Autism spectrum disorders may be due to cerebral toxoplasmosis
associated with chronic neuroinflammation causing persistent
hypercytokinemia that resulted in an increased lipid peroxidation,
oxidative stress, and depressed metabolism of endogenous and
exogenous substances
§
Joseph Prandota
*
Department of Social Pediatrics, Faculty of Health Sciences, University Medical School, 5 Bartla Street, 51-618 Wroclaw, Poland
Research in Autism Spectrum Disorders xxx (2009) xxx–xxx
§
Part of this article is based on the author’s presentation entitled: ‘‘Pathophysiology of vaccination-associated adverse events’’ at the International
Conference ‘‘Autism and vaccinations: Is there a link?’’ October 25–6, 2008, Warsaw, Poland (
www.ipin.edu.pl/autism/page08.html), organized by
Warsaw University, and sponsored by the European Commission and Ministry of Science and Higher Education, Poland.
* Tel.: +48 71 348 42 10; fax: +48 71 345 93 24.
E-mail address:
Prandota@ak.am.wroc.pl.
A R T I C L E I N F O
Article history:
Received 2 September 2009
Accepted 15 September 2009
Keywords:
Autistic spectrum disorders
Cerebral toxoplasmosis
Immune irregularities
Hypercytokinemia
Nitric oxide
Oxidative stress
Hypermetabolic state
Depressed enzyme activities
A B S T R A C T
Worldwide, approximately 2 billion people are chronically infected with
Toxoplasma
gondii
with largely yet unknown consequences. Patients with autism spectrum disorders
(ASD) similarly as mice with chronic toxoplasmosis have persistent neuroinflammation,
hypercytokinemia with hypermetabolism associated with enhanced lipid peroxidation,
and extreme changes in the weight resulting in obesity or wasting. Data presented in this
review suggest that environmental triggering factors such as pregnancy, viral/bacterial
infections, vaccinations, medications, and other substances caused reactivation of latent
cerebral toxoplasmosis because of changes in intensity of latent central nervous system
T.
gondii
infection/inflammation and finally resulted in development of ASD. Examples of
such environmental factors together with their respective biomarker abnormalities are:
pregnancy (increased NO, IL-1
b, TNF-a, IL-6, IL-10, prolactin; decreased IFN-g, IL-12),
neuroborreliosis (increased IL-1
b, sIL-1R2, TNF-a, IFN-g, IL-6, IL-10, IL-12, IL-18,
transforming growth factor-
b1 (TGF-b1)), viral infections (increased IL-1b, IL-6, IL-8,
TNF-
a, IFN-g/a/b, TGF-b1), thimerosal (increased IL-5, IL-13; decreased IFN-g, TNF-a, IL-
6, IL-12p70, NOS), and valproic acid (increased NO, reactive oxygen species; decreased
TNF-
a, IL-6, IFN-g). The imbalances in pro- and antiinflammatory processes could
markedly hinder host defense mechanisms important for immune control of the parasite,
such as the production of NO, cytokines, and reactive oxygen/nitrogen species, tryptophan
degradation by indoleamine 2,3-dioxygenase and/or tryptophan 2,3-dioxygenase,
limitation of the availability of intracellular iron to
T. gondii, and the mechanisms
mediated by an IFN-
g responsive gene family. These fluctuations could result in a
recurrent profuse multiplication of
T. gondii in the brain associated with persistent
neuroinflammation, chronic overproduction of pro- and antiinflammatory cytokines, and
NO causing increased oxidative stress, and significantly depressed activity of several
enzymes including cytochrome P450 monooxygenase family responsible for metabolism
of physiological substrates and xenobiotics, such as steroids, fatty acids, prostaglandins,
drugs, pollutants, and carcinogens, finally leading to development of ASD. This reasoning
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
Contents lists available at
ScienceDirect
Research in Autism Spectrum Disorders
Journal homepage: http://ees.elsevier.com/RASD/default.asp
1750-9467/$ – see front matter
2009 Elsevier Ltd. All rights reserved.
doi:
10.1016/j.rasd.2009.09.011
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
1.1. ASD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
1.2. Toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
2. Cerebral toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
3. Pathophysiology of Measles-Mumps-Rubella (MMR) vaccination-associated adverse effects, thimerosal (THIM) and
T. gondii
infection may participate in development of ASD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
3.1. Diphtheria-Tetanus-Pertussis (DTP) vaccinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
3.2. Thimerosal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
3.3. Gene polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
4. Excessive changes in the weight status of ASD participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
4.1. Overweight and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
4.2. Wasting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
5. Increased oxidative stress in ASD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
6. Hypercytokinemic and hypermetabolic responses to
T. gondii infection in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
6.1. Overproduction of IFN and other cytokines in patients with ASD and during chronic
T. gondii infection in mice . . 000
7. Overproduction of nitric oxide in patients with ASD and its important role in control of
T. gondii infection. . . . . . . . . . . . 000
7.1. Patients with ASD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
7.2.
T. gondii infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
7.3. Depressed metabolism of endogenous and exogenous substances in the patients with ASD probably is due to a
significantly diminished activity of several enzymes by hypercytokinemia and overproduction of
reactive oxygen species (ROS) during neuroinflammation caused by
T. gondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
1. Introduction
1.1. ASD
Autism is a complex developmental disorder characterized by abnormalities of verbal and non-verbal communication,
stereotyped restricted interests and repetitive behavioral patterns, and impairment of socialization (
American Psychiatric
Association, 1994; Autism Society of America, 2009
). The 10-fold increase in autism diagnosis in the last decade now
affecting about 1 in 150 children in the U.S. has justifiably raised great public health concern (
CDC, 2005; McCarthy &
Hendren, 2009
). Hughes (2008) reported that in one U.S. study (MMWR Surveillance Summary, 2007) involving 2685
patients with autism, the prevalence was 0.7%, relatively low in Alabama and high in New Jersey. Recently,
Baron-Cohen
(2007)
found that in the South Thames region about 1% of children have an ASD. Advanced parental age appeared to be a risk
factor for development of ASD (
Durkin et al., 2008). Autism is not usually diagnosed until approximately 18 months of age
despite evidence of prenatal changes in the brain, and affect over 400,000 people in the United States (
Bryson & Smith, 1998;
Gillberg & Wing, 1999
). In Great Britain, the costs of supporting children with ASDs amount to be 2.7 billion pounds each
year, while for adults these costs amount to 25 billion pounds each year (
Knapp, Romeo, & Beecham, 2009).
Despite efforts to clarify contributing factors, the etiology and pathophysiology of this clinically heterogenous group of
disorders remain poorly understand. Observations from case reports and small case series provided however some evidence
of the potential etiologic role of both prenatal and early or late postnatal environmental factors that plays an important role
in aberrant brain development resulting in ASD. These environmental exposures suggested to be associated with ASD
include pregnancy, viral/bacterial infections, vaccinations, certain medications and other substances including valproic acid,
thalidomide, ethanol, thimerosal (THIM) (
Arndt, Stodgell, & Rodier, 2005; Chess, 1971; Libbey, Sweeten, McMahon, &
Fujinami, 2005; London & Etzel, 2000; Miller et al., 2005; Shi, Tu, & Patterson, 2005
).
Recently,
Prandota (2009a) suggested that neuropathological changes and clinical features of ASD subjects are similar to
these reported in congenital and chronic cerebral toxoplasmosis (CT) in humans and mice, and proposed that development of
ASD may be due to the reactivation of latent CT. The aim of this work was therefore to compare clinical and molecular
biochemical abnormalities reported in ASD subjects with these found in animals and men with CT. As a result of these
may be supported by such abnormal metabolic events as: (1) patients with ASD have
significantly decreased melatonin levels caused by marked deficit in acetylserotonin
methyltransferase activity, possibly resulting from maternal and/or fetal/postnatal overproduction
of NO, characteristic for this clinical entity; (2) thimerosal inhibited both insulinlike
growth factor-1- and dopamine-stimulated methylation reactions, and depressed
methionine synthase activity, the metabolic events important for promoting normal
neurodevelopment; (3) valproic acid, a strong histone deacetylase inhibitor, have potent
anti-
T. gondii activity. Thus, patients with ASD should be tested for T. gondii infection.
2009 Elsevier Ltd. All rights reserved.
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
studies, several clinical, histopathological and molecular explanations of the pathophysiological disturbances characteristic
for ASD patients, have been proposed.
1.2. Toxoplasmosis
Toxoplasma gondii
usually infects 10–80% of inhabitants of various countries, depending on hygiene standards, eating
habits, profession, living in a town or rural location (
Boyer, Marcinak, & McLeod, 2007; Rorman, Zamir, Rilkis, & Ben-David,
2006; Tenter, Heckeroth, & Weiss, 2000
), with largely yet unknown consequences. In the U.S. between 15 and 33% of all
adults are seropositive for antibody to this parasite, while in France, the seroprevalence has been estimated as high as 60–
70% by the sixth decade of life (
Kasper et al., 2004). In animals, prevalence levels vary widely and may surpass 50% in dogs,
rabbits, and sea otters, 60% in mice, rats, and wild birds, and 70% in cats, bears, deer, and humans (
Webster, 1994; Webster &
Macdonald, 1995
). Serological studies have also identified infection rates of 50% or higher in domestic chickens, gees, cattle,
goats, pigs, and sheep, with the animals themselves usually being asymptomatic. Outbreak of toxoplasmosis was associated
even with municipal drinking water (
Bowie et al., 1997), and because recently, a novel migration route of T. gondii from liver
to bile and feces has been reported (
Piao, Aosai, Mun, & Yano, 2005), both these findings may partly explain high frequency of
infection with this parasite.
T. gondii
remains as chronic, cryptic, latent brain infection throughout the life of the host (Boyer et al., 2007). Even in an
immunocompetent patient persistent toxoplasma bradyzoite cysts without evidence of toxoplasmosis were found (
Pusch,
Romeike, Deckert, & Mawrin, 2009
). Several reports suggested that chronic T. gondii infection might alter human behaviors,
cognitive functions, and cause cryptogenic epilepsy, headaches, and onset of schizophrenia (
Flegr, Kodym, & Tolarova, 2000;
Flegr et al., 2003; Hermes et al., 2008; Palmer, 2007; Webster, Lamberton, Donnelly, & Torrey, 2006
).
T. gondii
infection in an early stage of pregnancy has often serious impacts on the health of infected offsprings, including
microcephaly, hydrocephalus, mental retardation, convulsions, cerebral calcifications, and chorioretinitis (
Koppe & Rothova,
1989; Remington, McLeod, Thulliez, & Desmonts, 2001
). Delayed neurologic sequele, including lower intelligence quotient,
retarded psychomotor development, and sensorineural deafness, have also been demonstrated in subjects who were
exposed in utero, even among those with subclinical infection during the neonatal period (
Remington et al., 2001).
