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Reversal of perfluorooctanesulfonate- caused immunotoxicity by glucan-resveratrol-vitamin C combination


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Perfluorinated compounds (PFCs) perfluoroctanic acid (PFOA) and perfluooctane sulphonic acid (PFOS) are environmentally widespread bioaccumulative chemicals known to induce profound effects on the immune system. In this study, two types of PFC, i.e., PFOS and PFOA, were administered orally (daily) a dose of 20 mg/kg/d. After exposure, all mice exhibited significant immunosuppressive effects upon both cellular (phagocytosis and NK cell activity) and humoral (antibody response) branches of their immune responses. The mice were then fed with a 4 mg/kg/d dose of a combination of resveratrol-glucan-vitamin C (RVB 300). The results showed that treatment with PFCs and RVB 300 resulted in significantly lower level of immunotoxic effects from PFCs. These outcomes suggest to us that RVB 300 can potentially be successfully used as a natural remedy against immunotoxicities induced by low-level exposure(s) to perfluorinated compounds.
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1 23
Oriental Pharmacy and Experimental
ISSN 1598-2386
Volume 13
Number 1
Orient Pharm Exp Med (2013) 13:77-84
DOI 10.1007/s13596-013-0105-7
Reversal of perfluorooctanesulfonate-
induced immunotoxicity by a glucan-
resveratrol-vitamin C combination
V.Vetvicka & J.Vetvickova
1 23
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Reversal of perfluorooctanesulfonate-induced
immunotoxicity by a glucan-resveratrol-vitamin C
V. Vetvicka &J. Vetvickova
Received: 21 August 2012 /Accepted: 10 January 2013 / Published online: 29 January 2013
#Institute of Oriental Medicine, Kyung Hee University 2013
Abstract Perfluorinated compounds (PFCs) perfluoroctanic
acid (PFOA) and perfluooctane sulphonic acid (PFOS) are
environmentally widespread bioaccumulative chemicals
known to induce profound effects on the immune system. In
this study, two types of PFC, i.e., PFOS and PFOA, were
administered orally (daily) a dose of 20 mg/kg/d. After expo-
sure, all mice exhibited significant immunosuppressive effects
upon both cellular (phagocytosis and NK cell activity) and
humoral (antibody response) branches of their immune
responses. The mice were then fed with a 4 mg/kg/d dose of
a combination of resveratrol-glucan-vitamin C (RVB 300).
The results showed that treatment with PFCs and RVB 300
resulted in significantly lower level of immunotoxic effects
from PFCs. These outcomes suggest to us that RVB 300 can
potentially be successfully used as a natural remedy against
immunotoxicities induced by low-level exposure(s) to per-
fluorinated compounds.
Keywords Glucan .Immunotoxicity .Phagocytosis .
Antibodies .PFOS .PFOA
Perfluorinated hydrocarbons (PFC) are used in thousands of
products. Two major classes are perfluorinated carboxylates
like perfluoroctanic acid (PFOA) and perfluoroctane sulfo-
nates such as perfluooctane sulfonic acid (PFOS). These com-
pounds have an extremely long lifetime in the environment
and are detectable in the blood of both animals and humans
(Lau et al. 2007). A study of people with high exposure to
perfluorinated compounds (as food contaminants) indicated
that there was a significant reduction in humoral immune
responses to immunizations (Grandjean et al. 2012). Animal
studies noted severe suppression of both humoral and cellular
immunity caused by exposure to these agents (Peden-Adams
et al. 2007). Among the reactions suppressed by chronic
exposure to this class of agents were: natural killer (NK) cell
activity; lymphocyte proliferation and antibody responses
(Dong et al. 2009; Brieger et al. 2011); release of tumor
necrosis factor (TNF)-α(Brieger et al. 2011); and, secretion
of interleukins (IL)-6 and 10 (Corsini et al. 2012). Wang et
al. (2011) reported induction of thymic and splenic hyper-
trophy. Qazi et al. (2009) indicated there was an exposure-
related decrease in levels circulating neutrophils. For a full
review of the immunotoxicologic effects of PFC compounds,
the reader is directed to De Witt et al. (2012).
While most of the current literature is focused on the
description of immunotoxic effects of PFCs, there is little
known about the possible blocking of these effects. Studies
showing the strong potential of glucans to help overcome
immunosuppressive effects induced by immuno- modulat-
ing processes such irradiation or chemotherapy (Patchen
and MacVittie 1982; Vetvicka et al. 2007b; Vetvicka and
Vetvickova 2009) led us to the hypothesis evaluated herein.
Specific- ally, this study sought to first compare the type-
s/degrees of immunosuppression caused by either PFOS or
PFOA and to then to evaluate if any suppression could be
reversed by a therapy that utilized a glucan-resveratrol-
Vitamin C combination.
The choice of this particular combination to examine was
based on our own earlier studies demonstrating significant
stimulation of both cellular and humoral branches of the
immune response using a combination of glucan and humic
acid (Vetvicka et al. 2010). Another study showed that both
glucan and resveratrol stimulated phagocytosis, increased
splenic cell expression of CD4, and induced restoration of
V. Vetvicka (*):J. Vetvickova
Department of Pathology, University of Louisville School of
Medicine, 511 S. Floyd, MDR Bldg., Room 224,
Louisville, KY 40202, USA
Orient Pharm Exp Med (2013) 13:7784
DOI 10.1007/s13596-013-0105-7
Author's personal copy
splenic population profiles after an experiment- ally-induced
leukopenia (Vetvicka et al. 2007a). When effects of all of
these substances on the expression of some important genes
(i.e., NF-κB2 and Cdc42) in breast cancer cells were mea-
sured, up-regulation of Cdc42 expression was evident only
with the use of both immuno- modulators combined
(Vetvicka et al. 2007b; Vetvicka and Vancikova 2010).
Follow-up studies showed that, compared to each compo-
nent alone, a glucan-resveratrol-Vitamin C combination
caused the strongest inhibition of experimentally-induced
lung and breast cancers (Vetvicka and Vetvickova 2012a).
Materials and methods
Female BALB/c mice (8-wk-old) were purchased from the
Jackson Laboratory (Bar Harbor, ME). All mice were
housed in a sterile animal facility that was maintained at
22 °C and at a 35 % relative humidity, with a 12-hr light/-
dark cycle. All mice had ad libitum access to standard
mouse chow and filtered water throughout the studies. All
mice were allowed to accli- mate for a minimum of 10 day
prior to any exposure being performed. All mice were
weighed prior to the first dosing and again daily to assure
that dosing volumes were constant over the course of the
treatments. All animal work was done according to a
University of Louisville IACUC protocol and with the ap-
proval of that committee IACUC #04050.
