cell
count in the HIV positive participants was 307.46±248.37 cells/μL with a
range of 37–1505 cells/μL. The mean CD4+ T-cell count in the HIV negative
participants was 1099.04±454.91 cells/μL (range 472–2099 cells/μL). Based on
the CDC classification system [30], 17 HIV positive patients were placed in
category A, 52 in category B, and 47 in category C.Table 1
Demographic and clinical
characteristics of HIV positive and HIV negative study participants.
CharacteristicHIV positiveHIV negativeHigh AF-ALBLow AF-ALBHigh AF-ALBLow AF-ALBTotal number of participants58584040Age (mean ± SD years)38.28±9.6538.22±9.3039.50±18.2542.08±16.86Gender number (%)Male24 (41.4)17 (29.3)23 (57.5)22 (55)Female34 (58.6)41 (70.7)17 (42.5)18 (45)Viral load (mean ± SD copies/ml)85049±23069670260±19316300CD4 count (mean ± SD cells/μL)316±266298±2301001±2091197±614CDC classification number (%)A10 (17.3)7 (12.1)B25 (43.1)27 (46.5)C23 (39.7)24 (41.4)AF-ALB (mean ± SD pmol/mg albumin)1.44±0.560.59±0.241.31±0.360.70±0.14Aflatoxin B1
albumin adducts (AF-ALB)—Mean ± SD =1.01±0.53 pmoL/mg albumin; median = 0.91 pmoL/mg albumin;
range = 0–3.48 pmoL/mg albumin.“High AF-ALB”
participants were≥0.91 pmoL/mg
albumin; “low AF-ALB” participants were <0.91 pmoL/mg albumin.SD = standard deviation.