2. Cerebral toxoplasmosis
Immunocompetent hosts infected with
T. gondii must develop a powerful immune response that has to be under tight
control (
Aliberti, 2005) and persistently maintained during their lifetime in all infected tissues to avoid life-threatening
toxoplasmic encephalitis after reactivation of latent parasites (
Bhopale, 2003; Israelski & Remington, 1993). However,
Gazzinelli, Eltoum, Wynn, and Sher (1993)
found that acute CT was induced by neutralization of TNF-a and correlated with
down-regulated expression of iNOS and other markers of macrophage activation. Reactivation of CT was also demonstrated
after 2 weeks administration of dexamethasone, which induced depression in T
H1 immune responses (Kang, Choi, Shin, &
Lee, 2006
) probably associated with a promotion of type 2 cytokine production (Agarwal & Marshall, 2001), and following
treatment of some diseases with biologic agents, such as, for example, etanercept, which inhibits TNF-
a function, or
infliximab (
Hansen, Gartlehner, Powell, & Sandler, 2007; Lassoued, Zabraniecki, Marin, & Billey, 2007; Young & McGwire,
2005
). Moreover, exogenous donors of NO induced apoptosis in T. gondii tachyzoites via a calcium signal transduction
pathway because it caused a gradual decrease of calcium (Ca
2+) in tachyzoites that resulted in a continuous decline in their
motility and cell survival (
Peng, Lin, Lin, Jiang, & Zhang, 2003). Thus, the imbalance in TH1/TH2 cytokines (Gomez Marin,
Pinon, Bonhomme, & Guenounou, 1997
) and other mediators of inflammation associated with hypoxia, or produced after
administration of several drugs and substances (including donors of NO), along the course of some diseases, and clinical
states probably resulted in reactivation of latent CT and finally manifested as various types of headaches, including migraine
(
Koseoglu, Yazar, & Koc, 2009; Prandota, 2007, 2009b, 2010a, 2010b). Therefore, we believe that development of ASD
triggered by pregnancy, viral/bacterial factors, vaccinations, administration of some drugs, chemical substances including
organic and inorganic mercury, THIM, dsRNA poly(I:C), and ethanol, was associated with disturbances of
T. gondii and/or the
host immune defense mechanisms. Marked immune irregularities and abnormalities in other laboratory indices due to these
environmental factors have been summarized in
Table 1. These disorders probably finally induced disturbances in the fetal
brain development in utero and/or reactivation of latent congenital/acquired postnatally CT because of changes in the
intensity of the CNS infection/inflammation caused by the parasite.
Many populations of both T and non-T cells are important sources of IFN-
g in resistance against various bacterial, viral,
and parasite infections.
T. gondii infects a variety of host cells, and IFN-g-mediated immune responses control the parasite in
both phagocytic and non-phagocytic cells through at least six different mechanisms depending on the types of cells
responding to this cytokine. Such effector functions involve:
(1) mechanisms mediated by an IFN-
g responsive gene family. Several of these proteins, including IGTP, may be involved in
the processing and trafficking of cytokines and/or antigens. IGTP is an essential mediator of specialized antimicrobial
activities of IFN-
g,
(2) production of NO by inducible NO synthase (iNOS),
(3) production of various cytokines (TNF-alpha, IFN-
g, IL-1b, etc.),
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Table 1
Pregnancy, viral/bacterial infections, vaccinations, medications, and other environmental triggering factors associated with development of autistic spectrum disorders (ASDs) probably because of reactivation of
latent cerebral toxoplasmosis due to changes in
T. gondii and/or host defense mechanisms that affected intensity of the CNS infection/inflammation finally causing abnormal maturation of the brain.
Postulated triggering
factors associated
with ASD development
Disturbances in NO and ROS/RNI production, NOS and/or cytokines activation/levels,
and changes in IDO/TDO enzymes and other mediator activities
References
Pregnancy
a
(
Davies
et al., 1994
)
During normal and particularly abnormal pregnancy plasma NO metabolite levels were markedly increased.
In all three trimesters of normal pregnancy, and also postpartum, the numbers of IFN-
g- and IL-4-secreting
cells were significantly higher compared with nonpregnant controls, and the numbers of these cells gradually
increased as the pregnancy progressed compared with postpartum. Pregnancy estradiol concentrations
(and higher) enhanced production of IL-10 and reduced IL-12, IFN-
g levels and IFN-g/IL-10 ratio in stimulated
whole blood cells. Because of the known IL-10 inhibitory actions on T
H1 cells and monocytes/macrophages
these high IL-10 levels keep T
H2 cytokines favored during pregnancy
(
Matalka, 2003; Matthiesen, Ekerfelt,
Berg, & Ernerudh, 1998;
SchroNcksnadel et al., 2003; Tang,
Dorotheo, Schiffman, & Bahrani,
2004; Ligam, Manuelpillai, Wallace,
& Walker, 2005; Veenstra van
Nieuwenhoven et al., 2002; Veith
& Rice, 1999; Faas et al., 2005;
Belo et al., 2005; Kawaguchi et al.,
2005; Benedetto, Folgore,
Romano-Carratelli, & Galdiero,
2001; Von Mandach, Lauth, &
Huch, 2003; Goodrum, Saade,
Belfort, Moise, & Jahoor, 2003;
Aboagye-Mathiesen, Toth,
Zdravkovic, & Ebbesen, 1995
)
During pregnancy, percentage NK cells, helper lymphocytes, and cytotoxic lymphocytes that produced
IFN-
g and this cytokine plasma concentration significantly decreased compared with women in the follicular
phase of ovarian cycle. There was also a marked decrease in the percentage of helper lymphocytes producing
IL-2 in pregnant women compared with nonpregnant women, while the percentage of IL-4-producing
lymphocytes was not affected. First-trimester extravillous trophoblast cultures produced greater than 5-fold
more IFN production than third-trimester villous trophoblast on a per cell basis, whereas term
syncytiotrophoblast produced twice as much IFN as term mononuclear villous trophoblast when stimulated
with the same inducer
Median values of CRP levels were found to be consistently elevated throughout pregnancy. Moreover, circulating
levels of neutrophil-activation products were higher in normal gestation compared with non-pregnant controls
During pregnant period, PRL serum levels are drastically elevated. PRL significantly enhanced IFN-
g-induced IDO
expression in CD14
+ cells (prepared from peripheral blood of healthy controls) at comparable to those seen in a
pregnant period. In addition, TNF-
a and PRL up-regulated expression of ICAM-1 and production of endogenous
IL-6 and IL-3 by microglia, which could induce anti-
T. gondii actions in the brain
Maternal/fetal
influenza virus
infection
(
Julkunen et al.,
2000; Libbey et al.,
2005; Shi, Fatemi,
Sidwell, &
Patterson, 2003
)
Influenza A virus infection resulted in an increased TNF-
a, IL-1b, IFN-a/b, IL-6, IL-8, IL-18 production by
monocytes/macrophages and IP-10, MIP-1
a, MIP-1b, MCP-1, MCP-3, RANTES, MIP-1a. NOS-2 mRNA levels in
the brains of influenza virus infected mice showed greater levels than in control animals. Following an experimental
neurotropic viral infection, the expression of type III NOS on reactive astrocytes intimately associated with endothelial
cells and neurons increased substantially, predominantly in virally infected regions of the brain. Human influenza
infection in utero increased expression of GFAP in exposed cortical and hippocampal cells, and the GFAP-positive cells
in prenatally exposed brains showed hypertrophy. This virus also induced apoptotic DNA fragmentation and moderate
overproduction of ROS in primary cultured chorion cells prepared from human fetal membranes. It is interesting that
e.g. maternal influenza infection was likely to alter fetal brain development indirectly because viral RNAs were not
detectable in fetal brains from infected mothers, probably involving the maternal inflammatory response
(
Barna, Komatsu, & Reiss, 1996;
Fatemi et al., 2002; Julkunen et al.,
2000; Lee et al., 2007; Shi et al.,
2003, 2005; Sladkova &
Kostolansky, 2006; Uchide,
Ohyama, Bessho, Yuan, &
Yamakawa, 2002; Van Reeth,
2000; Watanabe, Kawashima,
Takekuma, Hoshika, & Watanabe,
2008
)
Measles virus (MV)
infection (
Fombonne,
1999; Libbey et al.,
2007; Singh &
Jensen, 2003
)
In autistic children, the level of measles antibody directed against a protein of 74 kDa was significantly higher as
compared with controls (
P = 0.003). MV infection caused marked serum increase of IFN-g and a decrease of absolute
number of platelets, total lymphocyte counts, CD3
+, CD4+, CD8+ cell counts and the CD4/CD8 ratio. The IFN-g level was
correlated negatively with the peripheral lymphocyte, CD3
+, CD4+ cell counts and the CD4/8 ratio. Primary MV
infections led in human malignant glioma cell lines to the induction of IL-1
b, IL-6, IFN-b and TNF-a. Persistently
infected with MV astrocytoma cells synthesized considerably higher levels of IL-1
b and TNF-a than uninfected cells.
MV proteins strongly induced expression of
b-family chemokines mRNA in human embryonic astrocytes. MV infection
of primary human monocytes specifically down-regulated IL-12 production critical for generation of cell-mediated
immunity. Cytokines in supernatants from PBMC of children who received measles vaccination showed a
predominant T
H1 cytokine pattern with increased plasma levels of TNF-a, IFN-g, and sIL-2R
(
DeLong, Bean, & Brown, 1981;
Karp et al., 1996; Ohga, Miyazaki,
Okada, Akazawa, & Ueda, 1992;
Ovsyannikova et al., 2003;
Schneider-Schaulies, Schneider-
Schaulies, & Ter Meulen, 1993;
Singh & Jensen, 2003; Xiao
et al., 1998
)
4
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
Herpes simplex virus
(HSV) infection
(
DeLong et al., 1981;
Ghaziuddin, Tsai,
Eilers, & Ghaziuddin,
1992; Gillberg, 1986,
1991; Lipkin &
Hornig, 2003
)
Increased TNF-
a, IL-2, IL-4, IL-6, IL-10 by HSV type 1. Increased IL-12, INF-g by HSV type 2. HSV-1 induced apoptotic cell death
by increasing intracellular ROS. HSV-2 established latent infection in a different population of ganglionic neurons than HSV-1.