RVB 300 mixture containing resveratrol, yeast-derived glu-
can #300, and Vitamin C (at a ratio of 2:2:1 [w/w/w]) was
purchased from Restart Your Life (Lexington, SC). RPMI
1640 medium, Freunds adjuvant, tri-nitrophenol (TNP),
bovine γ-globulin, MTT ([3-(4,5-dimethyl- thiazol-2-yl)-
2,5-diphenyltetrazolium bromide]), bovine serum albumin
(BSA), Concanavalin A (ConA), lipopolysaccharide (LPS),
sodium dodecyl sulfate, perfluorooctanesulfonate (PFOS;
85 % purity), and perfluorooctanoic acid (PFOA; 96 %
purity) were all purchasedfromSigma(St.Louis,
MO). Fetal calf serum (FCS) was obtained from
Hyclone Laboratories (Logan, UT).
Each treatment group in these studies contained five mice.
Some mice received an oral (non-sedated gavage) dosing of
PFOS or PFOA in phosphate-buffered saline (PBS; pH 7.4) at a
dose of 20 mg/kg/day. Control mice received PBS only. Some
mice (with and without any PFC treatments) also received 4 mg
RVB 300/kg/d by oral gavage; these treatments occurred with
the PFC or PBS instillation on each day. All treatments were for
7 day (except the antibody production) and all experiments
were repeated three times. For all of the protocols outlined
below (except those pertaining to antibody formation), 1 day
after the final treatment, all mice were euthanized by CO
asphyxiation and their blood/organs (i.e., spleen, thymus) were
aseptically removed for analyses. Blood was isolated from the
orbital vein into 5 ml plastic tubes (Fisher, Pittsburgh, PA) and
then placed at room temperature to allow for clotting. Serum
was subsequently isolated and stored; the cells were then used
in some of the protocols outlined below. The organs were blot
dried, trimmed of extraneous tissue matter, and weighed; each
value was normalized to body weight and reported as somatic
index (i.e., 100 × organ weight/body weight).
The phagocytic activity of peripheral blood cells (phagocytes)
in the blood samples iso- lated above was assessed using a
technique described in Vetvicka et al. (1982,1988). Briefly,
peripheral blood cells (0.1 ml) were combined with 50 μlofa
suspension of 2-hydroxyethyl methacrylate particles (HEMA;
/ml) and then incubated 60 min at 37 °C, with intermit-
tent gentle shaking. Thereafter, the samples were prepared as
smears on glass slides that were, in turn, treated with Wright
stain. Cells were then examined using a CK 30 microscope
(Olympus, Pittsburgh, PA); cells with three or more HEMA
particles were considered as positive for phagocytic activity.
At least 200 cells in 60 high-power fields were examined per
slide; five slides/animal were analyzed.
Lymphocyte proliferation assay
Splenocytes were prepared from each harvested organ using a
standard protocol (Fernandez-Botran and Vetvicka 1995). Only
preparations containing cells with viability >95 % were used in
the experiments. Ultimately, isolated splenocytes were washed
three times in RPMI 1640 medium supplemented with 10 %
FCS and re-suspended at 5 ×10
cells/ml. An aliquot (100 μl)
of each suspension was added to triplicate wells of a 96-well
plate that contained either ConA or LPS (each at 10 μg/ml, final
concentration after cell addition) or medium (negative control).
The plates were then incubated for 48 h at 37 °C in a humidified
incubator. Thereafter, 10 μl of MTT (5 mg/ml) was
added to each well, and the plates were incubated a further 4 h
before 100 μl of 20 % sodium dodecyl sulfate (w/v) solution
was added to each well. After incubation overnight at 37 °C, the
plates were placed in an ELISA reader (Tecan, Research
Triangle Park, NC) and the absorbance values in each well
monitored at 570 nm (OD). The proliferation index was then
calculated as: (OD
-stimulated cells/OD non-stimulated
cells) or (OD
-stimulated cells/OD non-stimulated cells).
78 V. Vetvicka, J. Vetvickova
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Ex vivo cytotoxicity assay
Spleen cells from the above preparations were also used for
assessments of ex vivo cytotoxic activity. YAC-1 cells
(obtained from ATCC; Manassas, VA) were grown in
RPMI 1640 supplemented with 10 % FCS (without anti-
biotics) and then labeled as outlined in Vetvicka et al.
(2007a). Splenocytes (10
/ml stocks) were then placed as
100 μl aliquots into V-shaped 96-well microplates and in-
cubated with 50 μl of the labeled YAC-1 target cells, at an
effector- target ratio of 64:1. After gently centrifuging at 250
× g for 5 min, the plates were incubated for 4 h at 37 °C. The
cytotoxic activity of the cells was then determined using a
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega,
Madison, WI), following manufacturer instruc- tions.
Briefly, 10 μl of lysis solution was added to select wells (i.e.,
those for determination of maximum killing potential) 45 min
before the end of the incubation period. The plates were then
centrifuged at 250 × g for 5 min and 50 μl of culture superna-
tant from each well was then trans- ferred to corresponding
wells of a flat-bottom 96-well microplate. Kit-provided reac-
tion sub- strate (50 μl) was then added to each well, and the
plate was covered and incubated (in dark) for 30 min at room
temperature. The optical density (at 492 nm) in each well was
then determined using the ELISA plate-reader. Specific cell-
mediated cytotoxicity was ultimately calculated via the formu-
la: Percent-specific killing (% cytotoxicity) =100 × [(OD
)], where spontaneous release was
obtained from target cells incubated with medium alone and
maximum release was obtained from target cells lysed with the
solution provided in the kit.
Splenic and thymic cellularity
To determine cellularity in two key immune system organs in
the mice, spleen and thymuses were suspended in RPMI 1640
and single cell suspensions were prepared using a steel mesh.
The suspension was then washed twice with cold medium and
any erythrocytes present were lysed with a 0.74 % (w/v)
ammonium chloride lysing buffer. After washing via
centrifuga- tion, the final cells present in the pellet were re-
suspended in medium and counted in a hemo- cytometer.
Viability of the final populations were assessed via
trypan blue exclusion; only suspensions with a viabili-
ty>95 % were analyzed.
Cells were stained with monoclonal antibodies in 12×75-
mm glass tubes using standard techniques (Fernandez-Botran
and Vetvicka 1995). For these analyses, fluorescein isothio-
cyanate (FITC)-labeled anti-CD4, -CD8, and -CD49b anti-
bodies (BD Pharmingen, San Jose, CA) were employed.
Briefly, aliqouts of 5×10
cells were combined with 10 μlof
FITC-labeled anti- bodies (120 μg/ml in PBS) and placed on
ice (in dark) for 30 min. Thereafter, the cells were diluted with
ice-cold PBS, centrifuged to remove non-adherent antibody
materials, and then re-suspended in PBS containing 1 % BSA
and 10 mM sodium azide. Flow cytometry was then per-
formed using a FACScan (Becton Dickinson, San Jose, CA)
system. For each sample, a minimum of 10,000 events were
analyzed using Flowjo software (Tree Star, Ashland, OR).
