Immunological Abnormalities in Humans Chronically
Exposed to Chlorpyrifos
Jack D. Thrasher, Ph.D.
Sam-1 Trust
Alto, New Mexico
Gunnar Heuser, M.D., Ph.D.
NeuroMed and NeuroTox Associates
Agoura Hills, CA
Alan Broughton, M.D., Ph.D.
Antibody Assay Laboratories
Santa Ana, CA
Published in: Archives of Environmental Health, 2002, 57:181-187
ABSTRACT. Twenty-nine individuals with chronic health complaints following exposure to Chlorpyrifos
(CPS) were compared to three control groups (one positive and two negative) with respect to the following: (1) peripheral lymphocyte phenotypes; (2) autoantibodies (ANA, parietal cell, brush border, mitochondria, smooth muscle, thyroid gland, and CNS/PNS myelin); (3) mitogenesis to PHA and ConA. The data show an increase in CD26 expression, and decrease in per cent CD5 phenotype, decreased mitogenesis to PHA and ConA, and an increased frequency of autoantibodies. The alterations in these peripheral blood markers were unaffected by medications, age, sex or season. It is concluded that Chlorpyrifos, causes immunological changes.
INTRODUCTION. CPS (0,0 diethyl 0-[3,5,6-trichloropyridinol]) phosphorothionate) is a chlorinated organophosphate insecticide that is a moderate inhibitor via CPS oxon of acetylcholinesterase. The inhibition of this enzyme leads to signs and symptoms of overstimulation of the cholinergic system.1,2 The commercial product contains a number of chemicals, including but not limited to alkyl phosphates, alkyl phosphorothioates (sulfotepp), 3,5,6-trichloropyridinol (TCP) and solvents.3 Other adverse effects of CPS in animals and humans are reported as follows: 1)developmental neurotoxicity; 4-72) targeting of DNA, RNA, protein, nuclear transcription and cAMP signaling cascade in post natal brain neurogenesis;8-12 3) mitotic abnormalities, apoptosis and cytoxicity in rat embryos and midbrain micromass cultures;13,14 4) birth defects in humans;15 5) decreased T cell blastogenesis to PHA and ConA with increased expression of CD4 and CD8 surace makers in rats16 and, 6) generation of reactive oxygen species, DNA damage and lactate acid dehydrogenase leakage in rat brain and liver17
We previously reported a preliminary study on immunological alterations observed in twelve individuals chronically exposed to CPS.18 In this paper we confirm and extend our earlier observations on changes in peripheral blood immunologic phenotype frequencies and on the presence of autoantibodies after exposure to CPS. In addition, these data show that the observations on changes in peripheral blood phenotype frequencies are not effected by medications, age, season, and sex Finally, it is suggested that immune alterations, increased frequency of autoantibodies and increased rate of apoptosis are probably associated with chronic illness following exposure to CPS.
MATERIALS AND METHODS
Subjects and Controls. Twenty-nine individuals (10 males, 19 females) with chronic health complaints were evaluated for immunologic abnormalities following exposure to CPS. All subjects were diagnosed by their treating physicians with Multiple Chemical Hypersensitivity (MCS). Common symptoms were an initial flu-like illness, followed by multi-organ symptoms that included headaches, loss of memory, dizziness, gastrointestinal disturbances, arthralgia, menstrual irregularities, and fatigue and heightened olfactory sensitivity to low concentrations of chemicals. All were non-smokers. To the best of our knowledge none of the 29 individuals took either prescribed or over-the-counter medications. The data were compared to two negative (unexposed to CPS) and one positive control (exposed to CPS) groups. Group A controls were volunteer asymptomatic chiropractic students (29+9 y, 15 M, 13 F), who were exposed to formaldehyde in an anatomy course one year prior to immune testing. Group B controls were healthy volunteer home dwellers (54+19 y, 13 M, 16 F), who gave informed consent for immunological testing. The History of atopy (asthma, hay fever)was unknown for the students and previously reported for the home dwellers.