Primary astrocytes as well as neurons supported HSV-1 replication, but these cell types did not produce cytokines or
chemokines in response to HSV-1. Microglia responded to nonpermissive viral infection by producing marked amounts of TNF-
a,
IL-1
b, chemokine IP-10, RANTES, together with smaller amounts of IL-6, IL-8, and MIP-1a. In mouse macrophages, HSV-2
synergized with INF-
g in the induction of NO production. Acyclovir/corticosteroids suppressed increased iNOS mRNA
expression observed during acute and chronic HSV encephalitis
(
Baskin, Ellermann-Eriksen,
Lovmand, & Mogensen, 1997;
Ellermann-Eriksen, 2005; Gosselin
et al., 1992; Hukkanen, Broberg,
Salmi, & Eralinna, 2002; Kim et al.,
2008; Lokensgard et al., 2001;
Malmgaard, Paludan, Mogensen,
& Ellermann-Eriksen, 2000;
Margolis, Imai, Yang, Vallas, &
Krause, 2007; Meyding-Lamade
et al., 2002; Sweeten, Posey,
et al., 2004; Wei, Zhang, Mei,
& Dong, 2006
)
Cytomegalovirus
infection (CMV)
(
Ivarsson, Bjerre,
Vegfors, & Ahlfors,
1990; Stubbs, Ash,
& Williams, 1984;
Sweeten, Posey, et al.,
2004; Yamashita,
Fujimoto, Nakajima,
Isagi, & Matshuishi,
2003
)
About 40% of women with primary opportunistic CMV infection during gestation transmit the infection to their fetus. In pregnant
women with acute CMV infection, increased serum TNF-
a, IL-8, IgM, and IgG, hyperproduction of Fc-receptors of NK cells, and B
lymphocytes (CD19
+) were found. In syncytiotrophoblast and trophoblast like cells cultures infected with CMV increased
amounts of IL-6 were found. In congenitally infected neonates, a predominant T
H1 response, as evidenced by IL-2, IL-8, IL-12
and IFN-
g with concomitant lack of IL-4, was reported. Human CMV induced production of IL-6 and TNF-a from macrophages and
microglial cells. Certain CMV strains induced generation of high amounts of IL-8, which in turn enhanced CMV replication in the
placenta. In murine CMV persistent infection, T cells were modified to produce massive amounts of TNF-
a and IFN-g upon in vivo
stimulation with anti-CD3. CMV infection induced IL-1
b release and subsequent up-regulation of proinflammatory adhesion
molecules on noninfected
neighboring cells through a paracrine mechanism. On HUVEC, surface expression of VCAM-1 and E-selectin was induced
de novo
on CMV infection and ICAM-1 surface expression was increased
>200%, while on human smooth muscle cells, ICAM-1 surface
expression induced
de novo, although VCAM-1 and E-selectin were not changed. During early pregnancy, women with
CMV-mRNA positive and IgM positive
had markedly increased the expressive quantity of decidual iNOS-mRNA. In endothelial cells, CMV infection increased oxidative
stress and impaired eNOS pathway. It was found that NO increased amounts of the viral DNA in lungs and hearts of mice latently
infected with thevirus, which may be the initial step of viral reactivation from the latent state. In human smooth muscle cells,
CMV induced also intracellular ROS generation and then used these ROS to facilitate its own gene expression and replication. It is
noteworthy that a possible neuroimmunological link between
T. gondii and CMV infections and personality changes was suggested.
Moreover, in autistic patients, subependymal cysts were detected after birth, as well as abnormal intensity area in the
periventricular white matter suggestive of disturbed myelination, and both these regions of the brain were characteristic for
T. gondii
infection. It must be added that, for example, in contrast to HeLa cells, BeWo trophoblasts were unable to control
replication of
T. gondii, even in the presence of exogenous IFN-g, because indoleamine 2,3-dioxygenase-dependent mechanism
was not operant in these cells, which may facilitate progression of infection caused by this intracellular pathogen at the
maternal-fetal interface
(
Asrankulova, Rizopulu, &
Kurbanov, 2004; Dengler, Raftery,
Werle, Zimmermann, & SchoN nrich,
2000; Halwachs-Baumann,
Weihrauch, Gruber, Desoye, &
Sinzger, 2006; Hassan, Dooley,
& Hall, 2007; Kovacs, Hegedus, Pal,
& Pusztai, 2007; Novotna et al.,
2005; Okada, Tanaka, Noda,
Okazaki, & Koga, 1999; Oliveira
et al., 2006; Pulliam, Moore, &
West, 1995; Speir, Shibutani,
Yu, Ferrans, & Epstein, 1996;
Speir, 2000; Tanaka & Noda,
2001; Wang, Wen, & Ling, 2002;
Weis et al., 2004
)
Rubella virus infection
(
Chess & Fernandez,
1980; Chess, Fernandez,
& Korn, 1978; Chess,
1971, 1977; Ueda,
Nishida, Oshima, &
Shepard, 1979
)
In 11–13 years old girls, vaccination with live attenuated vaccine Rudivax resulted in a marked decrease of CD3 and CD4
lymphocytes, and a significant increase of plasma TNF-
a and IL-10 levels with maximum on the day 30 after immunization.
Simultaneously, a significant reduction in plasma IFN-
g accompanied by a marked elevation of IL-4 was found. This
evidence of immunosuppression persisted for at least 1 month after vaccination
(
Pukhalsky et al., 2003)
Measles–mumps–rubella
vaccinations (
Kaye et al.,
2001; Patja et al., 2000;
Taylor et al., 2002
)
Immunization with live measles virus vaccine caused a markedly increased production of IL-4, TNF-
a, accompanied by low levels
of IFN-
g, IL-1a, and PGE2. Similar constellation of cytokines was reported after measles–mumps–rubella-II (MMR-II) immunization.
This may suggest that T
H2 cells producing IL-4 are preferentially activated by measles vaccine and may contribute to the
immunologic abnormalities associated with measles and possibly other viral infections. It is interesting that single-nucleotide
polymorphisms (SNPs) within IL-2 gene were associated with high antibody and high lymphoproliferative responses, whereas SNPs
within IL-10 and IL-12R genes were associated with low antibody and lymphoproliferative responses to measles. Significant
associations were also found between SNPs and secreted cytokine levels
(
Dhiman et al., 2005, 2007;
Ovsyannikova et al., 2005;
Ward & Griffin, 1993
)
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Table 1 (
Continued )
Postulated triggering
factors associated
with ASD development
Disturbances in NO and ROS/RNI production, NOS and/or cytokines activation/levels,
and changes in IDO/TDO enzymes and other mediator activities
References
Bacterial/viral
vaccinations in infants
(
Duclos & Ward, 1998;
Fombonne, 1999; Kaye
et al., 2001; Patja et al.,
2000; Rimland, 2000;
Shoenfeld & Aron-Maor,
2000; Taylor et al., 2002;
Wakefield & Montgomery,
1999
)
BCG vaccination in rats caused an increase in NO synthesis that was maximal 10 days after vaccination and returned to initial
values after 20 days. Also routine influenza vaccination of hospital workers resulted in a significant rise in mean peak exhaled
NO between days 0 and 7 compared with controls, and it was accompanied by increased sputum lymphocytosis and
respiratory epithelial shedding. Administration of the whole cell DTP vaccination (plus LPS) increased mRNA expression/plasma
levels for IL-1
b, IL-2, IL-5, IL-6, TNF-a, IFN-g, and iNOS, as well as revealed latent urinary tract diseases in some genetically
predisposed children. Influenza virus and rubella virus vaccinations also markedly increased serum IFN-
a and IFN-g levels.
Rubella viral antigen-antibody complex (VAAC) caused the release of superoxide anion (O
2
) from human polymorphonuclear
leukocytes proportional to the amount of VAAC. In chronic active hepatitis B there was a generation of type 1 cytokine profile.
Interferon substantially induced IDO and increased
L-tryptophan metabolism in human peripheral blood monocytes
(
Ansher & Thompson, 1994;
Blood-Siegfried et al., 1998;
Kimura et al., 1997; Meredith
et al., 1985; Ozaki, Edelstein,
& Duch, 1987; Penn &
Williams, 1984; Prandota,
2004a, 2004b; Ryan et al.,
1997; Shandrenko & Dmitrenko,
2001; Tetteh et al., 2003;
Thomas, Ng, Elsing, &
Yates, 1999
)
In children, vaccine-modified measles was associated with an early up-regulation of T
H1 cytokine production (increased plasma
levels of IFN-
g, IL-2, IL-12) and a down-regulation of TH2 cytokine production (IL-4, IL-10). Kinetically, IL-4 levels increased from
day 0 to days 14 and 60, while IFN-
g and IL-10 decreased consistently from day 0 to days 14 and 60
Lyme neuroborreliosis
(
Bransfield, Wulfman,
Harvey, & Usman, 2008
)
In patients with Lyme disease serum concentration of TNF-
a, IL-1b, IL-1Ra, IL-6, sIL-6Ra, sgp130, and IL-15 were significantly
higher than those of control group. PBMC of these patients showed an increased IFN-
g and decreased IL-4 production. Borrelia
burgdorferi
induced also NO production by macrophages and neural cells. Rat isolated Kuppfer cells stimulated by this
pathogen induced ROS and iNOS production
(
Bransfield et al., 2008;
Jablonska & Marcinczyk, 2006;
Seiler, Vavrin, Eichwald, Hibbs,
& Weis, 1995; Tatro, Romero,
Beasley, Steere, & Reichlin,
1994; Marangoni et al., 2006
)
Thimerosal (THIM),
organic and inorganic
mercury (
Amin-Zaki et al.,
1979; Bernard, Enayati,
Redwood, Roger, & Binstock,
2001; Rimland, 2000
)
THIM, a preservative added to MMR and other vaccines, in a concentration-dependent manner inhibited LPS-induced
proinflammatory cytokines TNF-
a, IL-6, and IL-12p70 from human monocyte-derived dendritic cells. These THIM-exposed
dendritic cells induced T
H2 (IL-5 and IL-13) and decreased TH1 (IFN-g) cytokine secretion from T cells in absence additional
THIM added to coculture. THIM exposure of dendritic cells led to the depletion of intracellular glutathione (GSH). Furthermore,
THIM, in a concentration- and time-dependent manner, induced apoptosis in T cells via mitochondrial pathway by inducing
oxidative stress and depletion of GSH. Neuronal cell THIM-induced apoptosis was associated with generation of ROS, including
H
2O2, and release of cytochrome c and apoptosis-inducing factor from mitochondria to cytosol. Hydrogen peroxide alone
appeared to be responsible for THIM-mediated oxidative stress-induced apoptosis. THIM increased free arachidonic acid levels,
elicited endothelium-dependent vasodilation that was associated with increased release of epoxyeicosatrienoic acids (EETs)
(EETs are cytochrome P450-derived metabolites of arachidonic acid). THIM inhibited both IGF-1- and dopamine-stimulated
methylation with IC
50 of 1 nM and eliminated methionine synthase activity. A blood mercury level of 29 nM has been
recommended by the Environmental Protection Agency as a reference value for defining toxic exposure and inorganic