Antibody formation assay
Formation of antibodies was evaluated using ovalbumin
(OVA) as antigen. Mice were injected twice (2 week apart,
IP) with 0.1 mg of ovalbumin (in 100 μl volume) and the
serum was collected 7 day after the second injection. Total
level of specific antibodies against OVA was detected using
an ELISA assay. As positive control, a combination of OVA
and Freunds adjuvant (with no co-treatments with any
PFCs) was used. All test groups here received daily admin-
istration of the PFCs (alone or in combination with RVB) or
PBS for the entire 3-wk period of OVA exposures.
Measurement of IgM antibodies
Serum levels of IgM antibodies directed specifically against
TNP hapten was measured by ELISA in 96-well plates with
wells that had been coated with 100 μlTNP(10μg/ml) conju-
gated to bovine γ-globulin by incubation overnight, followed
by blocking with 1 % (w/v) BSA. Briefly, 100 μl of test serum
was added to each well and the plate incubated for 120 min at
37 °C. After washing with PBS-Tween to remove non-adherent
proteins, goat anti-mouse IgM conju- gated with alkaline phos-
phatase (1:2000, Sigma) was added and the plate incubated a
further 120 min at 37 °C. After another round of washing to
remove the detection antibody, p-nitrophenyl phosphate sub-
strate (diluted according to manufacturer instruction; Sigma)
was added and the absorbance in each well was measured
using the ELISA plate-reader. Wells containing standard- ized
amounts of mouse anti-TNP (IgM) antibody (BD Biosciences,
San Jose, CA) were analyzed in parallel to generate standard
curves from which levels of anti-TNP IgM in each test sample
could then be extrapolated. All test groups here received daily
administration of the PFCs (alone or in combination) or PBS
for the entire 3-wk period of antigen exposures.
All experiments were repeated three times with at least 5
mice/group (15 mice total). Initially, a one-way analysis of
variance (ANOVA) was used to determine if there were
signifi- cant differences between groups for a given mea-
sured endpoint. If significance was noted, a Studentst-test
was then used to compare all treatment groups to the con-
trols. Significance in each analysis was assigned when a p-
value0.05 was obtained.
Reversal of perfluorooctanesulfonate-induced immunotoxicity 79
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Natural trans-resveratrol with a purity of 99.62 % (HPLC)
was used in these experimens. Vitamin C was pharmaceuti-
cal grade, 1,3/1,6-D-β-glucan #300 was originally from
Transfer Point and has a purity over 85 % (Vetvicka et al.
2007b; Vetvicka and Vetvickova 2012a). At the beginning
of the experiment, there were no significant differences in
body weights among the mice in the various groups (Fig. 1).
However, from Day 4 and thereafter, there was a significant
deterioration in body weights in mice in the PFOS and
PFOA groups. Simultaneous addition of PFCs and RVB
300 produced no changes in body weight. On the last day
of the treatment (i.e., Day 8), body mass was significantly
decreased and liver mass significantly elevated in both the
PFOS and PFOA groups (Table 1). Treatment with RVB
300 returned the body mass to normal levels and signifi-
cantly improved the liver mass as well.
With regard to cellularity, 7 days of exposure to PFOA
caused only an insignificant decrease in this value in the
spleen, but a strong (i.e., >50 %) decrease in this parameter
in the thymus. Exposure to PFOS resulted in a significant
decrease in cellularity in both organs. Co-treatment with
RVB 300 either returned the number of cells to normal
levels (spleen and PFOS + RVB 300 in thymus) or at least
significantly increased the number of cells in comparison to
levels in PFOA-RVB 300 in thymus (Fig. 2). In light of this
finding of altered cellularity in the spleen, any changes in
CD4, CD8 and CD49b sub-populations were also evaluated
(Fig. 3). Whereas there were no changes in CD4 or CD8
sub-population levels after any of the various test treat-
ments, RVB 300 alone caused significant increase in levels
of NK (CD49b
) cells.
To evaluate the effects of the PFOS and PFO - and the
possible mitigation of same by the test agents - on lympho-
cyte proliferation, mitogen-induced proliferation of B- and
T-lympho- cytes was evaluated. Figure 4illustrate how each
PFC significantly suppressed proliferation of T-
lymphocytes. PFOA exposure also inhibited proliferation
of B-lymphocytes. In both cases, addition of RVB 300
returned cell proliferation activities to normal levels.
Effects of each PFC on phagocytosis (here, by peripheral
blood neutrophils) were also evaluated using a well-
established model of synthetic microspheres. Data in
Fig. 5reflect the fact that on one hand, both PFCs signifi-
cantly inhibited phagocytosis. On the other hand, the stim-
ulation of phagocytic activity caused by RVB 300 was
strong enough not only return the reducedphagocytosis
to control values, but to a level even significantly higher
than that noted with cells from the PBS control hosts.
Potential effects on cellular immunity were also evaluat-
ed by monitoring NK activity in cells harvested from the
exposed hosts. The results of these studies showed almost
the same results as with phagocytosis. Both PFOA and
PFOS significantly inhibited NK cell activity and co-
treatment with RVB 300 not only restoredactivity, but
increased it to levels above those of cells from the PBS-
treated control mice (Fig. 6).
To assess effects on humoral immunity, an experimental
model of mice immunized with ovalbumin (OVA) was
employed. Mice were injected twice (14 days apart) with
OVA and the serum was collected 7 days after the second
Body weight (g)
Fig. 1 Body weight changes in mice treated with the test substances.
Body weights were measured daily. Values shown are mean ± SD
Table 1 Body mass change and organ mass change after PFOA, PFOS, and RVB 300 treatments
Body mass change
Spleen mass
Thymus mass
Liver mass
PBS 1.74± 0.24 0.44± 0.03 0.29 ±0.01 6.12± 0.10
1.11± 0.20 0.42± 0.03 0.30±0.01
8.11± 0.11
1.02± 0.23 0.40± 0.02 0.27 ±0.02
9.73± 0.14
RVB 300 1.75± 0.27 0.45± 0.04 0.28± 0.01 6.13± 0.12
PFOA + RVB 300
1.60± 0.22 0.44± 0.05 0.29 ±0.02
6.77± 0.12
PFOS + RVB 300
1.71± 0.20 0.43± 0.04 0.30 ±0.01
6.40± 0.11
Final mass (g) start mass (g)
Calculated as: [organ mass (g)/body mass (g)]×100
Significantly different at
p<0.05 from PBS values,
p<0.05 from PFOA alone,
p<0.05 from from PFOS alone
80 V. Vetvicka, J. Vetvickova
Author's personal copy
injection. In these studies, mice were being concur- rently
injected with the various test materials over the entire 3-wk
study period. The results indicate that both PFC caused
significantly decreased antibody formation and that RVB
300 co- treatment helped to significantly mitigate only the
reduction in antibody formation induced by PFOA (Fig. 7);
RVB 300 led to an increase of 48 % vs. that from PFOS
alone, but this change was not significant. These results
were further confirmed by evaluation of the IgM formation
using immunization with a different antigen (i.e., TNP).
Once again, both PFOA and PFOS inhibited IgM formation
and RVB 300 co-treatment more than doubled this produc-
tion (Fig. 8).