18
In some cases measurements of the concentrations of CPS were taken. The detection of CPS in the homes following commercial application was done by either OSHA method 62 or EPA method 8080 at independent commercial laboratories. Most of the subjects were exposed in either their home or work place following commercial mis-application of the insecticide. One experienced a CPS spill in his truck, while two males were exposed to wet CPS treated lumber while working at a lumber mill. Two individuals (male and female) were exposed by a sprayer that left puddles of insecticide and vegetation concentrations of 0.013 to 0.074 ppm. Testing of five different houses revealed the following concentrations: 1) swab samples (n = 4), 50.7+57.6 nanograms/in2; 2) swab samples (n = 15) ranging for n.d, to 150 ppm (mean, 18.9+38.5 ppm); 3) swab samples (n = 7) ranging from n.d. to 128.7 ppm (mean = 22.2+44.7 ppm; 4) two carpet samples containing 16 and 316 mg/kg; and, 5)attic bulk samples(n = 6) ranging from 3.9 to 190 mg/kg (mean = 37.4+90 mg/kg, wipe samples (<1.6 ug/kg) and air samples (<0.008 mg/m3). These houses totaled 6 individuals (2 males, 4 females). The houses were tested up to 4 years after application of CPS. In addition, all subjects were healthy prior to exposure, expressing only common health problems, e.g colds, hay fever, etc. Exposure to CPS was verified through records maintained by the subjects. No attempt was made to correlate symptoms with the data, because it was beyond the scope of this report. However, it has been ascertained that at least three individuals (two males one female) were subsequently diagnosed with LE-like disease, while a fourth (female) died from complications of chronic seizures believed to be kindling of the CNS.
Blood Collection. Venous blood samples were drawn from all individuals 1 to 4.5 years following exposure. All samples, including the controls, were collected under the supervision of an attending physician in silicon-treated sodium heparinized glass evac-tubes. The blood samples were transported to the laboratory and were used within 24 hours with a cell viability of 90 % or greater. All samples were assigned a computer-generated number. Quality assurance was performed by positive and negative controls, which were run simultaneously with the unknown samples. The mononuclear cells were isolated using Ficoll-isopaque density gradient centrifugation.
19
Lymphocyte Markers. All subjects had the following immunologic tests performed: Phenotyping for surface makers CD4, CD5, CD8, CD19, CD25, CD26 and CD4/CD8 as previously reported.18 These phenotypes were selected for comparison to our previous observations.18 These data were analyzed by ANOVA statistics for the effects of age, sex and seasonal variation on lymphocyte markers.
Autoantibody Determinations. Autoantibodies against smooth muscle (ASM), nucleic acids and nucleoprotiens (ANA), mitochondria (AMA), parietal cells (APC) and brush border (ABB) were performed using standard indirect immunofluorescence method.20 Antibodies to myelin sheath were detected using frozen spinal cord (CNS) and sciatic nerve (PNS) as substrates with an indirect immunofluorescence technique using antihuman immunoglobulin conjugated to fluorescein.
21
Thyroid antibodies were measured by an immunoradiographic procedure using thyroglobulin and purified thyroid peroxidase in eleven subjects.22
Mitogenesis. Mitogenesis to Phytohemagglutinin (PHA) and Concanavillin (ConA) were performed on peripheral lymphocytes from 13 individuals utilizing the colorimetric MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay.23 Viable cells activate the MTT, which is measured colorimetrically at 570 nm. Mononuclear cells were isolated, suspended at in 0.1 ml of RPM medium at 106 cells. They were cultured in RPMI 1640 medium, 10 % fetal calf serum and antibiotics (penicillin and streptomycin). Cells from each individual were tested at 3 different concentrations of the mitogens for optimum stimulation. The tests were performed in triplicate at the optimum concentration and reported as the average of the three.
RESULTS
Effects of Age, Sex and Season on Peripheral Lymphocyte Markers
The effects of age, sex and season on peripheral blood phenotypes were analyzed. ANOVA tests comparing the three age groups showed no significant differences for total counts or percentages of each cell type (Table 1). All data were then pooled and analyzed for effects of sex and season on the mean and percent cell counts. The data were not affected by either sex or season (data not shown).
Table 1. This table presents the mean absolute counts (cells/mm3) of lymphocyte phenotypes according to the three age groups. ANOVA tests (F values) revealed that age had no effects on the mean cell counts.
|
Group 1
|
Group 2
|
Group 3
|
||
|
Age(y)
|
27+7.5
|
45.7+3.8
|
65.8+3.1
|
F-values
(p=0.05) |
|
WBC
|
7000+1574
|
7670+954
|
5820+643
|
2.636 (N.S.)
|
|
lymphs
|
2968+553
|
2996+589
|
2195+420
|
2.843 (N.S.)
|
|
CD5
(%) |
2126+553
(68+11) |
2043+505
(69+12) |
1989+538
(70+10) |
1.897 (N.S.)