mercury inhibited IGF-1-stimulated methylation with an IC
50 of 15 nM
(
Agrawal, Kaushal, Agrawal,
Gollapudi, & Gupta, 2007;
Chen, Jiang, & Quilley, 2003;
Makani et al., 2002; 1997
Mercury Study Report to
Congress, 2009; Mian et al.,
2008; Waly et al., 2004; Yel,
Brown, Su, Gollapudi, &
Gupta, 2005
)
Thalidomide (THAL)
(
Miller & StroNmland,
1993; StroNmland,
Nordin, Miller,
Akerstrom, &
Gillberg, 1994
)
THAL embryopathy affected fetal development early in pregnancy, probably on days 20–24 after conception. THAL enhanced
proliferation of CD8
+ T cells, NK cells in PHA-stimulated PBMC from healthy subjects, and increased markedly secretion
of IL-6 and decreased secretion of IFN-
g from these cells. The drug inhibited also TNF-a, VEGF, and bFGF secretion, and
exhibited weak NOS-inhibitory activity. When pregnant rats were exposed to THAL on embryonic day 9 (E9), a dramatic shift
of the distribution of serotonergic neurons in the dorsal raphae nucleus was observed on postnatal day 50. The exposure
to THAL on E9 resulted in an increase of hippocampal serotonin, frontal cortex dopamine, and hyperserotoninemia
(
Melchert & List, 2007;
Miyazaki, Narita, & Narita,
2005; Narita et al., 2002;
Shimazawa, Sano, Tantani,
Miyachi, & Hashimoto,
2004; StroNmland et al.,
1994; Yang et al., 2006
)
6
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
Valproic acid (VPA)
(
Arndt et al., 2005;
Christianson, Chesler,
& Kromberg, 1994;
Moore et al., 2000;
Schneider & Przewłocki,
2005; Trottier,
Srivastava, & Walker,
1999; Williams & Hersh,
1997; Williams et al.,
2001
)
VPA significantly induced production of IL-1
a (the cell associated form), IL-1b (the readily secreted form), IL-6, ROS, NO, and
monocyte chemoattractant protein-1 (MCP-1). [Nb.] MCP-1, a member of the
b-chemokine subfamily exhibits potent
chemotactic activity toward monocytes, macrophages, and T lymphocytes attracting them to sites of inflammation, and
induces production of superoxide anions and cytokines in monocytes. When pregnant rats were exposed to VPA on
embryonic day 9 (E9), a dramatic shift of the distribution of serotonergic neurons in the dorsal raphae nucleus was
observed on postnatal day 50. The exposure to VPA on E9 resulted in an increase of hippocampal serotonin, frontal cortex
dopamine, and hyperserotoninemia. Gender-specific behavioral and immunological alterations have been reported especially
in male rats prenatally exposed to VPA. The male VPA rats had lower sensitivity to pain, increased repetitive/stereotypic-like
activity, higher anxiety, decreased level of social interaction, increased basal level of corticosterone, decreased weight of
the thymus, decreased splenocytes proliferative response to Con-A, lower IFN-
g/IL-10 ratio, and increased production of
NO by peritoneal macrophages. Female VPA rats exhibited only increased repetitive/stereotypic-like activity and
decreased IFN-
g/IL-10 ratio
(
Karabiber, Yakinci, Durmaz,
Temel, & Mehmet, 2004; Kawai
& Arinze, 2006; Miyazaki et al.,
2005; Narita et al., 2002;
Phiel et al., 2001; Rollins,
1996; Strobl, Cassel, Mitchell,
Reilly, & Lindsay, 2007;
Verrotti et al., 2001; Wu, Koga,
Martin, & Meydani, 1999
)
It must be emphasized that VPA is an effective inhibitor of histone deacetylase (HDAC) (a family of enzymes that participate
in the regulation of chromatin structure, gene expression, and cell signaling), with an IC
50 (0.4 mM) well within the
therapeutic range of VPA (0.35–0.7mM in serum).
T. gondii expresses a HDAC class I enzyme homologous to human hdac3,
and VPA inhibited the parasite tachyzoite proliferation at concentrations only a few times greater than its respective IC
50
Ethanol (ETH)
(
Greenbaum, Nulman,
Rovet, & Koren, 2002;
Harris, MacKay, &
Osborn, 1995; LoN ser,
2000; Nanson, 1992;
Riley & McGee, 2005
)
Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. ETH significantly
enhanced TNF-
a, IL-6, and TGF-b1 in vitro production by various cells. Chronic ETH treatment resulted in a significant
increase of serum IL-10, TNF-
a, IFN-g, TGF-b1, VEGF-A levels after 12 weeks. Also serum nitrite levels and hemolysate TBARS
level were increased, while total antioxidant status and GSH content in whole blood hemolysate decreased from 4th week
onwards of exposure. ETH exposure during embryonic cerebral cortical neuroepithelial proliferation prevented the-early
differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion. Among active
alcoholics without liver disease, a significantly increased spontaneous production of IL-1
b, IL-6, IL-12, and TNF-a by peripheral
blood monocytes was observed. In developing rat cerebellum ETH treatment on postnatal day 4 resulted in ROS increases while
exposure on P14 produced consistent decreases in ROS production. Long-term alcohol ingestion impaired both eNOS-dependent
and nNOS-dependent reactivity of cerebral arterioles in male rats, but surprisingly nNOS-dependent dilatation of cerebral
arterioles in female rats was not impaired. It is interesting that alcohol exposure resulted also in cerebellar Purkinje cell number
loss (most consistent finding in autism) and density reduction, and a decrease of cerebellar lobule I volume. ETH potently
inhibited basal- and IGF-1-stimulated (IGF-1) methionine synthase activity, reduced folate-dependent methylation, and blocked
the ability of IGF-1 to increase DNA methylation. The IC
50 for ETH inhibition of methylation (8 mM) corresponded to blood levels
produced by only one or two drinks, thus indication its potential for hindering methylation events from only moderate drinking
(
Camarillo, Kumar, Bake,
Sohrabji, & Miranda, 2007; Das
et al., 2009; Heaton, Madorsky,
Paiva, & Mayer, 2002; Heaton,
Paiva, Mayer, & Miller, 2002;
Jeong, Hong, Park, An, & Kim,
2005; Kern, 2003; Laso,
Vaquero, Almeida, Marcos,
& Orfao, 2007; Lee, Rowe,
Eskue, West, & Maier, 2008;
McVicker, Tuma, Kharbanda,
Kubik, & Casey, 2007; Sun &
Mayhan, 2005; Waly
et al., 2004
)
Organophosphate
poisoning (
Pasca
et al., 2006
)
Paraoxonase is the enzyme responsible for organophosphate detoxification in humans. In North America, autism has been
associated with variants in the paraoxonase gene which can decrease the activity of this enzyme by 50%. After
N. brasiliensis
infection in Wistar rats, a significant reduction in serum paraoxonase and arylesterase activity was found.
N. brasiliensis infection
also increased serum concentrations of proinflammatory IL-1, IL-6, and TNF-
a cytokines, which are known to inhibit synthesis of
PON1 mRNA. Thus, the observed reduction in PON1 activity during
N. brasiliensis infection was likely associated with
inflammatory reactions against the parasites. Moreover, in PON1(/) mice, a twofold increase in leukocyte adhesion vs. wild-type
controls was reported. This finding correlated with a significant increases in aortic mRNA levels of P-selectin, upregulation in
VCAM-1 and ICAM-1, and an increase in aortic superoxide production rate. Furthermore, PON1 mRNA expression by HepG2 cells
was decreased within 3 hours of stimulation by IL-
b or TNF-a. Finally, it was found that serum PON1 activity and serum NO levels
were significantly decreased in patients with hepatosteatosis, and PON1 activity correlated positively with serum NO
levels (
r = 0.51, P < 0.001)
(
Atamer et al., 2008; D’Amello
et al., 2005; Farid, Nakahara,
Murakami, Hayashi, & Horii,
2008; Kumon et al., 2002;
Ng et al., 2008; Saemundsen,
Ludvigsson, Hilmarsdottir, &
Rafnsson, 2007; Worth, 2002
)
a
Pregnancy induces production of enhanced amounts of various proinflammatory cytokines, and thus may reactivate latent toxoplasmosis and facilitate transmission of T. gondii tachyzoites to the fetus. GFAP,
glial fibrillary acidic protein; PGE2, prostaglandin E2, RANTES, regulated on activation, normal T-cell expressed, and secreted.
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
(4) tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase,
(5) limiting the availability of intracellular iron to the parasite, and
(6) production of reactive oxygen/nitrogen species/intermediates, ROS/RNI (
Butcher et al., 2005; Denkers & Gazzinelli, 1998;
Fujigaki et al., 2002; Halonen, Chiu, & Weiss, 1998; Suzuki, 2002
).
Reactivation of chronic toxoplasmosis resulting in toxoplasmic encephalitis is a common event in AIDS patients (
Butcher
& Denkers, 2002; Derouich-Guergour et al., 2002; Mathew & Chandy, 1999; Reiter-Owona et al., 2000; Suzuki, 2002
).
Conversion from
T. gondii bradyzoites to tachyzoites is a prerequisite for reactivation. The control of multiplication of
tachyzoites was found to be largely dependent on endogenous IFN-
g with partial involvement of TNFRp55 and iNOS, and
characterized by a dramatic increase in tachyzoite-specific antigen SAG-1 (
Gross et al., 1997; Kim & Boothroyd, 2005; Reiter-
Owona et al., 2000; Seng et al., 2004; Silva, Tafuri, Alvarez-Leite, Mineo, & Gazzinelli, 2002
).
The presence of external stress factors, such as IFN-
g-mediated NO formation has been identified to stabilize the cyst
stage, and reactivation of chronic toxoplasmosis thus might occur in the absence of these factors, as it has been observed in
AIDS patients with decreased IFN-
g levels (Gross et al., 1997). Reiter-Owona et al. (2000) demonstrated that in AIDS subjects
this transformation process appeared to be nonsynchronous and the manifestation of toxoplasmic encephalitis was
dependent on the degree and site of tissue destruction. Cyst rupture as the first event in the process of reactivation was not
seen and it was suggested that tachyzoites could invade by dissemination across the blood–brain barrier. The authors
suggested that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical
symptoms occur (
Luft & Remington, 1992). When a chronically infected host eventually becomes immunodeficient,
bradyzoites reactivate to tachyzoites causing neurological diseases (
Luft & Remington, 1992; Prandota, 2009b, 2010a,
2010b
). T. gondii can control the regulation of the expression of TNF-a receptors on human cells in vitro (Derouich-Guergour
et al., 2002
) and this mechanism may influence the role of TNF-a in clinical course of toxoplasmosis. Moreover, effects the
parasite on IL-12 and TNF-
a production are nonidentical, with T. gondii exerting a longer-lasting suppression of the latter
because mechanism of entry determines the ability of the parasite to inhibit macrophage proinflammatory cytokine
production (
Butcher & Denkers, 2002).