In the last decade, considerable attention has been paid to
PFOS and related compounds, due to their presence in the
environment and subsequently animal and human systems.
Several important papers have discussed the immunosup-
pressive role of these compounds (Peden-Adams et al. 2007;
Dong et al. 2009;Qazietal.2009;Zhengetal.2009;
Brieger et al. 2011; Corsini et al. 2012). Among immune
system-related reactions that have been documented as ad-
versely impacted by oral exposure to PFOS and PFOA are
cell proliferation, antibody formation, inflam- mation, and
cytokine production. Even more attention was focused on
the toxic and immunosup- pressive effects of these com-
pounds after reports surfaced of reduced humoral responses
to routine immunization among children who had been
exposed to PFCs (Grandjean et al. 2012).
Total cellularity (x106)
* *
Fig. 2 Spleen and thymus cellularity after 7 days of exposures to the
test substances. Values shown are mean ± SD.
Value significantly
different from control (PBS only) at p0.05.
Significant differences
between PFOA and PFOA + RVB group at p0.05
T cells B cells
Proliferation index
* *
Fig. 3 Proliferation index of lymphocytes after 7 days of exposures to
the test substances. Unstimulated data were not different between
groups; therefore the data are presented as the proliferation index.
Value significantly different from control (PBS only) at p0.05
CD4 CD8 CD49b
% of positive cells
Fig. 4 Splenic populations in mice treated for 7 days.
Value signifi-
cantly different from control (PBS only) at p0.05
% of positive neutrophils
* *
Fig. 5 Effects of 7 days of exposure to the test substances on phago-
cytosis by peripheral blood cells. Values shown are mean ± SD.
significantly different from control (PBS only) at p0.05
Reversal of perfluorooctanesulfonate-induced immunotoxicity 81
Author's personal copy
However, despite all the current knowledge about immuno-
suppression caused by PFCs, no study about the possible
reversal of these effects exists in the literature. This, together
with our previous report showing that simultaneous treatment
of mercury with glucan (which resulted in significantly low-
ered immunotoxic effects of the metal; Vetvicka and
Vetvickova 2009) suggested to us that glucans can be used as
a natural remedy against low-level exposures to immunosup-
pressants. Since those earlier observations, we have determined
that a combination of glucan-resveratrol-Vitamin C had even
superior effects to that derived from the glucan alone (Vetvicka
and Yvin 2004). Among some of those observations, it was
noted that both glucan and resveratrol stimulated phagocytosis
of blood leukocytes, caused increased expression of CD4 on
spleen cells, and potentiated a restoration of splenic normal
status after experimental induction leukopenia (Vetvicka et al.
2007a; Vetvicka and Vetvickova 2012b). Thus, the aim of this
study was to determine if this combination could be used to
overcome the immunosuppressive effects induced by PFCs.
For the current study, a 7-day oral exposure to PFOS or
PFOA - as originally described by Zheng et al. (2009) - was
employed. As expected, decreases in body weight were fol-
lowed by changes in organ mass (Dong et al. 2009). However,
co-treatment with RVB 300 (the glucan- resveratrol-Vitamin
C combination used here) returnedboth weights body and
liver mass to almost control levels. At the level of the primary
immune organs, cellularity in the spleen and thymus was most
impacted by PFOS. Again, co-treatment with RVB 300
returnedthe number of cells to near normal. In all instances,
RVB 300 itself had no effects on cellularity in either organ.
The effects of each PFC on proliferation are less clear.
Some studies documented suppression of ConA-induced T-
lymphocyte proliferative activity (Zheng et al. 2009). Others,
in contrast, showed an insignificant increase in T- and B-
lymphocyte proliferative capacities (Peden-Adams et al.
2007). Here we demonstrated that, whereas the effects of both
compounds on T-lymphocytes were profound, those on B-
lymphocytes suggested a greater degree of resist- ance. No
effects on the expression of surface markers CD4/CD8/CD49b
were noted, suggesting that PFCs do not affect the migration
of lymphocytes into the spleen or change the development and
maturation of thymocytes.
In one portion of this study, the focus was upon the
effects of the PFCs on cellular immunity. The effects on
Fig. 6 Splenic NK cell activity following 7 days exposure to tested
substances. Each value represents the mean ± SD. *Significant differ-
ence at p 0.05 from control (PBS) group
ODat 405nm
Fig. 7 Effects of tested substances glucans on formation of antibodies
against ovalbumin. Mice were injected twice (2 weeks apart) with
antigen and the serum was collected 7 days after the final injection.
The level of specific antibodies against ovalbumin was detected using
ELISA. *Value significantly different between control (ovalbumin and
Freund adjuvant) and samples at p0.05
50 100 200 400 800
Dilution of the serum
OD at 405 nm
Fig. 8 Effects of tested substances glucans on formation of IgM anti-
bodies against TNP. Mice were injected twice (2 weeks apart) with
antigen and the serum was collected 7 days after the final injection. The
level of specific antibodies against ovalbumin was detected using
ELISA. *Value significantly different between control (TNP and
Freund adjuvant) and samples at p0.05
82 V. Vetvicka, J. Vetvickova
Author's personal copy
phagocytic activity were assessed using 2-hydroxyethyl
methacrylate particles that have only a slight negative
charge and thus do not non-specifically adhere to the cell
surface and significantly lowers the chance of false negative
outcomes (Vetvicka et al. 1982). The results revealed that
both PFOS and PFOA inhibited phagocytosis to a level
equal to 30 % of normal values. The co-treatment with
RVB 300, well known to strongly stimulate this activity,
restoredphagocytosis by harvested cells to the levels noted
in mice that received RVB 300 alone.
Some previous studies have noted a dose-dependent de-
crease in NK cell activity as a result of PFOS exposure
(Zheng et al. 2009). However, as with the lymphocyte
proliferative capacity analyses, other studies reported no
effects at all (Nelson et al. 1992). This discrepancy some-
times can be ascribed to mouse strain or gender differences
(as female B
mice were less sensitive than male mice;
Peden-Adams et al. 2007). The results of the present study
indicated that both PFOS and PFOA imparted strong inhib-
itory effects on the activity of NK cells in the hosts. As with
many of the other endpoints analyzed here, the co-treatment
with RVB 300 once again led to a significant restoration of
this immune function.
In order to also assess the impact of each PFC (and any
mitigating effects of the RVC 300) on humoral responses in
the treated hosts, effects of PFOS and PFOA exposure on
antibody responses were analyzed. Using two different anti-
gens (i.e., ovalbumin and TNP), it was observed that both
PFCs (particularly PFOA) imparted strong inhibitory effects
on both total immunoglobulin formation and on IgM for-
mation. Once again, RVB 300 helped to restoresome of
the antibody formation activity; however, with neither PFC
was the combination co-treatment able to fully restore the
antibody formation capacities in the exposed hosts.
Two major conclusions were reached in this study. The first
was that since both PFOS and PFOA significantly sup-
pressed both branches of immune reactions even after short
7-d exposure - when taken together with findings of signif-
icant loss of body weight and hepato- megaly - these com-
pounds clearly present a significant danger to exposed hosts.