## 3.2. T, B, and NK Cell Phenotypes of Intergroup
and Intragroup Comparisons

Nominally,
significant inter- and intragroup differences are summarized in Figure1 and
Table 2. When intergroup comparisons were made, HIV associated differences were
mostly similar among both high AF-ALB and low AF-ALB groups. Relative and
absolute CD4+ T-cells were significantly decreased in HIV positive participants
compared to HIV negative controls (Figure 1(a)) and the proportions of CD8+ T-cells
were higher in HIV positive participants than in the negative controls (P=.000, Figure 1(b)). Differences in
lymphocyte antigen expression that were evident in the HIV positive group were
the CD28+ and HLA-DR+CD38+ percentages within both the CD4+ and CD8+ lymphocyte
populations (all P≤.001, Table 2, Figures 1(c), 1(d), 1(i),
and 1(j)). There were fewer CD4+CD25+
and CD4+CD25+CD45RO+ regulatory T-cells in HIV positive participants than HIV
negative controls among both high and low AF-ALB groups (Table 2, Figure 1(k). The difference was statistically
significant among the high AF-ALB group (P=.000) and was not statistically
significant among the low AF-ALB group (P=.061).Table 2
Summary
of the immune indices in which significant differences (P value) were
identified by intra- and intergroup comparisons among high and low aflatoxin B1 albumin adduct (AF-ALB) and HIV positive and HIV negative participants.
Cell subset (percentage)Intergroup comparisonIntragroup comparisonHigh AF-ALBLow AF-ALBHIV positiveHIV negativeHIV positive versus negativeHIV positive versus negativeHigh versus Low AF-ALBHigh versus Low AF-ALBLymphocytes (count)↓ 0.003↓ 0.045CD4+ cells↓ 0.000↓ 0.000CD4+ cells (count)↓ 0.000↓ 0.000CD4+CD28+↓ 0.000↓ 0.000CD45RA+CD62L+CD4+↓ 0.029CD45RO+CD45RA-CD4+CD4+HLA-DR+CD38+↑ 0.000↑ 0.000CD4+CD25+↓ 0.001↓ 0.016CD4+CD25+CD45RO+↓ 0.000↓ 0.061↓ 0.009CD4+CD25-CD45RO-↑ 0.000↑ 0.000CD4+CD25-CD45RO+↑ 0.000↑ 0.000CD8↑ 0.000↑ 0.000CD8+CD28+↓ 0.000↓ 0.000↑ 0.022CD45RA+CD62L+CD8+↓ 0.017↓ 0.003CD45RO+CD45RA-CD8+↑ 0.006↑ 0.002CD8+HLA-DR+CD38+↑ 0.000↑ 0.000CD8+CD27-CD45RA+↑ 0.038CD8+CD27+CD45RA+↓ 0.03CD8+CD27-CD45RA-CD8+Perforin+↑ 0.000↑ 0.000↓ 0.012↓ 0.008CD8+Granzyme A+↑ 0.000↑ 0.003CD8+Granzyme A+Perforin+↑ 0.000↑ 0.002↓ 0.010CD3-CD19+↓ 0.03CD19+CD69+↓ 0.000↓ 0.027CD3+CD3+CD69+↓ 0.024CD3-CD56+CD3-CD16+CD56+↑ 0.052CD3+CD56+PerforinPercentages of T-, B-, and NK-cells in PBMCs in relation to aflatoxin B1 albumin adduct (AF-ALB) levels and HIV
infection. The
percentages are shown for HIV positive high AF-ALB(n=58) and low AF-ALB (n=58) groups, and HIV negative high AF-ALB (n=40) and
low AF-ALB (n=40) groups.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)HIV infection was associated with less naive
CD8+ (CD45RA+CD62L+CD8+) T-cells (Table2, Figure 1(f)), and more memory CD8+ (CD45RO+CD45RA-CD8+)
T-cells (Table 2, Figure 1(h)) among both high AF-ALB (P=.017,P=.006) and low AF-ALB
groups (P=.003,P=.002). There were more CD8+CD27-CD45RA+ cells
in HIV positive participants than HIV negative controls (Table 2, Figure 1(l)) among
the low AF-ALB group (P=.038) and less CD8+CD27+CD45RA+ cells among the
high AF-ALB group (P=.03, Table 2). When CD8+ cells were analyzed for
the presence of intracellular perforin and granzyme A without stimulation, we
found that the percentages of CD8+ T-cells containing both perforin and
granzyme A were statistically significantly higher in HIV positive participants
with high AF-ALB (P=.000) and low AF-ALB (P≤.003) compared with HIV negative
controls (Table 2, Figures 1(m) and 1(n)).There was lower CD69 expression on
CD19+ B-cells in HIV positive participants among both high and low AF-ALB group
(Table2, Figure 1(p)), but this difference was only statistically significant
in the low AF-ALB group (P=.000).
No significant HIV associated difference in CD3-CD56+ and perforin expressing
NK-cells were apparent by intergroup comparison among either the high AF-ALB or
low AB-ALB groups. With the exception of CD19+CD69+,
CD8+CD27-CD45RA+, and CD8+CD27+CD45RA+ cells, many of the significant T-cell
perturbations that were associated with HIV infection in high AF-ALB HIV
positive participants were present in their low AF-ALB counterparts.When intragroup comparisons were made,
aflatoxin associated differences depended in part on the HIV serostatus of the
participants. Among both the HIV
positive and HIV negative participants, higher AF-ALB was associated with lower
expression of perforin on CD8+
T-cells (Table2, Figure 1(m)). Lower perforin and granzyme A expressing CD8+ T-cells also were seen in both groups (Table 2, Figure 1(n)), but the difference
was only statistically significant for the HIV negative groups (P=.01).
Additional aflatoxin associated differences among HIV positive participants
included lower percentage of
CD4+CD25+CD45RO+ regulatory T-cells (P=.009), which was associated with HIV
infection in both high and low AF-ALB
HIV positive participants (Table 2, Figure 1(k)), and lower percentage
of naive CD4+ (CD4+CD45RA+CD62L+) T-cells (Table 2, Figure 1(e)) in the high
AF-ALB group.AF-ALB
associated reduction in the B-cells was apparent in HIV positive participants (P=.03)
but not in HIV negative participants (Table 2, Figure 1(o)). High AF-ALB associated
differences among HIV negative controls included less CD69 expression on both
CD3+ T-cells (P=.024) and CD19+ B-cells (P=.027) (Table 2, Figure 1(p)).No significant aflatoxin-related differences in NK (CD3-CD56+
or CD3-CD56+CD16+) cells and perforin expressing NK (CD3-CD56+Perforin) cells were
apparent by intragroup comparisons among either the HIV positive or the HIV
negative participants (Table2, Figures 1(q) and 1(r)).When we conducted correlation analyses
between AF-ALB and the immune parameters among HIV positive participants, we
found significant correlations between AF-ALB and perforin-expressing CD8+ T-cells(r=−0.170;P=.045), T-regulatory cells (r=−0.395;P=.002),
and B-cells (r=−0.212;P=.012). These findings are similar to our
findings above. Also our results were consistent with those presented above for
the entire study group and for HIV negative participants.