3. Pathophysiology of Measles-Mumps-Rubella (MMR) vaccination-associated adverse effects, thimerosal (THIM) and
T.
gondii
infection may participate in development of ASD
During a 14-year prospective follow-up afterMMRvaccination,
Patja et al. (2000) reported several adverse events such as
neurological, allergic, and miscellaneous reactions and one death, with febrile seizures being the most common side effect.
Wakefield et al. (1998)
demonstrated eight children with autism whose first symptoms appeared within 1 month after
receiving an MMR vaccine, and an association between autism and the MMR vaccine has been sometimes proposed (
Kaye,
del Mar Melero-Montes, & Jick, 2001; Taylor et al., 2002; Wakefield & Montgomery, 1999
). Recently, Gerber and Offit (2009)
suggested that although epidemiologic studies did not support the association between MMR vaccine or THIM and autism,
the simultaneous administration of multiple vaccines could overwhelm or weaken the immune system of the vaccinated
child. It seems however that the innate immune molecular abnormalities caused by these vaccines and their components,
such as THIM and aluminium, may have contributed to the risk of autism, ADHD and other developmental disorders (
Waly
et al., 2004
).
3.1. Diphtheria-Tetanus-Pertussis (DTP) vaccinations
Wilson (1967)
argued that in some instances immunization could shorten the incubation period of certain diseases or
convert a latent infection/inflammation into clinically apparent disease. The necessary precondition for such an occurrence
was the presence of latent infection or asymptomatic colonization (
Wilson, 1967). Prandota (2004a, 2004b) suggested that in
infants and young children, DTP vaccination revealed some urinary tract diseases, such as acute renal failure, nephrotic
syndrome, or pyleonephritis (
Table 2). It was proposed that cytokine irregularities and down-regulation of several
cytochrome P450 enzymes induced by the vaccine uncover latent diseases in genetically predisposed individuals (
Prandota,
2004a, 2004b
). In animal models, large doses of pertussis toxin caused hyperinsulinemia and hypoglycemia as well as
leukocytosis with a predominant lymphocytosis (
Munoz, Arai, Bergman, & Sadowski, 1981). Studies in mice showed that
administration of the whole-cell DTP vaccine caused dose- and time-dependent marked increases in the hepatic mRNA
expression for IL-6, IL-1, and TNF, as well as a significant depression lasting for about 7 days in the expression of mRNA and
activities of liver isoenzymes of cytochrome P450 (
Ansher & Thompson, 1994; Ansher, Thompson, Bridgewater, & Snoy,
1993; Fantuzzi et al., 1994
). Both spleen and liver weights of mice were increased for 7–14 days following DPT vaccine
administration, and histopathologic tissue examination showed random multifocal inflammation with hepatocyte necrosis
(
Ansher, Thompson, Snoy, & Habig, 1992). DTP vaccine also caused marked induction of INF-g coincident with the maximal
inhibition of CYP450 levels (
Ansher et al., 1993), and increased inducible NOS mRNA expression (Blood-Siegfried, Crabb
Breen, Takeshita, & Martinez-Maza, 1998
). Blood-Siegfried et al. (1998) suggested that the whole-cell pertussis present in
DTP vaccine produce symptoms reminiscent of biological responses to circulating proinflammatory monokines such as IL-
1
b, TNF-a, and IL-6. The whole-cell vaccine in vitro induced significantly more these cytokines production than did the
acellular pertussis or diphtheria-tetanus-only vaccine. They believed that although pertussis endotoxin was a major inducer
8
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
Table 2
Clinical and laboratory data of children with urinary tract diseases revealed after DTP vaccination (
Prandota, 2004a, 2004b).
Pt Age (months), sex Patient’s history/physical
examination findings
DTP vaccination dose;
time from vaccination
to apparent clinical
symptoms (h)
Clinical symptoms before admission Laboratory, ultrasonographic, and voiding
cytourethrographic data
1 3, M Syncope 1 month earlier 3rd; 5 Syncope, tachycardia ESR 50/h, UTI, pyuria, bilateral VUR
2 3, M 9 pts Apgar, unrest 3 weeks
earlier, atopic dermatitis
2nd; 8.5 Dysuria, unrest, crying, bending legs,
fever 38
8C, right OM, urethritis, rhino
ESR 21/h, CRP 40.7 mg/L, WBC 22.3
109/L,
PLT 536
109/L, serum K+ 5.89 mmol/L, UTI, pyuria
3 4, M Dysuria from 2 months,
atopic dermatitis
2nd; 4 days Vomitus, fever 39
8C, brown urine ARF (Ckr 30 mL/min), UTI, pyelonephritis, bilateral VUR,
BP 160/100 mmHg, Hb 0.93 mmol/L, WBC 16,2
109/L,
pyuria, erythrocyturia
4 4, F 9 pts Apgar, right-sided
lower tonus of muscles
from the 1st month of age
2nd; 2 Edema of legs, dysuria, fever 38
8C,
unrest, cyanosis from 2 weeks during
rehabilitation with Vojta method
ESR: 15/h, Hb 1.66 mmol/L, WBC 16.8
109/L, PLT
487
109/L, CRP 44.6 mg/L, IgG 0.69 g/L (n = 1.9–8.6);
IgM
< 0.17 g/L (n = 0.25–1.2 g/L), UTI, pyuria
5 5, F UTI and pyuria at the 2nd
month of age
3rd; few Vomitus, fever 40
8C ESR: 25/h, UTI, left VUR, pyuria
6 5, F 8 pts Apgar 2nd; 10 days Fever, dysuria, unrest ESR 5/h, serum K
+ 5.88 mmol/L, bilateral VUR, right
pyelon and ureter duplex, UTI, pyuria
7 5, M Atopic dermatitis 1st; 6 days Green, watery stools with mucus ESR 7/h, UTI, pyuria
8 7, M Atopic dermatitis, UTI at
the 3rd month of age
3rd; 4.5 Temp. 38.9
8C, unrest, groaning,
urethritis, rhino, right OM
ESR: 20/h, CRP 28.8 mg/L, WBC 15.7
109/L, serum
K
+ 4.01 mmol/L, UTI, pyuria
9 20, F 8 pts Apgar 2nd; few Temp. 38
8C, pharyngitis ESR: 37/h, UTI, pyuria
10 21, M Purulent angina twice in
the last month
4th; few Edema of the extremities, ascites ESR: 90/h, UTI, pyuria
11 21, F Purulent angina 4th; 3 Drowsiness, perioral cyanosis ESR: 15/h, UTI, pyuria
12 24, F Atopic dermatitis 4th; 4 days Oliguria, and generalized edema SSNS with ARF, pyuria
13 65, M SSNS, atopic dermatitis 5th; 7 days Pharyngitis First proteinuria 2.1 g/L, and then full blown
symptoms of SSNS
UTI, urinary tract infection; VUR, vesicoureteral reflux; SSNS, steroid-sensitive nephrotic syndrome; ARF, acute renal failure; GLN, glomerulonephritis; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein;
WBC, white blood cells; PLT, platelets; BP, arterial blood pressure; rhino, rhinopharyngitis; OM, otitis media.
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of IL-6, other components of
Bordetella pertussis also contributed to this cytokine production by monocytes (Blood-Siegfried
et al., 1998
). In other studies performed in infants, antigen-stimulated peripheral blood mononuclear cells of responsive
whole-cell pertussis vaccine recipients were characterized by an elevated production of INF-
g (which inhibits the growth of
T
H2 cells), and IL-2, low to minimal production of IL-5, and no production of IL-4 (Ausiello, Urbani, la Sala, Lande, & Cassone,
1997; Ryan, Gothefors, Storsaeter, & Mills, 1997; Ryan et al., 1998
). In addition, B. pertussis vaccine decreased phenytoin
hydroxylation and elimination rate by depressing CYP450 levels in hepatic endoplasmic reticulum (
Renton, 1979). Tables 3–
6
presented effects of DTP vaccinations on the liver and spleen weights, tissue histopathologic changes, pathophysiologic
functions, biomarkers, and changes in T
H1 and TH2 cells cytokine profiles in mice and infants (Ausiello et al., 1997; Blood-
Siegfried et al., 1998; Ryan et al., 1997, 1998
).
Mordue, Monroy, La Regina, Dinarello, and Sibley (2001)
found that lethal infections caused by type I (RH) or type II (PTG)
strain of
T. gondii infections were accompanied by extremely elevated serum levels of TH1 cytokines, including IFN-g, TNF-a,
IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced
during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by
neutralization of IL-18, but not TNF-
a or IFN-g. Nonlethal infections with a low dose of type II PTG strain parasites were
characterized by a modest induction of T
H1 cytokines that led to control of infection and minimal damage to host tissues.
Thus, for the host protection overstimulation of immune responses are normally necessary (
Mordue et al., 2001).
Gavrilescu and Denkers (2001)
found that infection with the RH strain of T. gondii led to widespread parasite
dissemination and rapid death of mice, while mice survived low virulence strain ME49 infection, and tachyzoite
dissemination was much less extensive. Furthermore, massive apoptosis and disintegration of the splenic architecture was
characteristic of RH, but not ME49, infection. In addition, hyperinduction of IFN-
g and lack of NO production were found
during RH, in contrast to ME49 infection. It must be noted that in a cell culture environment RH Ankara strain tachyzoites
markedly decreased their average size from 3
mm 5.7mm prior to the first inoculation to 1mm 2.1mm after 2 months
(
DoNskaya et al., 2006). The preliminary results of virulence showed that as the size of cell culture-derived tachyzoites
Table 5
Changes of some biomarkers after administration of different doses of DTP vaccine and its components in mice (
Ansher et al., 1993).
DTP vaccination elicited dose- and time-dependent alterations in hepatic drug metabolism, i.e.: CYP450 levels became depressed more than 50%
at 7 days following a single injection of PT (pertussis toxin) mixed with DT (diphtheria and tetanus toxoids) or acellular pertussis (AP) vaccine
adsorbed
DT combined with 125 ng of PT was required to produce this effect, 16 ng of PT combined with APDT vaccine produced similar effect
Alterations of hepatic CYP450 levels were associated with increased quinone reductase activity, but with no changes in plasma IL-6 or TNF levels
Endotoxin caused alterations in hepatic drug metabolism within 24 h but these effects had resolved by 7 days
DTP vaccine and PT preparations caused a marked induction of IFN-
g coincident with maximal inhibition of CYP450 levels
Table 3
Reactions to different types of DTP vaccination in mice (
Fantuzzi et al., 1994).