Our study, therefore, provides additional support to regula-
tory agencies seeking to eliminate/minimize PFOA and
PFOS from pollutant emissions and other products. The
second is that, for the first time, PFC-induced immunosup-
pression can be at least partly restored/mitigated by oral
admini- stration of a combination of glucan-resveratrol-
vitamin C. From our data, it could be imagined that RVB
300 might be used in the prophylactic treatment of PFC-
based poisonings. Experi- ments evaluating the mechanisms
of the RVB 300-based restoration of immunosuppression
seen here are currently under way. Furthermore, the clinical
importance of this observation deserves further study.
Declaration of interest The authors have no financial interests in
any company mentioned in this study and report no conflicts of
interest. The authors alone are responsible for the content of this
Brieger A, Bienefeld N, Hasan AR, Goerlich R, Haase H (2011)
Impact of perfluorooctanesulfonate and perfluorooctanoic adic
on human peripheral leukocytes. Toxicol In Vitro 25:960968
Corsini E, Sangiovanni E, Galbiati V, Viviani B, Marinovich M, Galli
CL et al (2012) In vitro characterization of the immunotoxic
potential of several perfluorinated compounds (PFC). Toxicol
Appl Pharmacol 258:248255
De Witt JC, Peden-Adams MM, Keller JM, Germolec DR (2012)
Immunotoxicity of perfluorinated compounds: recent develop-
ment. Toxicol Pathol 40:300311
Dong GH, Zhang YH, Zheng L, Liu W, Jin YH, He QC (2009) Chronic
effects of perfluorooctanesulphonate exposure on immunotoxicity
in adult male C57BL/6 mice. Arch Toxicol 83:805815
Fernandez-Botran R, Vetvicka V. (Eds.) 1995. Flow cytometry. In:
Methods in Cellular Immunology, Boca Raton, FL. CRC Press,
pp. 2946.
Grandjean P, Andersen EW, Budtz-Jergensen E, Nielsen F, Molbak K,
Weihe P et al (2012) Serum vaccine antibody concentrations in
children exposed to perfluorinated compounds. JAMA 307:391397
Lau C, Anitole K, Hodes C, Lai D, Pfahles-Hutchens A, Seed J (2007)
Perfluoroalkyl acids: a review of monitoring and toxicological
findings. Toxicol Sci 99:6694
Nelson DL, Frazier DE, Ericson JE, Tarr MJ, Mathes LE (1992) The
effects of perfluorodecanoic acid (PFDA) on humoral, cellular,
and innate immunity in Fischer-344 rats. Immunopharmacol
Immunotoxicol 14:925938
Patchen ML, MacVittie TJ (1982) Use of glucan to enhance hemopoi-
etic recovery after exposure to Co
irradiation. Adv Exp Med
Biol 155:267272
Peden-Adams MM, EuDaly JG, Dabra S, EuDaly A, Heesemann L,
Smythe J et al (2007) Suppression of humoral immunity follow-
ing exposure to the perfluorinated insecticide sulfluramid. J
Toxicol Environ Health 70:11301141
Qazi MR, Bogdanska J, Butenhoff JL, Nelson BD, DePierre JW (2009)
High-dose, short-term exposure of mice to perfluorooctanesulph-
onate (PFOS) or perfluoro- octanoate (PFOA) affects the number
of circulating neutrophils differently, but enhances the inflamma-
tory response of macrophages to lipopolysaccharide (LPS) in a
similar fashion. Toxicology 162:207214
Vetvicka V, Vancikova Z (2010) Synergistic effects of glucan and
resveratrol. Afr J Biochem Res 4:105110
Vetvicka V, Vetvickova J (2009) Effect of glucan on immunosuppres-
sive action of mercury. J Med Food 12:10981104
Vetvicka V, Vetvickova J (2012a) Combination of glucan, resveratrol
and Vitamin C demonstrates strong anti-tumor potential.
Anticancer Res 32:8288
Vetvicka V, Vetvickova J (2012b) Comparison of immunological prop-
erties of various bioactive combinations. Biomed Papers (In
Reversal of perfluorooctanesulfonate-induced immunotoxicity 83
Author's personal copy
Vetvicka V, Yvin JC (2004) Effects of marine β-glucan on immune
reaction. Int Immunopharmacol 4:721730
Vetvicka V, Fornusek L, Kopecek J, Kaminkova J, Kasparek L,
Vranova M (1982) Phagocytosis of human blood leukocytes: a
simple micromethod. Immunol Lett 5:97100
Vetvicka V, Holub M, Kovaru H, Siman P, Kovaru F (1988) α-Fetoprotein
and phagocytosis in athymic nude mice. Immunol Lett 19:9598
Vetvicka V, Dvorak B, Vetvickova J, Richter J, Krizan J, Sima P et al
(2007a) Orally administered marine (13)-β-D-glucan phycarine
stimulates both humoral and cellular immunity. Int J Biol
Macromol 40:291298
Vetvicka V, Volny T, Saraswat-Ohri S, Vashishta A, Vancikova Z,
Vetvickova J (2007b) Glucan and resveratrol complex possible
synergistic effects on immune system. Biomed Pap Med Fac
Vetvicka V, Baigorri R, Zamarreno AM, Garcia-Mina JM, Yvin JC
(2010) Glucan and humic acid: synergistic effects on the immune
system. J Med Food 13:863869
Wang Y, Wang L, Liang Y, Qui W, Zhang J, Zhou Q et al (2011)
Modulation of dietary fat on the toxicological effects in thymus
and spleen in BALB/c mice exposed to perfluoroooctane sulfo-
nate. Toxicol Lett 204:174182
Zheng L, Dong GH, Jin YH, He QC (2009) Immunotoxic
changes associated with a 7-day oral exposure to perfluor-
ooctane sulfonate (PFOS) in adult male C57BL/6 mice. Arch
Toxicol 83:679689
84 V. Vetvicka, J. Vetvickova
Author's personal copy
... Rodent studies have demonstrated that both PFOA and PFOS are capable of suppressing the TDAR, with lower bound benchmark dose estimates of 1.75 mg/kg/day, which supports the epidemiologic findings that these PFAS elicit immunosuppression [130][131][132]. In vivo animal studies evaluating the immunosuppressive potential of other PFAS are sparse. ...
... Animal Bioassay In Vitro PFOA Association: [123]; [125]; [126]; [127] Association: reviewed in [141]; [132]; [130] Association: [135]; [136] Long-chain PFAS a PFOS Association: [123]; [125]; [127] Association: reviewed in [141]; [131]; [132] Association: [135]; [136] PFHxS Association; [125]; [ ...