## 4. Discussion

To identify possible correlates that
may underlie the interaction of AF-ALB with HIV disease progression, we sought
to identify immune perturbations that are common to both conditions. This study
demonstrated, for the first time, associations of aflatoxin with immune
parameters in HIV-infected people.The changes observed in CD3+, CD4+, and
CD8+ T-cell phenotypes, CD19+ B-cells and CD3-CD56+ NK-cells in HIV positive
compared to HIV negative participants (intergroup comparison) in this study are
consistent with previously well-characterized, HIV-associated changes in these
cells. HIV-associated immune perturbations were largely similar in participants
with high and low AF-ALB levels. HIV infection induced a decrease in CD4+ T-cell
numbers and concomitantly activated the immune system. HIV infection was
associated with greater expression of HLA-DR/CD38 and lower expression of CD28
on CD4+ and CD8+ T-cells. We found that the surface expression of HLA-DR/CD38
in both CD4+ and CD8+ T-cells was significantly increased in HIV positive participants.
The means of CD4+ and CD8+ cells expressing HLA-DR/CD38 progressively increased
with advancing clinical disease as determined by CDC stage (data not shown).
Also there was a strong negative correlation between both CD4+ T-cell
percentage and CD4+ T-cell count with HLA-DR+CD38+ expressing CD4+ T-cells
(data is not shown). This activated immune phenotype has been extensively
validated in prior studies and demonstrates T-cell activation to be a strong
prognostic indicator for progression to AIDS [31–33]. T-cell activation is
believed to be the major cause of CD4+ T-cell depletion in HIV infection,
through a progression of activation-induced cell death (AICD) [34–37]. The
increased immune activation together with increased viral replication causes
severe depletion of CD4+ T-cells, eventually leading to the development of
AIDS. Therefore, our data suggest that HLA-DR/CD38 could be used as a
progression marker in HIV-infected Ghanaians as in HIV-infected North Americans
[38].In the present study, the principal
costimulatory molecule CD28 has been uniformly downmodulated in CD4+ and CD8+ T-cells
in HIV infected participants. Engagement of the CD28
molecule on CD8+ T lymphocytes in HIV positive individuals during activation
has been
reported to increase CD8+ T-cell proliferation and differentiation and to
prevent apoptosis [39–41]. T-cell receptor stimulation in the absence of CD28
often leads to anergy and to cell death via apoptosis in HIV-infected patients
[42]. Many studies have suggested that HIV induces dysfunction of CD4+ and CD8+
cells by CD28 downregulation [43, 44]. The
loss of CD28 expression on CD4+ and CD8+ cells in HIV infected participants may
be associated with the functional defect of the T-cells and progression to
AIDS.CD8+ T-cells are very important
lymphocyte subsets in the immune response against HIV infection. Naive CD8+ T-cells
can differentiate into effector-type CD8+ T-cells after they have recognized
MHC-matched antigens and then express cytolytic molecules, such as perforin and
granzymes which are stored in intracellular granules. Therefore, perforin is
expressed in antigen-primed CD8+ T-cells with a cytolytic activity potential
that contribute to the inhibition of pathogen spread through immediate lysis of
infected cells [45]. In our study, the percentage of naive CD8+ T-cells in HIV
infected participants was lower than in HIV negative controls, and the
percentage of perforin expressing CD8+ T-cells was significantly increased in
the HIV-infected participants than in HIV
negative controls. Our observation is consistent with other reports [33, 34].
Overexpression of perforin in HIV-infected participants may be the consequence
of CD8+ T-cell hyperactivation and expansion as a part of feedback regulation of
anti-HIV cytotoxic T-lymphocyte (CTL) activity [46, 47]. However, among both
the HIV infected and HIV negative control groups, those with high AF-ALB showed
a lower percentage of perforin expressing CD8+ T-cells compared to those with
low AF-ALB levels. This may indicate that CD8+ T-cells synthesizing perforin to
enhance the CTL response are impaired in individuals with high AF-ALB [22].
Thus cellular immune function against infectious diseases, such HIV infection
will be affected.T-regulatory cells (Tregs) represent 5–10%
of peripheral CD4+ T-cells in healthy individuals and are characterized by
constitutive expression of CD25+ and CD45RO+. Tregs (CD4+CD25+CD45RO+)
have been implicated in controlling responses to chronic pathogens [48–50]
and are known to profoundly inhibit both CD4+ and CD8+ T-cell activation,
proliferation, and effector function, although the mechanism of this inhibition
remains unclear. Thus Tregs may play a critical role in limiting
immunopathology that results from persistent high level immune stimulation from
chronic viral infections [51]. We evaluated this population of cells in HIV
positive and HIV negative participants for both high and low AF-ALB
groups. We found that CD4+CD25+CD45RA+
Tregs showed a tendency to decrease in HIV
infected participants with both high and low AF-ALB level compared to HIV
negative controls. In addition, we found that the HIV positive group with high
AF-ALB had the lowest percentage of Tregs of all the groups suggesting that
there is a loss of Tregs in HIV infected participants with high AF-ALB. This
loss may facilitate the immune hyperactivation associated with HIV and lead to