0.005 (N.S.) |
|
CD4
(%) |
1423+373
(48.6+6.2 |
1479+288
(50+9.9) |
1197+282
(43.3+3.8) |
0.191 (N.S.)
0.021 (N.S.) |
|
CD8
(%) |
794+304
(26.8+6.8) |
765+291
(25.1+6.8) |
515+173
(24.9+9.2) |
1.653 (N.S.)
0.021 (N.S.) |
|
CD4/CD8
|
1.89+0.42
|
2.15+0.65
|
2.58+0.94
|
2.170 (N.S.)
|
|
CD25
(%) |
67+47
(2.1+1.4) |
105+61
(3.8+1.6) |
69+39
(3.2+2) |
1.441 (N.S.)
1.339 (N.S.) |
|
CD26
(%) |
490+233
(17.4+6.2) |
513+292
(16.9+9.3) |
369+173
(17.3+8) |
0.532 (N.S.)
0.008 (N.S.) |
|
CD19
(%) |
159+139
(4.9+3.2) |
287+173
(9.8+4.7) |
141+93
(9.2+4.5) |
2.377 (N.S.)
0.939 (N.S.) |
Lymphocyte and White Cell Counts
Table 2 summarizes the results on lymphocyte markers for the 29 subjects, the 12 positive controls (exposed to CPS) and two control groups previously reported.18 No significant differences were observed in the absolute and per cent counts of each cell types except as follows: 1) The per cent CD5 were significantly lower than the two control groups (p = < 0.05) and no different than the positive control; 2) Both the per cent and absolute numbers of CD26 cells were significantly greater than that of the two control groups (p = <0.001) and no different than the positive control.
Table 2. This table summarizes the mean cell counts (cells/mm3) for lymphocyte phenotypes in the exposed (n = 29) as compared to the positive control (n = 12) and two negative control groups; A ( n = 28) and B (n = 27).
|
Cell
Type |
Exposed
|
Control
|
Control A
|
Control B
|
p
Values |
|
(n = 29)
|
(n = 12)
|
(n = 28)
|
(n = 27
|
||
|
WBC
|
6887+754
|
7011+1584
|
6824+1742
|
6886+1527
|
N.S.
|
|
Lymphs
|
2703+754
|
2845+650
|
2392+707
|
2517+550
|
N.S.
|
|
CD5
(%) |
1876+469
(69+11) |
1901+533
(66.6+9.6) |
1772+576
(74.1+7.7) |
1906+462
(75.6+5.6) |
N.S.
<0.05 |
|
CD4
(%) |
1342+367
(50+7.6) |
1336+381
(47.9+9.6) |
1234+421
(51.3+5.9) |
1336+324
(53.1+4.7) |
N.S.
N.S. |
|
CD4/CD8
|
2.1+0.7
|
2.3+0.76
|
2.2+0.72
|
2.3+0.59
|
N.S.
|
|
CD25*
(%) |
78+55
(2.8+1.8) |
47.3+64.4
(1.5+1.8) |
71.2+45.6
(3.3+2.7) |
61.8+42.4
(2.4+) |
N.S.
N.S. |
|
CD19*
(%) |
205+257
(7.4+4.8) |
196+167
(7.0+5.0) |
143+103
(6.1+4.5) |
202+65
(8.0+2.0) |
N.S.
N.S. |
|
CD26
(%) |
438+265
(16.1+9) |
372+392
(13+13.4) |
122+93
(5.1+3.6) |
52+43
(2.1+1.8) |
<0.001
<0.001 |
*n = 26 for CD25 and CD19
N.S. = Not Significant
Mitogenesis
The results of stimulation of peripheral lymphocytes by PHA and ConA in thirteen subjects are presented in Table 3. Five individuals had stimulation indices below expected
values for both PHA and ConA, one had a lower value for PHA and one for ConA. The average lowered stimulation indices were 73+24 % (PHA) and 65+19 %(ConA) as compared to expected of 96-195 % (PHA) and 94-354 % (ConA). The absolute numbers and per cent CD26 cells and autoantibodies were not significantly different between those individuals with abnormal mitogenesis(n = 7) versus those with normal stimulation (n = 6).