Full-text available
Per- and polyfluoroalkyl substances (PFAS) constitute a large class of environmentally persistent chemicals used in industrial and consumer products. Human exposure to PFAS is extensive, and PFAS contamination has been reported in drinking water and food supplies as well as in the serum of nearly all people. The most well-studied member of the PFAS class, perfluorooctanoic acid (PFOA), induces tumors in animal bioassays and has been associated with elevated risk of cancer in human populations. GenX, one of the PFOA replacement chemicals, induces tumors in animal bioassays as well. Using the Key Characteristics of Carcinogens framework for cancer hazard identification, we considered the existing epidemiological, toxicological and mechanistic data for 26 different PFAS. We found strong evidence that multiple PFAS induce oxidative stress, are immunosuppressive, and modulate receptor-mediated effects. We also found suggestive evidence indicating that some PFAS can induce epigenetic alterations and influence cell proliferation. Experimental data indicate that PFAS are not genotoxic and generally do not undergo metabolic activation. Data are currently insufficient to assess whether any PFAS promote chronic inflammation, cellular immortalization or alter DNA repair. While more research is needed to address data gaps, evidence exists that several PFAS exhibit one or more of the key characteristics of carcinogens.
... While systemic stress likely plays a role in TDAR suppression at relatively high doses, such as 15 mg PFOA/kg body weight/ day and above [28,29], adrenalectomized animals still exhibited TDAR suppression, indicating lack of systemic stress at lower doses [30]; this reference was not included in the Chang et al. [10] review). Similarly to PFOA, PFOS administered orally also suppresses the TDAR, albeit at a much wider range of doses and with less consistent responses, from 0.002 to 40 mg of PFOS/kg body weight/ day when given during development or from seven to 60 days in duration [27,[31][32][33][34][35][36][37]. Two of these studies reported that PFOS did not suppress the TDAR. ...
In this perspective, we evaluate key and emerging epidemiological and toxicological data concerning immunotoxicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) and seek to reconcile conflicting conclusions from two reviews published in 2016. We summarize ways that immunosuppression and immunoenhancement are defined and explain how specific outcomes are used to evaluate immunotoxicity in humans and experimental animals. We observe that different approaches to defining immunotoxicological outcomes, particularly those that do not produce clinical disease, may lead to different conclusions from epidemiological and toxicological studies. The fundamental point that we make is that aspects of epidemiological studies considered as limitations can be minimized when data from toxicological studies support epidemiological findings. Taken together, we find that results of epidemiological studies, supported by findings from toxicological studies, provide strong evidence that humans exposed to PFOA and PFOS are at risk for immunosuppression.
... The same protocol that was previously described (Vetvicka et al., 2013) was used throughout this study. Briefly, a randomized, double-blind, placebo-controlled trial compared β-glucan #300 and placebo in children. ...
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The role of glucan in stimulation of immune reactions has been studied for decades. In this report we focused on the effects of orally administered glucan in children with chronic respiratory problems. We measured the physical endurance using a 6MWT test and the levels of eNO in 50 children aged 8-12 years and evaluated the effects of 100 mg/d oral dose of glucan. We found significant improvements in physical indurance and exhaled nitric oxide in glucan-treated children. In addition, strong improvements in general conditions were found. Short-term oral application of natural immunomodulator β-glucan enhancers the overall health and regulation of energetic metabolism in children with chronic respiratory problems.
... Similarly, addition of probiotics into the food resulted in reduction of cholesterol and blood sugar levels in people with diabetes (Moroti et al., 2012) and reduced total or LDL cholesterol in hypercholesterolaemic patients (Larkin et al., 2009). Both our studies and studies by others using several bioactive substances showed that, in many cases, a well prepared combination of individual, highly active biomodulating molecules offered even better results (Vetvicka and Vetvickova, 2013;Ditteova et al., 2003;Vetvicka et al., 2010). These findings led us to study the activities of a Cinnulin/LactoSpore combination. ...
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The search for an optimal combination of natural immunomodulators led us to study the biological effects of the combination of a cinnamon extract Cinnulin PF and probiotic LactoSpore. We found that this combination has strong synergetic effects on phagocytosis and on regulation of cholesterol and blood sugar levels. In addition, the Cinnulin/LastoSpore combination also reduced intestinal damage in mouse model of colitis.
... First, recent studies suggested that β-glucan can restore some immune functions depressed by stress [6,9,20]. Second, the β-glucan-resveratrol-vitamin C combination has superior immunostimulating properties, including anti-cancer effects [13,21]. ...
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Stress has repeatedly been found to reduce the abilities of the immune system to fight against individual attacks. The current dissatisfaction with classical medications has led to more attention being focused on natural molecules. As recent studies have suggested that some bioactive molecules can have synergistic effects in stimulation of immune system and reduction of stress, we have evaluated the stress-reducing effects of the resveratrol-β-glucan-vitamin C combination. We found that compared to its individual components, this combination was the strongest reducer of stress-related symptoms, including corticosterone levels and IL-6, IL-12 and IFN-γ production.
Perfluorinated and polyfluorinated compounds (PFASs) are a class of synthetic chemical substances that are widely used in human production and life, such as fire-fighting foams, textiles and clothing, surfactants, and surface protective agents. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are the most abundant and common perfluorinated compounds in biota and humans. Currently, PFOA and PFOS have been listed in the Stockholm Convention on Persistent Organic Pollutants, and their production has been halted in many countries. However, because the high-energy carbon-fluorine bond can make it resistant to hydrolysis, photolysis, microbial degradation, and vertebrate metabolism, PFOA and PFOS show environmental persistence and bioaccumulation and hence, are of great concern to humans and wildlife. PFOA and PFOS have toxic effects on the immune system of the body. This article reviewed the effects of PFOA and PFOS on immune organs such as the spleen, bone marrow, and thymus of mice and zebrafish, and the effects on non-specific immune functions such as the skin barrier, intestinal mucosal barrier, and humoral immunity. We also reviewed the influence of specific immune functions based on cellular immunity, and further summarized the possible immune toxicity mechanisms such as AIM2 inflammasome activation, gene dysregulation, and signal pathway disorders caused by PFOA and PFOS. The aim of this review was to provide a reference for further understanding of the immunotoxicity and the responsible mechanism of PFOA and PFOS.
Perfluoroctanesulfonate (PFOS) belongs to a larger family of compounds known as Per- and polyfluoroalkyl substances (PFAS). The strength of the carbon–fluorine bond makes PFOS extremely resistant to environmental degradation. Due to its persistent nature, research has been directed to elucidating possible health effects of PFOS on humans and laboratory animals. Here we have explored the effects of PFOS exposure on immune development and function in mice. We exposed adult mice to 3 and 1.5 μg/kg/day of PFOS for 2 and 4 weeks, respectively, and examined the effects of PFOS exposure on populations of T cells, B cells, and granulocytes. These doses of PFOS resulted in serum levels of approximately 100 ng/ml with no weight loss during exposure. We find that PFOS does not affect T-cell development during this time. However, while PFOS exposure reduced immune cell populations in some organs, it also led to an increase in the numbers of cells in others, suggesting possible relocalization of cells. We also examined the effect of PFOS on the response to influenza virus infection. We find that exposure to PFOS at 1.5 μg/kg/day of PFOS for 4 weeks does not affect weight loss or survival, nor is viral clearance affected. Analysis of antibody and T cell specific antiviral responses indicate that at this concentration, PFOS does not suppress the immune cell development or antigen specific immune response.