Type of DTP vaccine Reactions to various types of DTP vaccinations in mice
Wild-type DTP vaccines Decreased liver microsomal CYP450 levels by 50%, high level of IL-6, prolonged hexobarbital-induced
sleeping time
Mutated whole-cell vaccine Decreased liver microsomal CYP450 levels by 30% paralelled by the prolongation of hexobarbital-induced
sleeping time, increased serum IL-6 induction compared with the wild-type DTP vaccine
Acellular vaccine mutated No effect on liver drug metabolism, hexobarbital-induced sleeping time, or IL-6 serum levels
Table 4
Sequence of biochemical markers changes observed after the whole-cell DTP vaccination in mice (
Ansher & Thompson, 1994; Fantuzzi et al., 1994).
Time after DTP vaccination (h) Biochemical markers changes found in mice after the whole cell vaccination
1–2 3–6-fold increases in mRNA expressions for IL-1, IL-6 and TNF
4 Peak of the increase of inducible NO synthase mRNA expression
8–12 Markedly reduced mRNA expressions for liver isoenzymes CYP 1A2, and 2E1
Table 6
Changes in the spleen and liver weights, tissue histopathologic changes, pathophysiologic functions, and cytokine levels following DTP vaccination and
endotoxin in mice and infants (
Ansher et al., 1992; Blood-Siegfried et al., 1998; Ryan et al., 1997).
A single human dose of 0.5 mL of DTP vaccine increased hexobarbital-induced sleep times to 1.6–1.8-fold above those of controls both in the
endotoxin-responsive (ER) and NR mice and this effect persisted for 7 days
Hepatic CYP450 levels were decreased by 30–40% 24 h after DTP vaccine administration
Both spleen and liver weights of the ER and NR mice were increased during 7–14 days following DTP vaccine injection, and histopathologic tissue
examination showed random multifocal inflammation with hepatocyte necrosis
Plasma IL-6 and TNF levels in ER mice were markedly increased after DTP and LPS treatment, while NR mice had reduced increases
Peripheral blood monocytes from vaccinated infants produced IL-2, IL-5, and IFN-
g, while the spleen cells produced IL-5
10
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
decreased markedly, the virulence in mice also diminished (
DoNskaya et al., 2006). Thus, these data demonstrated that
Toxoplasma
strain characteristics and tachyzoites size exerted a profound effect on the host immune response and that both
these features are crucial determinants in parasite virulence and its eventual detecting in the brain.
3.2. Thimerosal
King, Lindsay, Holladay, and Ehrich (2003)
demonstrated that there was a significant increase in mice brain tissue cyst
counts within the group exposed to bothmethylmercury (MeHg) and
T. gondii vs. T. gondii alone, indicating that bothMeHg and
T. gondii
had synergistic effects,with effects of MeHg especially on the immune system. THIM is an ethylmercury derivative of
thiosalicylate for a long time used as a preservative to block the growth of contaminating organisms in biological products.
THIMinhibited insulin-like growth factor-1 (IGF-1) and dopamine-stimulatedmethylation reactions in the body important for
growth factors and the promotion of normal neurodevelopment (
Waly et al., 2004). Moreover, THIM has been reported to
activate apoptosis in T cells (
Makani, Gollapudi, Yel, Chiplunkar, & Gupta, 2002) and induce DNA breaks, membrane damage,
and cell death in cultured human neurons and fibroblasts (
Baskin, Ngo, & Didenko, 2003). Clinical importance of these findings
may be supported by the observation that a single THIM-containing vaccination produced ethylmercury blood levels of 10–
30nM(
Stajich, Lopez,Harry, & Sexson, 2000), and blood samples in 2-month-old infants, obtained 3–20 days after vaccination,
contained 3.8–20.6 nM ethylmercury (
Pichichero, Cernichiari, Lopreiato, & Treanor, 2002).
3.3. Gene polymorphisms
Adenosine deaminase activity was found to be reduced in autism (
Stubbs et al., 1982) and a polymorphism in the
adenosine deaminase gene resulting in a lower activity of the enzyme is over-represented in autism (
Bottini et al., 2001;
Persico et al., 2000
). This may lead to higher adenosine levels and enhanced build-up of preblock intermediates, such as Sadenosylhomocysteine,
that may affect physiological neurodevelopment.
Polymorphisms in genes encoding cytokines have been shown to have association with parasitic diseases. The IFN
g
+ 874T/A gene polymorphism was found to be associated with susceptibility to retinochoroiditis toxoplasmosis (
De
Albuquerque et al., 2009
). In addition, the polymorphism IL-10-592*A gene, as well as the constellation of TNF-a and IL-6
genetic variants may also predispose some infants to a more than usually intense inflammatory response after various
vaccinations (
Summers et al., 2000). Thus, it seems convincing that several environmental triggering factors, includingMMR
vaccination and THIM, may uncover
T. gondii infection and favour development of ASD in certain predisposed individuals
(
Table 1). Before administration of a MMR vaccine it is therefore advised to take a careful history from both the child and his
parents in order to avoid serious clinical mishaps.
4. Excessive changes in the weight status of ASD participants
4.1. Overweight and obesity
Children with autism had a serious prevalence of at-risk-for overweight and overweight (
Curtin, Bandini, Perrin, Tybor, &
Must, 2005; Sugiyama, 1991; Takeuchi, 1994; Xiong et al., 2009
). Xiong et al. (2009) found that among 380 boys and 49 girls
with ASD, the prevalence of at-risk-for or being overweight were 31.8 and 17% in 2–5 years old group, and 37.9 and 21.8% in
6–11 years old group. Other authors (
Curtin et al., 2005) reported that the prevalence of at-risk-for-overweight was highest
in the 12–17.9 years old group, and in a large study of 20,031 Japanese children and adolescence with mental retardation that
included 413 children with autism, the prevalence of obesity was found to be 22% in boys and 11% in girls (
Takeuchi, 1994).
Proinflammatory cytokines, such as IL-1, TNF-
a, sTNFR-1 and sTNFR-2 have been shown to be elevated in obese patients and
to decline with weight loss (
Himmerich et al., 2006; Zimmermann, Kraus, Himmerich, Schuld, & PollmaNcher, 2003), in
general population (
Himmerich et al., 2006), as well as in obese prepubertal children (Table 7; Aygun, Gungor, Ustundag,
Gurgoze, & Sen, 2005; Kapiotis et al., 2006
). In these children statistically significant positive correlations were found
between leptin and IL-2, IL-1
b, IL-6 or TNF-a serum concentrations (Aygun et al., 2005). There was also a significant negative
Table 7
Serum proinflammatory cytokines and leptin concentrations in obese children at prepubertal age compared with healthy children of the same age.
Parameters Obese children Controls
P
Leptin (ng/mL)
a 19.9 7.4 7.9 5.1 <0.001
IL-1
b (pg/mL) 33 8.9 3.6 1 <0.001
IL-2 (U/L) 0.4
0.1 0.9 0.1 <0.01
IL-6 (pg/mL) 45.2
11.8 13.1 3.9 <0.001
TNF-
a (pg/mL) 9.2 2.3 3.9 1 <0.001
E-selectine (ng/mL) 78
38 59 29 <0.01
hsCRP (mg/L) 4.1
4.8 0.9 1.5 <0.001
Results are mean
SD; CRP, C-reactive protein; hs, high-sensitivity.
a
Leptin treatment was found to increase energy expenditure (oxygen consumption), as well as increased thermogenic marker uncoupling protein-1 and
type II deiodinase mRNA levels 1.7- and 3-fold, respectively, in mice (
Asensio et al., 2008).
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
correlation between serum leptin and IL-2 concentrations (
Aygun et al., 2005). Psychotropic drugs that induce weight gain in
psychiatric patients also clearly activated the TNF-
a system (PollmaNcher, Schuld, Kraus, Haack, & Hinze-Selch, 2001).
4.2. Wasting
Mouridsen, Rich, and Isager (2002)
examined 117 childrenwith autismand found that bodymass index (BMI) formaleswas
significantly lower than the agematched reference population but not for females. Also
BoN lte,Ozkara, and Poustka (2002) found
that among 103 analyzed individualswith autismand Asperger’s syndrome 28% had a BMI in the 5th percentile or below. A low
BMIwas also found in 13 childrenwithAsperger’s syndrome (
Hebebrand et al., 1997).Moreover, itwas found that symptoms of
ASD are overrepresented in the patients with anorexia nervosa who evidence a chronic course (
Zucker et al., 2007), and some
symptoms may overlap in the expression of repetitive behaviors and interests in individualswith anorexia nervosa, obsessive–
compulsive personality disorder, and autism (
Zucker & Losh, 2008; Zucker et al., 2007). Central nervous system mechanisms
contribute to the development of cachexia–anorexia syndrome that occurs in chronic pathophysiologic processes including
infections and parasitic disease (
Plata-Salaman, 2000). IL-1, IL-6, IFN-g, TNF-a, and brain-derived neurotrophic factor have been
associated with various cachectic–anorexic conditions resulting from interactions among cytokines, peptides/neuropeptides,
andneurotransmittersasmediatorsofneurologicandneuropsychiatricmanifestationsofdisease(
Cerami, Ikeda,LeTrang,Hotez,
& Beutler, 1985;Matthys & Billiau, 1997; Plata-Salaman, 2000; Reichenberg et al., 2002
). For example, in rheumatoid arthritis,
excess production of the catabolic cytokines IL-1
b and TNF-a by peripheral bloodmononuclear cells drive cachexia, increased
resting energy expenditure (REE), and protein catabolism but without overt weight loss (
Roubenoff et al., 1994, 1997). Also in
HIV-infected men, loss of lean body mass (LBM) often accompanied by elevated REEwas common andwas driven by excessive
productionof thecytokinesTNF-
a(promoteshypermetabolism—elevatedREE) andIL-1b(bothTNF-aandIL-1bare responsible
for loss of LBM) (
Roubenoff et al.,2002). In these subjects, serumfree testosterone concentrationswere inversely associated with
TNF-
a production but was not an independent predictor of either loss of LBM or REE. Both TNF-a and IL-1b production by
peripheral bloodmononuclearcellspredictedlossofLBM(
Roubenoffetal.,2002).EvenasymptomaticHIV-infectedpatientswith
normal absolute CD4
+ T-cell numbers had bothmarkedly (+8%, P< 0.05) higher rates of REE and fat-oxidation rates than control
subjects (
Hommes,Romijn,Endert,&Sauerwein,1991).Hommeset al. (1991)suggestedthatcytokines, likeTNF, IL-1,andIL-6are
involved in themechanismof the hypermetabolismbecause TNF-
a and IL-1 exerted metabolic effects related to tissuewasting
(
Evans, Argiles, &Williamson, 1989) and IL-6 induced the hepatic acute phase response to inflammation (Kishimoto, 1989). It
must be noted that in HIV-infected patients with a CD4 count less than 100, cerebral toxoplasmosis is the most common
opportunistic infection,with the probability of reactivated infection in subjects not receiving prophylaxis being approximately
30% (
UptoDate, 2007).Moreover, in one study among 505 ofHIV/AIDS patients, 44.4% showed Toxoplasma seropositivity with or
without toxoplasmic encephalitis (
Nissapatorn et al., 2004). Finally, NO has been shown to potently reduce testosterone
production in vivo and to directly suppress Leydig cells in vitro (
Adams, Meyer, Sewing, & Cicero, 1994; Del Punta, Charreau, &
Pignataro, 1996; Weissman et al., 2005
). DNA array assays showed a low level of expression of endothelial NOS, while the
neuronal and inducible isoforms of NOS were below detection levels (
Adams et al., 1994).