Table 3. This table lists the values following mitotic stimulation with PHA and ConA for 13 individuals that were tested. The six individuals with normal values had a stimulation index of 111+9% (PHA) and 123+38% (Con A). Five of the remaining seven had abnormally low values for both PHA and Cona, while one each had a value below expected for PHA and ConA, respectively. Comparison of the 6 normal vs the 7 abnormal individuals revealed no difference in CD26 and CD5 cells as well as autoantibodies.
|
PHA
|
PHA
|
ConA
|
ConA
|
|
Normal
|
Below
|
Normal
|
Below
|
|
114
|
81
|
94
|
62
|
|
128
|
74
|
138
|
65
|
|
106
|
27
|
130
|
75
|
|
107
|
74
|
169
|
25
|
|
104
|
85
|
103
|
76
|
|
117
|
95
|
103
|
77
|
|
103
|
77
|
||
|
111+9 %
|
73+24 %
|
123+38 %
|
65+19 %
|
Expected Values: PHA (96-195%); ConA (95-354%)
Notes: The 6 individuals with normal values had a stimulation index of 111+ 9% (PHA and 123 + 36 % (ConA). Five of the remaining 7 individuals had abnormally low values for PHA and ConA, where as each had a value below expected for PHA and ConA, respectively. Comparison of the 6 noprmal vs 7 abnormal individuals revealed no difference in CD26 and CD5 cells, or in autoantibodies
Autoantibodies Detected in the Peripheral Blood
Autoantibodies that were detected in the peripheral blood are presented in Tables 4 through 7. Odds ratios for ANA, ASM and APC were significantly different from the unexposed control subjects and no different from the exposed group. The odds ratio could not be determined for AMA because of zero values for the controls (Table 4). In addition, the odds ratios for individuals with multiple autoantibodies, i.e. at least 1, 2 or 3 different autoantibodies were significantly different from the non-exposed controls (Table 5).
Table 4. This table summarizes the number and percentage of autoantibodies found in the exposed, positive control and negative controls (A & B). Odds ratio at 95% C.I. were calculated and are presented for exposed vs controls A & B.
|
Autoantibody
|
Exposed
(n = 27) No.(%) |
Pos.Contr.
(n = 12) No.(%) |
Contr. A
(n = 28) No.(%) |
Contr. B
(n = 27) No.(%) |
Odds
Ratio Exp/A |
Odds
Ratio Exp/B |
|
ANA
|
5 (19)
|
3(25)
|
1(3.6)
|
0
|
5.63
(1.76, 23.58) |
----*
|
|
ASM
|
14(50)
|
5(42)
|
4(14.3)
|
4(14.8)
|
6.14
(2.96, 13.28) |
6.14
(2.96, 13.18) |
|
AMA
|
1(23.8)
|
0
|
0
|
0
|
----*
|
----*
|
|
APC
|
7(26)
|
4(33)
|
1(3.6)
|
1(3.7)
|
8.88
(2.88, 1318) |
8.88
(2.88, 13.18) |
|
ABBA
|
9(33.3)
|
7(58)
|
4(14.3)
|
7(26)
|
3.31
(1.57, 7.19) |
1.71
(0.88, 3.33 |
*Odds ratio coul not be calculated because of zero values in Controls A and B
Table 5. This table summarizes the percentage of individuals who had 1, 2, and 3 autoantbodies as compared to the positive control and negative controls (A & B). The odds ratio at 95 % C.I. for exposed vs controls A & B are presented.
|
Autoantibody
|
Exposed
No. (%) |
Pos. Contr.
No. (%) |
Control A
No. (%) |
Control B
No. (%) |
Odds Ratio
Exp/A |
Odds Ratio
Exp/B |
|
1 pos.
|
19 (70)
|
9 (83.3)
|
5 (14.8)
|
4 (14.54)
|
14.33
(6.72, 31.31) |
14.33
(7.73, 31.31) |
|
2 pos.
|
16 (58)
|
6 (50)
|
2 (7.1)
|
1 (3.7)
|
18.35
(7.42, 50.89) |
33.14
(11.01, 131.3) |
|
3 pos.
|
3 (12)
|
3 (25)
|
0
|
1 (3.7)
|
----*
|
13.5
(1.91, 583.79) |
*Odds ratio could not be calculated because of zero value for Control A
Antithyroid autoantibodies detected in ten of 26 individuals are presented in Table 6. Ten (38.5%) had antibodies directed towards the thyroid gland. Four had both antithryoglobulin and antimicrosomal antibodies, three had only antimicrosomal antibodies and three had only antithyroglobulin antibodies.