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The European Commission asked EFSA for a scientific evaluation on the risks to human health related to the presence of perfluoroalkyl substances (PFASs) in food. Based on several similar effects in animals, toxicokinetics and observed concentrations in human blood, the CONTAM Panel decided to perform the assessment for the sum of four PFASs: PFOA, PFNA, PFHxS and PFOS. These made up half of the lower bound (LB) exposure to those PFASs with available occurrence data, the remaining contribution being primarily from PFASs with short half-lives. Equal potencies were assumed for the four PFASs included in the assessment. The mean LB exposure in adolescents and adult age groups ranged from 3 to 22, the 95th percentile from 9 to 70 ng/kg body weight (bw) per week. Toddlers and ‘other children’ showed a twofold higher exposure. Upper bound exposure was 4- to 49-fold higher than LB levels, but the latter were considered more reliable. ‘Fish meat’, ‘Fruit and fruit products’ and ‘Eggs and egg products’ contributed most to the exposure. Based on available studies in animals and humans, effects on the immune system were considered the most critical for the risk assessment. From a human study, a lowest BMDL10 of 17.5 ng/mL for the sum of the four PFASs in serum was identified for 1-year-old children. Using PBPK modelling, this serum level of 17.5 ng/mL in children was estimated to correspond to longterm maternal exposure of 0.63 ng/kg bw per day. Since accumulation over time is important, a tolerable weekly intake (TWI) of 4.4 ng/kg bw per week was established. This TWI also protects against other potential adverse effects observed in humans. Based on the estimated LB exposure, but also reported serum levels, the CONTAM Panel concluded that parts of the European population exceed this TWI, which is of concern.
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The European Commission asked EFSA for a scientific evaluation on the risks to human health related to the presence of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) in food. Regarding PFOS and PFOA occurrence, the final data set available for dietary exposure assessment contained a total of 20,019 analytical results (PFOS n = 10,191 and PFOA n = 9,828). There were large differences between upper and lower bound exposure due to analytical methods with insufficient sensitivity. The CONTAM Panel considered the lower bound estimates to be closer to true exposure levels. Important contributors to the lower bound mean chronic exposure were ‘Fish and other seafood’, ‘Meat and meat products’ and ‘Eggs and egg products’, for PFOS, and ‘Milk and dairy products’, ‘Drinking water’ and ‘Fish and other seafood’ for PFOA. PFOS and PFOA are readily absorbed in the gastrointestinal tract, excreted in urine and faeces, and do not undergo metabolism. Estimated human half‐lives for PFOS and PFOA are about 5 years and 2–4 years, respectively. The derivation of a health‐based guidance value was based on human epidemiological studies. For PFOS, the increase in serum total cholesterol in adults, and the decrease in antibody response at vaccination in children were identified as the critical effects. For PFOA, the increase in serum total cholesterol was the critical effect. Also reduced birth weight (for both compounds) and increased prevalence of high serum levels of the liver enzyme alanine aminotransferase (ALT) (for PFOA) were considered. After benchmark modelling of serum levels of PFOS and PFOA, and estimating the corresponding daily intakes, the CONTAM Panel established a tolerable weekly intake (TWI) of 13 ng/kg body weight (bw) per week for PFOS and 6 ng/kg bw per week for PFOA. For both compounds, exposure of a considerable proportion of the population exceeds the proposed TWIs.
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The role of glucan in stimulation of immune reactions has been well-established. In this report, we focused on the antibody production in glucan-supplemented children with chronic respiratory problems. We measured the levels of salivary IgA, IgM and IgG in 40 children aged 8-12 years and evaluated the effects of 100 mg d −1 oral dose of glucan. We found a significant increase in production of all tested antibodies in the glucan-stimulated group, but a decrease of antibody production in the control group. A thirty-day oral application of yeast-based natural immunomodulator β-glucan strongly stimulated the mucosal immunity of children with chronic respiratory problems.
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Recent data showing that glucan stimulates defense reactions in plants through synthesis of resveratrol, led us to study the possible synergetic effects of a glucan-resveratrol complex on immune reactions in mice. We measured phagocytic activity, expression of CD4 marker on spleen cells, IL-2 secretion and antibody response. In all cases we confirmed the stimulatory effects of glucan. Resveratrol alone had either limit or has no effect. However, a combined preparation showed very strong synergetic effects. Our data support further studies of these two natural immunomodulators.
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Lately, more and more preparation of various cocktails or mixtures of bioactive modulators have been introduced. Their true activity is, however, rarely tested. To compare six commercially available, glucan-based immunostimulators. Immunological effects of tested combinations were measured by evaluation of phagocytosis of synthetic particles by peripheral blood neutrophils, production of IL-2 by mouse splenocytes, production of superoxide anion and nitrite oxide, antibody response to imunization with ovalbumin, and NK cell activity. Our results showed that with the exception of the highest doses (phagocytosis) and superoxide anion and nitrite oxide production, only RVB 300 showed significant immunostimulative activity. Based on our results, we can conclude that most of the tested natural immunomodulators have limited, if any, biological effects. Only RVB 300 significantly stimulated all six tested immunological reactions.
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Perfluorinated compounds (PFCs) have emerged as important food contaminants. They cause immune suppression in a rodent model at serum concentrations similar to those occurring in the US population, but adverse health effects of PFC exposure are poorly understood. To determine whether PFC exposure is associated with antibody response to childhood vaccinations. Prospective study of a birth cohort from the National Hospital in the Faroe Islands. A total of 656 consecutive singleton births were recruited during 1997-2000, [corrected] and 587 participated in follow-up through 2008. Serum antibody concentrations against tetanus and diphtheria toxoids at ages 5 and 7 years. Similar to results of prior studies in the United States, the PFCs with the highest serum concentrations were perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). Among PFCs in maternal pregnancy serum, PFOS showed the strongest negative correlations with antibody concentrations at age 5 years, for which a 2-fold greater concentration of exposure was associated with a difference of -39% (95% CI, -55% to -17%) in the diphtheria antibody concentration. PFCs in the child's serum at age 5 years showed uniformly negative associations with antibody levels, especially at age 7 years, except that the tetanus antibody level following PFOS exposure was not statistically significant. In a structural equation model, a 2-fold greater concentration of major PFCs in child serum was associated with a difference of -49% (95% CI, -67% to -23%) in the overall antibody concentration. A 2-fold increase in PFOS and PFOA concentrations at age 5 years was associated with odds ratios between 2.38 (95% CI, 0.89 to 6.35) and 4.20 (95% CI, 1.54 to 11.44) for falling below a clinically protective level of 0.1 IU/mL for tetanus and diphtheria antibodies at age 7 years. Elevated exposures to PFCs were associated with reduced humoral immune response to routine childhood immunizations in children aged 5 and 7 years.