5. Increased oxidative stress in ASD
Peroxidation of lipidswas found to be increased in plasma of autistic children as compared to their developmentally normal
siblings, suggesting a vital role inthe pathology of autism(
Chauhan,Chauhan,Brown,&Cohen, 2004).Undernormal conditions,
a dynamic equilibrium exists between the production of ROS (superoxide anion, hydroxyl radical, singlet oxygen, hydrogen
peroxide) and the antioxidant capacity of the cell. Stress and injury to cells occur when redox homeostasis is altered, and ROS
generation overpowers the biochemical defenses of the cell. Lipid peroxidation reflects a chain reaction between
polyunsaturated fatty acids and ROS producing lipid peroxides and hydrocarbon polymers that are both highly toxic to the
cell (
Arita et al., 2001; Horton & Fairhurst, 1987; Jain, 1984; Li et al., 1996; Tappel, 1973). The oxidative stress reported in ASD
may be explained by amarkedly increased production ofNOin those individuals (
Sweeten, Posey, Shankar, & McDougle, 2004;
Zorog˘lu et al., 2003
). These high concentrations of NO and its derivatives, such as nitrosothiols and strong oxidants NO2 and/or
N
2O3, probably caused reversal and/or irreversal inhibition ofmultiple mitochondrial respiratory complexes, largely mediated
by NO inhibition of cytochrome
c oxidase (complex IV), as well asmitochondrial aconitase, complexes I, II, V, and opening the
mitochondrial permeability transition pore (peroxynitrite) (
Brown, 1999, 2001; Borutaite & Brown, 1996). In PC12 cells NO
donors inhibited oxygen consumption, decreased mitochondrial membrane potential, decreased cellular ATP, and increased
lactate production (
Bal-Price & Brown, 2000). Moreover, serum levels of transferrin (iron binding protein) and ceruloplasmin
(copper binding protein) were found to be significantly reduced in autistic children as compared to controls (
Chauhan et al.,
2004
). Transferrin acts as an antioxidant by reducing the concentration of free ferrous ion (Chauhan et al., 2004), and
ceruloplasmin acts as ferroxidase and superoxide oxidase, and protects polyunsaturated fatty acids in red blood cell
membranes from active oxygen radicals (
Arnaud, Gianazza, & Miribel, 1988; Sass-Kortsak, 1965). The importance of these
disturbancesmay be supported by the significant correlation observed between reduced serumlevels of these proteins and loss
of previously acquired language skills in children with autism (
Chauhan et al., 2004).
Table 8
(Rossignol, 2007; with own modification) summarized changes in enzyme activities and concentrations of some
biologicmolecules reportedinautistic subjects.Oxidativestresswas implicatedas amajorupstreamcomponent inthe signalling
cascade involved in activation of redox-sensitive transcription factors and proinflammatory gene expression leading to
12
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
Table 8
Changes of antioxidant enzyme activities and concentration of other biomarkers in autistic subjects.
Enzyme/biologic molecule/endogenous
substance/cell
Biological activities Activities decreased (
#) or
increased (
") (references)
Glutathione peroxidase Antioxidant enzyme
# (Yorbik, Sayal, Akay, Akbiyik,
& Sohmen, 2002
)
Superoxide dismutase Antioxidant enzyme
# (Yorbik et al., 2002)
Heme-oxygenase 1
a Antioxidant, anti-inflammatory, and antiapoptotic
enzyme
"
(?) (Min, Yang, Kim, Jou,
& Joe, 2006
)
Catalase
b Antioxidant enzyme # (Zorog˘lu et al., 2004)
Paraoxonase Antioxidant enzyme; organophosphate detoxification
# (D’Amello et al., 2005;
Pasca et al., 2006
)
a
1-Antichymotrypsin Antiproteolytic enzyme " (Chauhan et al., 2006)
HSP-70 Cellular protection against oxidative stress
# (Purcell, Jeon, Zimmerman,
Blue, & Pevsner, 2001
)
Malondialdehyde Marker of oxidative stress and lipid peroxidation
" (Chauhan et al., 2004)
Homocysteine Marker of oxidative stress and lipid peroxidation
" (Pasca et al., 2006)
Ceruloplasmin Antioxidant protein
# (Chauhan et al., 2004)
Transferrin Antioxidant protein
# (Chauhan et al., 2004)
Apolipoprotein B-100 Transporter protein found on VLDL
c " (Corbett et al., 2007)
Glutathione Antioxidant
# (James et al., 2004)
Zinc Antioxidant
# (Yorbik et al., 2004)
Copper plasma zinc/serum copper ratio Metal(s) (
Adams & Holloway, 2004)
#
(Faber, Zinn, Kern, &
Kingston, 2009
)
Melatonin Antioxidant hormone
# (Melke et al., 2008)
Methionine synthase, betaine homocysteine
methyltransferase, methionine adenosyltransferase
Methionine cycle, redox-sensitive enzymes
# (James et al., 2004)
Uroporphyrinogen decarboxylase Enzyme of the heme synthesis pathway and heavy
metal and non metal agents inhibition target
#
(Nataf et al., 2006)
Coproporphyrinogen oxidase Enzyme of the heme synthesis pathway and heavy
metal and non metal agents inhibition target
#
(Nataf et al., 2006)
Free and total serum carnitine Carnitine is responsible for transport of free
fatty acids to mitochondria
#
(Filipek, Juranek, Nguyen,
Cummings, & Gargus, 2004
)
Lactate (L) and pyruvate (P) plasma levels
d Mitochondrial cytopathy markers
(impaired aerobic glycolysis)
"
(L), # (P) (Correira et al., 2006;
Filipek et al., 2004; Oliviera
et al., 2005
)
Serum ammonia and alanine levels Mitochondrial dysfunction biomarkers
" (Filipek et al., 2004)
Plasma glutamic acid, phenylalanine
e, aspargine,
tyrosine, alanine, lysine, and glutamine (G) levels
Dysregulated amino acid metabolism
f " (Aldred, Moore, Fitzgerald,
& Waring, 2003
); # (G)
Serum complement C3 and C4 levels
g C3 and C4 complement proteins facilitate
immunological and inflammatory responses
"
(Chauhan et al., 2004, 2005;
Seeber, 2000
)
Plasma docosahexaenoic acid (DHA; 22:6n-3) DHA is found in high abundance in the
phospholipids of the brain and retina
h
#
(Wiest, German, Harvey,
Watkins, & H, 2009
)
Phospholipase A2 activity (PLA
2)i Decreased levels of arachidonic acid, docosatetraenoic
acid and DHA in RBC membranes from autism subjects
could be caused by increased activity of RBC type
IV PLA
2
"
(Bell et al., 2004)
Lymphoblasts from autistic patients had maximal
respiratory rates that were 40–50% higher than
lymphoblasts from no autistic relatives
This abnormality may be an adaptation to partial
inhibition of ATP synthesis
"
(Benzecry, Deth, & Holtzman,
in press; Holtzman, 2008
)
RBC, red blood cells; VLDL, very low density lipoproteins; HSP-70, heat shock protein-70.
a
T. gondii activates hypoxia-inducible factor 1 (HIF1) already at physiologically relevant oxygen levels and requires HIF1 for growth and survival (Spear
et al., 2006
).
b
Catalase converts hydrogen peroxide to water and molecular oxygen, thereby reducing the amount of free hydroxyl radical formation (Chance, 1954);
NO causes inhibition of this enzyme activity (
Brown & Borutaite, 1999).
c
Apos are involved in the transport of lipids, cholesterol and vitamin E (Spear et al., 2006).
d
It is interesting that the lactic and pyruvic acid plasma levels were significantly higher than those of controls also in migraine and tension-type
headache (
Okada, Araga, Takeshima, & Nakashima, 1998), which is consistent with the suggestion of Prandota (2007) that T. gondii infection plays and
important role in the pathophysiology of headaches and in ASD development (
Prandota, 2009a, 2009b, 2010a, 2010b).
e
It must be noted that phenylalanine derivatives are active against T. gondii brain cysts in mice (Sarciron, Walchshofer, Paris, Petavy, & Peyron, 1998).
f
Melatonin have modulating effect on the production of several amino acids and NO in the brain (Bikjdaouene et al., 2003), and its concentration in ASD
individuals is markedly diminished (
Melke et al., 2008).
g
This increase may reflect a natural immune and antiinflammatory defense of the host reaction because complement has a membrane lytic activity
directed against the extracellular stage of
T. gondii (Seeber, 2000). A strong correlation was observed between increased C3/C4 levels and (a) severity of
autism, and (b) language disability (
Chauhan, Chauhan, & Cohen, 2005).
h
DHA contributes to membrane structure and function, eicosanoid signalling, and gene expression modulation; it also plays a role in inhibition of
neuronal apoptosis and in regulating neuronal excitability through gamma aminobutyric acid receptors (
Wiest et al., 2009).
i
It must be noted that PLA2 was found to be implicated in T. gondii host cell invasion through increasing their penetration (Saffer, Long Krug, &
Schwartzman, 1989
).
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
inflammatory response (
Chauhan & Chauhan, 2006; Parola, Bellomo, Robino, Barrera, & Dianzani, 1999; Uchida et al., 1999).
Oxidative stress and increased ROS production contribute to endoplasmic reticulumstress, protein misfolding, and induction of
the unfolded protein response (
Zhang & Kaufman, 2006). On the other hand, the increased oxidative stress characteristic for
autism (
Chauhan et al., 2004) may also reflect a defense of the host against T. gondii infection because it was reported that
prooxidant dietprovidedprotectionagainst infectionwiththisparasite (
McCarthy&Davis,2003). Itappearedthatmice feda diet
deficient in vitamin E and seleniumshowed the lowestmean numbers of tissue
T. gondii cysts and very little evidence of tissue
pathology during chronic infection. In contrast, the increased dietary supplementationwith these two antioxidants resulted in
trends toward increased tissue cyst number, tissue pathology, and weight loss (
McCarthy & Davis, 2003).