The titers in 6 were sufficient as to be indicative of autoimmune thyroiditis. None were tested for thyroid hormone abnormalities.
Table 6. This table presents the observations on antithyroid autoantibodies detected in 10 of the 26 individuals. Two of the subjects were tested by another laboratory. Six of the indivdiuals had sufficient titers as to be indicative of autoimmune thyroiditis*. None were tested for thyroid hormone abnormaliteis.
|
Antithyroglobulin
|
Antimicrosomal
|
|
0.33
|
0
|
|
0
|
*110
|
|
2.6
|
*16
|
|
0.8
|
*1.6
|
|
0.32
|
0
|
|
0
|
*11
|
|
1.9
|
*12
|
|
0.35
|
0
|
|
0
|
*80a
|
|
22
|
*1671b
|
* Six of the individuals had sufficient antibodies indicative of autoimmune thyroiditis, either clinical or subclinical. None were tested for thyroid disease.
Individuals 1-8 were tested by Antibody Assay Laboratories, Santa, CA as previously described
18
. Expected values in MRC are 0.0-0.33 (anthyroglobulin) and 0.0-0.35 (antimicrosomal).
aIndividual 9 was tested by Immunosciences Lab, Inc., Beverly Hills, CA utilizing rabbit RBC agglutination. Expected titers are <1:20 (anthyroglobulin) and <1:80 (antimicromsomal)
bIndividual 10 was tested by Immunodiagnostic Laboratories, San Leandro, CA utilizining FAMA (Fluorescence-Activated Microsphere Assay). The expected values are ,1 for both anthyroglobulin and antimicrosomal
.
Table 7 summarizes the percentage of individuals with antimyelin autoantibodies to central and peripheral myelin in the exposed versus controls. Antimyelin antibodies were considered positive at titers of 1:8 or greater. The critical Z values were all significant at p = <0.05 (1.645).
Table 7. This table summarizes the percentage of individuals with autoantibodies for each isotype (IgG, IgM, IgA) against myelin of the central (CNS) and peripheral (PNS) nervous system in the exposed vs. a control group. Critical Z values are given. All Z values are signficant at p = 0.05 or greater.
|
Exp.
|
Exp
|
Contr
|
Contr
|
|
||
|
Isotype
|
CNS
% |
PNS
% |
CNS
% |
PNS
% |
Z
Value* |
Z
Value* |
|
IgG
|
76
|
35
|
28
|
15
|
3.532
|
1.698
|
|
IgM
|
35
|
44
|
15
|
20
|
1.698
|
1.891
|
|
IgA
|
53
|
50
|
20
|
21
|
2.513
|
2.313
|
*Critical Z Values (two-sided significance level) at p = 0.1, 0.05,
0.02, 0.01 are 1.645, 1.960, 2.326 & 2.576, respectively.
DISCUSSION
We have previously pointed out that lymphocyte activation markers and autoantibodies can occur as a result of chronic illness, e.g. autoimmune disease as well as toxic exposure.18,24,25 However, it must be considered that each of the subjects in this study were healthy, leading a normal life prior to the exposure CPS. Concomitant to the exposure they developed multi-organ symptoms that have persisted for several years. Most common among these were an initial flu-like illness. A flu-like illness is a common symptom following exposure to organophosphates as well as to toxins general (for review, 24). Although some of the individuals in this study were eventually diagnosed with a LE-like illness and other may have an autoimmune thyroiditis,25 their initial illness and the symptoms began concomitant to the exposure to CPS. Thus, it is concluded that the immunologic observations in this and as previously reported are a result of the exposure, leading to subsequent health problems.