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β-Glucans are naturally occurring carbohydrates found in plants, fungi and some bacterial species, and currently are well-established and powerful immunomodulators with beneficial properties in cancer therapy. Recent studies suggested that some additional bioactive molecules have synergistic effects when combined with glucan. In the current study, we evaluated the anticancer properties of glucan, resveratrol, vitamin C combination. We found that compared to the individual components, the combination was the strongest activator of phagocytosis and antibody formation. Using two different models of cancer treatment, our results demonstrated that the combination strongly suppressed the growth of breast and lung tumors, most likely due to the stimulation of apoptosis.
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Perfluorinated compounds (PFCs) are environmentally widespread, persistent, and bioaccumulative chemicals with multiple toxicities reported in experimental models and wildlife, including immunomodulation. The two most commonly detected compounds, which also generally occur in the highest concentrations in environmentally exposed organisms, are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). PFOA and PFOS have been reported to alter inflammatory responses, production of cytokines, and adaptive and innate immune responses in rodent models, avian models, reptilian models, and mammalian and nonmammalian wildlife. Mounting evidence suggests that immune effects in laboratory animal models occur at serum concentrations below, within the reported range, or just above those reported for highly exposed humans and wildlife. Thus, the risk of immune effects for humans and wildlife exposed to PFCs cannot be discounted, especially when bioaccumulation and exposure to multiple PFCs are considered. This review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA, PFOS, and other PFCs in rodent models, alternative laboratory models, and wildlife.
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Humic acids are compounds resulting from decomposition of organic matter. Despite their common presence, our knowledge of their biological effects is limited, and current findings are controversial. We decided to evaluate the immunological effects of two different types of humic acids, differing in source and biochemical characteristics. Using both components either alone or in combination with the well-established yeast-derived immunomodulator glucan, we measured their effects on both the cellular (phagocytosis and tumor suppression) and humoral (antibody production and cytokine secretion) branches of immune reactions. In summary, our results suggest that humic acids are biologically active immunodulators affecting both the humoral and cellular branches of immune reactions. In addition, the two humic acids studied here are working in synergy in stimulation of the immune reaction, supporting further studies of these natural immunomodulators.
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Global cycling of mercury results in the presence of mercury salts in the environment. The well-established negative effects of mercury on the immune system led us to the study whether natural immunomodulator glucan can overcome the immunosuppressive effects of mercury. Two types of mercury, thimerosal and mercury acetate, were administered in a dose of 2-8 mg/L of drinking water to mice. After 2 weeks, all mice exhibited profound suppression of both cellular (phagocytosis, natural killer cell activity, mitogen-induced proliferation, and expression of CD markers) and humoral (antibody formation and secretion of interleukin-6, interleukin-12, and interferon-gamma) responses. The mice were then fed with a diet containing a standard dose of glucan. Our results showed that simultaneous treatment with mercury and glucan resulted in significantly lower immunotoxic effects of mercury, which suggests that glucans can be successfully used as a natural remedy of low-level exposure to mercury.
We have previously shown that PFOA and PFOS directly suppress cytokine secretion in immune cells, with different mechanisms of action. In particular, we have demonstrated a role for PPAR-α in PFOA-induced immunotoxicity, and that PFOS has an inhibitory effect on LPS-induced I-κB degradation. These studies investigate the immunomodulatory effects of four other PFCs, namely PFBS, PFOSA, PFDA, and fluorotelomer using in vitro assays. The release of the pro-inflammatory cytokines IL-6 and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes (hPBL) and in the human promyelocytic cell line THP-1, while the release of IL-10 and IFN-γ was evaluated in phytohemagglutinin (PHA)-stimulated hPBL. All PFCs suppressed LPS-induced TNF-α production in hPBL and THP-1 cells, while IL-6 production was suppressed by PFOSA, PFOS, PFDA and fluorotelomer. PFBS, PFOSA, PFOS, PFDA and fluorotelomer inhibited PHA-induced IL-10 release, while IFN-γ secretion was affected by PFOSA, PFOS, PFDA and fluorotelomer. Leukocytes obtained from female donors appear to be more sensitive to the in vitro immunotoxic effects of PFCs when their responses are compared to the results obtained using leukocytes from male donors. Mechanistic investigations demonstrated that inhibition of TNF-α release in THP-1 cells occurred at the transcriptional level. All PFCs, including PFOA and PFOS, decreased LPS-induced NF-κB activation. With the exception of PFOA, none of the PFCs tested was able to activate PPARα driven transcription in transiently transfected THP-1 cells, excluding a role for PPARα in the immunomodulation observed. PFBS and PFDA prevented LPS-induced I-κB degradation. Overall, these studies suggest that PFCs affect NF-κB activation, which directly suppresses cytokine secretion by immune cells. Our results indicate that PFOA is the least active of the PFCs examined followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer.
Perfluorooctane sulfonate (PFOS) can cause atrophy of the immune organs in rodents, but the mechanism underlying this action is not completely understood. In this study, BALB/c mice were fed a regular (RD) or high-fat diet (HFD). They were then exposed to PFOS (0, 5, and 20mg/kg/day) for 14 days. In the RD-exposure group, body weight significantly decreased and the immune organs showed considerable atrophy. Histopathological analyses showed that the corticomedullary junction of the thymus was indistinguishable, and sinus expansion in the spleen was observed. Transmission electron microscopy (TEM) results showed that lipofuscin granules and vacuoles appeared in the thymus and spleen. Increased apoptosis of thymocytes was observed. In the HFD group, all of these phenomena were not eliminated. More serious atrophy was seen in the immune organs under TEM. Even more adipocytes were in the lobules of the thymus in the HFD 20mg/kg/day PFOS groups. Expression of the proliferator-activated receptor-alpha and interleukin-1 beta were upregulated in the thymus and spleen in all exposure groups. These results suggest that PFOS may indirectly attack the immune organs by interfering with lipid metabolism, leading to co-senescence of the thymus and spleen. These data may aid understanding of how PFOS affects the immune system.
Perfluorinated compounds (PFCs), such as perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA), are xenobiotics that can be detected worldwide in the environment, wildlife, and humans. So far, the immunotoxicity of PFCs has only been investigated in rodents, but not in humans. In this study, we explore the impact of PFOS and PFOA on selected functions of human leukocytes in vitro. PFOS induced a significant decrease of natural killer-cell activity and reduced the release of the pro-inflammatory cytokine TNF-α following lipopolysaccharide (LPS)-stimulation. Furthermore, the plasma PFOS concentrations (2.09-8.98 ng/ml) found in our study subjects correlated positively with the LPS-stimulated IL-6 release. PFOA augmented significantly calcitriol-induced monocytic differentiation of the HL-60 cell line. Additionally, there was a significant linear relationship between LPS-stimulated TNF-α and IL-6 release, and the plasma PFOA (1.20-6.92ng/ml) concentrations of the study subjects. In conclusion, the investigated PFCs affect human immune cells mainly with regard to natural killer-cell cytotoxicity and the pro-inflammatory cytokine release by stimulated macrophages.