more severe disease in those with high aflatoxin levels.The activation molecule CD69 is a
costimulatory molecule for lymphocyte proliferation. It is expressed early on
the membranes of T- and B-lymphocytes through the stimulated antigen
receptor/CD3 complex or cross-linking of surface immunoglobulins, respectively.
T-lymphocyte activation via CD69 will progress to proliferation, thus
amplifying immune responses [52]. In our present study, the levels of CD69
expression on CD3+ subsets did not differ between the HIV positive and HIV
negative control groups. This supports the observation by Krowka et al. and
Böhler et al. [53, 54] that unstimulated T-cells from HIV- infected
and negative control individuals exhibited similar levels of CD69 expression [53, 54]. The reduced proportions of T-lymphocytes coexpressing CD69 were seen only
upon mitogen
stimulation in HIV-infected adults [53]. Among HIV negative controls, the
significantly decreased percentage of CD69 on CD3+ T-cells was found in the
high AF-ALB group compared to the low AF-ALB group as reported before [22].
This could partially contribute to the inability of these cells to mount
appropriate immune responses. However, this tendency was not seen when we
compared the high AF-ALB group to the low AF-ALB group among HIV positive
participants. This finding  probably
reflects the coexistence of immune activation as shown by increased HLA-DR and
CD38 expressing CD4+ and CD8+ T-cells, and immune deficiency as shown by reduced
numbers of CD4+ lymphocytes, and impaired T- lymphocyte proliferation in
HIV-infected individuals [55]. It has
been reported that there is the tendency for CD69 expression on unstimulated
peripheral blood T-cells to increase with plasma HIV viral load [56]. Therefore, the variability in plasma HIV viral load level may also contribute
to our observation.In keeping with findings of a previous
study [22], we found that high AF-ALB was associated with the reduced
percentages of unstimulated CD19+ B-cells coexpressing CD69 in both HIV
infected and control groups, but only the difference for the HIV negative
groups reached statistical significance. HIV infection was also associated with
decreased CD19+CD69+ B-cells in both groups but only the difference for the low
AF-ALB group reached statistical significance. No previous data exist
concerning the CD69 expressing B-cell in HIV-infected adults. However, there
are some reports that have shown that HIV-induced B-cell hyperactivity is associated
with reduction of CD21 [57], and CD27 [58, 59] expression in HIV
infected patients. Ginaldi et al. [60] reported that B-cells from HIV infected
patients show a significantly lower number of HLA-DR molecule per cell compared
to normal controls. Our study shows that
the ability of B-cell to express CD69 has been impaired in both high AF-ALB
exposure and HIV infection. This could suggest that AF-ALB may accentuate the
changes in HIV-related immune parameters.High aflatoxin appeared to accentuate
some HIV-associated changes in T-cell phenotypes and B-cells in HIV-infected
participants. Because of the cross-sectional design of this study, however, we
cannot exclude the possibility that these intragroup differences may simply
reflect an imbalance in the severity of HIV disease, with more advanced disease
(not evident by our CDC classification) in the high AF-ALB group. However,
despite these limitations, we believe that this study has a potentially
significant impact on understanding the effects of aflatoxin in HIV disease
progression.A better understanding of the
interaction of high aflatoxin with HIV disease might provide insight into key
mechanisms that underlie the immunopathogenesis of both processes. Potential
immune correlates of this interaction may include reduced Tregs, impaired CD8+
T-cell function, plus impaired B-cell CD69-expressing ability. In HIV-infected
patients with higher AF-ALB, loss of Tregs, and persistent immune activation
might lead to exhaustion of the naive CD8+ T-cell pool, uniform downmodulated
of the principal costimulatory molecule CD28
on CD8+ and CD4+ cells, impaired function of CD8+ T-cells synthesizing perforin
(which results in failure of cytolysis by CD8+ T-cells), plus impaired ability
of B-cell to express CD69. This will result in increased viral replication and
increase in the occurrence of viral escape mutants. Because of viral escape,
viral load will increase resulting in further loss of functional HIV specific
CD4+ T-cell and a progressive deterioration of the immune system.Given
these observations, high aflatoxin may promote more rapid HIV disease
progression in HIV-infected Ghanaians. Our findings should be considered
exploratory, given the cross-sectional design of this study and
because there may be other variables not considered that may
affect immune cell distribution and function. Further clarification of these
and other possible immune correlates of the interaction of high aflatoxin with
HIV disease progression might be afforded by additional investigation.


## 5. Conclusion

High AF-ALB appeared to accentuate some
HIV-associated changes in T-cell phenotypes and B-cells in HIV positive
participants. These results may indicate that CD8+ T-cells synthesizing
perforin to enhance the CTL response are impaired in individuals with high AF- ALB.
The loss of Tregs in HIV positive participants with high AF-ALB may facilitate
HIV associated immune hyperactivation and lead to more severe disease.



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*Source: 790309-2008-08-05.xml*