Additionally, the data on lymphocyte phenotypes present in the peripheral blood of these individuals corroborates and extends previous observations on humans with chronic illness following exposure to CPS.18 Also, the data show that the lymphocyte markers are not effected by age, sex, season and either over-the-counter or prescription medicines. Thus, the increased in CD26 cells, the decreased percent CD5 cells as well as the presence of multi-organ autoantibodies are associated with a chronic exposure to the organophosphate, CPS.
Multi-organ autoantibodies and an increased expression of CD26 and other activation markers on peripheral lymphocytes have been reported for other xenobiotics. These include formaldehyde,26,27 chlordane/heptachlor, 28,29 PCP,30 industrial solvents,31 silicone breast implants32 and MTBE/benzene.33 From these observations, it appears that immune activation and the development of multi-organ autoantibodies is a common manifestation of exposure to xenobiotics. Such immune alterations, e.g.. appearance of immune activation markers, abnormal mitogenesis, and multiple autoantibodies, may suggest the presence of an atypical autoimmune disease process and/or immune dysregulation.31-35 Several, recent observations on altered immune parameters in chronically ill individuals may shed some light on possible causative mechanisms, which require further examination and research. These are: 1) Gulf War Veterans; 2) apoptosis; and 3 respiratory burst.
Gulf War veterans with chronic fatigue have higher levels of T cells, MHC II+T cells, Il-2, IL-10, IFN-gamma, and TNF-alpha and decreased NK cells when compared to healthy controls.36 Also, ill veterans with neurological symptoms have a lower than expected activity of PON1 (paraoxonase) Type Q (Type A) arylesterase.37 PON1 hydrolyzes several organophosphates, including CPS, sarin, diazinon, and soman.37,38 Moreover, Au et al39 have shown that farmers with "unfavorable" versions of polymorphic metabolizing enzymes (cytochrome P4502E1, glutathione S-transferases mu and theta, and paroxonase genes) expressed greater cytogenetic effects following exposure to mixed pesticides when compared to individuals with "favorable" genes. Thus, it appears that the association between genetic susceptibility and alterations in various immune parameters in relation to toxic exposure needs further research.
Second, seven of the 13 individuals tested had abnormal low mitogenesis to stimulation of peripheral blood lymphocytes by PHA and ConA. Abnormal mitogenesis and an increased rate of apoptosis in some individuals may also be an underlying mechanism in altered immune parameters in humans following chemical exposure.33,34 Individuals with chronic fatigue induced by chemical exposure have an increase in serum TNF-alpha and an increased rate of apoptosis of peripheral lymphocytes.33 Humans exposed to gasoline contaminated water also have increased rates of apoptosis.33 CPS in rat embryo cultures is a mitotic spindle poison and causes apoptosis,and affects neurogenesis, in vivo and in vitro.8, 10,13,14 Thus, immune dysregulation caused by xenobiotics, including CPS, may occur via an increased rate of apoptosis. Finally, it is believed that an increase in lymphocyte surface marker expression and a decrease lymphocyte blastogenic response following CPS exposure may result in a compensatory shift of the immune response towards certain stimuli.16 Thus, the increased expression of CD26, decreased per cent CD5, and multiple autoantibodies in these individuals are compatible with these observations.
Third, polychlorinated pesticides, including the OPs and organochlorines, cause both in vitro and in vivo generation of reactive oxygen species.17,40 These species, apparently are capable of causing damage to single stranded DNA, increased leakage of lactate dehydrogenase from mitochondria and an apparent increased rate of apoptosis in rat brain and liver.17 CPS is a chlorinated compound and 3,5,6-trichlorpyridinol is the major metabolite.41 Thus, the role of reactive oxygen species, mitochondrial damage and leakage of mitochondrial proteins (e.g.lactate acid dehydrogenase, cytochrome c, etc), TNF-alpha have in apoptosis and the release of self-antigens. Particularly, since mitochondria are damaged by cytotoxic agents, releasing several proteins, including cytochrome c, that leads to cell death.42
In conclusion, it is becoming increasingly apparent that certain individuals are more susceptible to the adverse effects of xenobiotics, experiencing chronic adverse health effects following exposure. Altered frequency and function of biomarkers, such as certain genes, enzymes, lymphocyte phenotypes, lymphokines, multiple autoantibodies as well as increased apoptosis appear to be associated with exposure to xenobiotics and subsequent chronic health problems. In addition, three potential mechanisms that may be responsible for these immune alterations are discussed.
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