6. Hypercytokinemic and hypermetabolic responses to
T. gondii infection in mice
Arsenijevic, Girardier, Seydoux, Chang, and Dulloo (1997)
and Arsenijevic et al. (2001) found that in mice with T. gondii
infection, during days 1–7 postinfection food intake was unaltered, but energy expenditure was markedly increased, and this
was associated with elevated serum levels of TNF-
a, IL-1, IL-5, and IFN-g (Arsenijevic et al., 1997). This hypermetabolic state
persisted during subsequent anorexia condition, whose onset coincided with elevated serum IL-2 concentration, and at the
end of the acute phase of cachexia, the dual anorexia and hypermetabolic states were associated with the elevation of TNF-
a,
IL-1
b, IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-g levels (Arsenijevic et al., 1997). In the chronic phase of the infection, the mice
showed either partial weight recovery (Gainers, G; these mice had probably an endergonic oxidation reactions with
formation of ATP (
Mayes, 1973)), or no weight regain (NonGainers, NG; these mice had probably exergonic oxidation
reactions (
Mayes, 1973)). [Nb.] it must be noted that ATP activates a ROS-dependent oxidative stress response and secretion
of proinflammatory cytokines in macrophages (
Cruz et al., 2007). T. gondii is capable of relocating its main source of energy
between its cytoplasma and pellicle in response to exit from or entry into host cells and this ability allows the parasite to
optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for
growth and replication of intracellular parasites (
Pomel, Luk, & Beckers, 2008). Arsenijevic et al. (1997) found that the
infected G, though still hypophagic, were no longer hypermetabolic, and their cytokine mRNA was no longer elevated, except
for TNF-
a and IL-10. In contrast, the infected NG continued to show both anorexia and hypermetabolism, which were
associated with serum elevations of all cytokines studied and particularly those of the T
H2 type (IL-4 and IL-5) and IL-6.
Chronic murine infection with
T. gondii modified the response to an LPS challenge by prolonging hypermetabolic response
and potentiating hypophagic phase, and by enhancing concentration of circulating TNF-
a and IL-10, which unlike serum IL-
4, were found to be greater in the NG and G groups of animals than in the controls (
Arsenijevic et al., 1997, 1998). The blood–
brain barrier permeability was found to markedly increase only in the infected NG mice, which may allow a greater bidirectional
passage of cytokines and other neuroimmunologically active substances (
Arsenijevic et al., 1998).
The group of
Arsenijevic et al. (1997, 2001) reported that in mice infected with T. gondii hypermetabolic state was
associated with high lipid oxidation as estimated by a low respiratory quotient, which suggested an important extramitochondrial
mechanism of lipid oxidation. Increased lipid peroxidation was detected especially in serum, lung, spleen and
liver, i.e. tissues rich in macrophages. Following the infection, peritoneal macrophages exhibited an enhanced capacity to
produce ROS. Macrophage oxidative burst probably accounted for a substantial component of the increase in oxygen
consumption. The authors (
Arsenijevic et al., 1997, 2001) found that T. gondii infected IFN-g knockout mice showed a marked
reduction in their hypermetabolic-like response and there was a decrease in their ROS production by peritoneal
macrophages and attenuated lipid peroxidation in the acute phase of infection prior to the cachectic phase.
It is known that IFN-
g is amajor regulator of ROS production in macrophages (Nathan,Murray, Wiebe, & Rubin, 1983) and
enhanced lipid entry into macrophage-type cells requires this particular cytokine (
Whitman et al., 1999). Moreover, IFN-g
increased lipoprotein lipase expression (
Garner, Baoutina, Dean, & Jessup, 1997). In the chronic phase of the infection in theNG
mice high IFN-
g levels were associated with hyperlipid peroxidation and cachexia (Nathan et al., 1983). The blunted
hypermetabolic state in the IFN-
g knockout mice may not only be due to decreased ROS production but also to reduced lipid
uptake by phagocytic cells. Itmust be noted that also cerebral ischemia was accompanied by a strong inflammatory response
associated with a hypermetabolic state (
Astrup, Rehcrona, & Siesio, 1980; McCarthy, O’Saughnessy, & Rothwell, 1989) and
enhancedlipid peroxidation component (
Coppi, 1995).Asynergy between proinflammatory cytokines andoxidative stress that
trigger common signal transduction pathways leading to amplification of the inflammatory cascade has been reported also in
other clinical states, such as acute pancreatitis (
Pereda et al., 2006), and hyperthyroidism (Makay et al., 2009). Melatonin
treatment suppressed the hyperthyroidism-induced damage, as well as the exaggerated TNF-
a response (Makay et al., 2009).
This is not surprising because melatonin activated cellular immunity by stimulating CD4
+ and CD8+ cells production, upregulated
type T
H1 cells immune response by increasing production of TNF-a, IFN-g, IL-2, IL-12, and increased leukocyte
numbers and macrophage count (
Baltaci, Bediz, Mogulkoc, Kurtoglu, & Pekel, 2003; Santello et al., 2007; Santello, Frare,
Caetano, Alonso Toldo, & do Prado, 2008
). Orally administered melatonin reduced oxidative stress and the neuroinflammatory
response in the brain induced by amyloid-
b peptide in rats (Rosales-Corral et al., 2003). It must be noted that in ASD serum
melatonin levels are markedly decreased because of a significant deficit of acetylserotonin methyltransferase (ASMT) activity,
which is responsible for conversion of N-acetyloserotonin to melatonin (
Melke et al., 2008). One may suggest that the ASMT
deficit is secondary in nature, i.e. caused by maternal or fetal/postnatal overproduction of proinflammatory cytokines and/or
NO during neuroinflammation due to reactivation of latent CNS
T. gondii infection. This speculation may be supported by the
finding that NO dose- and time-dependently inhibited O
6-methylguanine-DNA-methyltransferase activity, which
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
corresponded to total quantity of NO released (
Laval & Wink, 1994), as well as irreversibly inhibited several cytochrome P450
enzymes (
Khatsenko, Gross, Rifkind, & Vane, 1993; Minayamiyma et al., 1997), and ASD individuals have NO overproduction
(
SoNguNt et al.,2003). Melatonin significantly decreased nitrite content in the brain (Bikjdaouene et al., 2003) and this effectwas
probably mediated via NO/
L-arginine pathway by constitutively expressed NOS (Yahyavi-Firouz-Abadi, Tahsili-Fahadan, Riazi,
Ghahremani,&Dehpour, 2006
).Moreover, oxidative stress associatedwith hypercytokinemia (characteristic for ASD) alsowas
found to be responsible for down-regulation of cytochrome P450 (
Morel & Baroukis, 1998). Interestingly, maternally
administered melatonin significantly attenuated LPS-evoked elevation of TNF-
a in fetal brain and increased IL-10 in fetal liver
(
Xu, Wang, Ning, Zhao, & Chen, 2007).
Shrestha, Tomita, Weiss, and Orlofsky (2006)
found that a distinct population of ROS(+) inflammatory macrophages
increased progressively in frequency during the course of
T. gondii infection, and was inversely correlated with the degree of
cell parasitization. These data indicated that ROS-producing cells contained anti-
Toxoplasma activity, and the non-infected
ROS-producing inflammatory macrophages were resistant to infection in vivo. In addition, macrophages infected with
T.
gondii
in vitro and then briefly transferred to acutely infected mice upregulated ROS production in a manner that was again
inversely correlated with the degree of intracellular parasitization. It was suggested that both ROS-associated anti-
Toxoplasma
activity and parasite-driven inhibition of ROS production underlie the observed pattern of ROS production. ROS
function and parasite evasion of this function may therefore contribute markedly to the balance between host defense and
disease progression during infection (
Shrestha et al., 2006).
6.1. Overproduction of IFN and other cytokines in patients with ASD and during chronic T. gondii infection in mice
Patients with autism have significantly increased concentrations of several proinflammatory cytokines, chemokines, and
differentiation factors in the brain tissue homogenates and in the cerebrospinal fluid (
Chez, Dowling, Patel, Khanna, &
Kominsky, 2007; Jyonouchi, Sun, & Le, 2001; Li et al., 2009; Vargas, Nascimbene, Krishnan, Zimmerman, & Pardo, 2005;
Zimmerman et al., 2005
). Li et al. (2009) also found that proinflammatory cytokines (TFN-a, IL-6, IFN-g, and granulocyte
macrophage-colony stimulating factor), and chemokine IL-8 were markedly increased in the brain cortex of autistic patients
compared with controls, but the T
H2 cytokines (IL-4, IL-5, and IL-10) showed no significant difference. Similar persistent
overproduction of these important biomolecules resulting in hypermetabolic state has been reported also in chronic murine
toxoplasmosis (
Arsenijevic et al., 1997, 1998, 2001; Hermes et al., 2008). Bartha et al. (2004) reported that children with
abnormal neurodevelopment outcome (neonatal encephalopathy) also had higher neonatal levels of IL-1
b, IL-6, IL-8, and
lower levels of IL-12, and that elevated inflammatory cytokines were associated with impaired cerebral oxidative
metabolism. Changes in the proinflammatory and antiinflammatory cytokine levels in autistic individuals and during
T.
gondii
infection are presented in Table 9 (Rossignol, 2007; with own modification).
PBMCs from ASD patients produced significantly greater amounts of TNF-
a, IL-1b, and/or IL-6 compared with PBMCs of
control subjects (
Jyonouchi et al., 2001). With stimulants of phytohemagglutinin, tetanus, IL-12p70, and IL-18, PBMCs from
47.9 to 60% of analyzed ASD patients produced markedly higher values of TNF-
a than controls depending on stimulants.
Thus, excessive innate immune responses in a number of ASD children may be most evident in an increased TNF-
a
production (
Jyonouchi et al., 2001).
Children with autism had a significant elevation of TNF-
a in the cerebrospinal fluid (CSF) compared with other patients
studied(means104.10 pg/mL vs.2.78 pg/mL;
Chez et al., 2007). The ratio of theCSFTNF-alevels to serumlevels averaged53.7:1,
respectively. This may suggest an inflammatory mechanism that may contribute to the onset of autism (
Chez et al., 2007).
Miller, Wen, Dunford, Wang, and Suzuki (2006)
found that in T. gondii infected mice both CD4+ and CD8+ immune T cells
produced large amounts of IFN-
g, in response to either infected macrophages or tachyzoite lysate antigens (TLA), but the
CD4
+ T cells produced greater amounts of the cytokine than did the CD8+ T cells with both stimulations. Both T cell
populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4
+ T cells did when stimulated
with TLA (
Miller et al., 2006). CD4+ immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with
infected macrophages, but CD8
+ T cells did not. These results indicated that CD4+ immune T cells produced IFN-g, IL-2, IL-4,
and IL-10 in response to infected macrophages, whereas CD8
+ immune T cells produced predominantly IFN-g and IL-2. Since
IL-4 and IL-10 could suppress IFN-
g-mediated protective mechanisms against the parasite, the production of these cytokines
by CD4
+ T cells in response to infected cells could negatively affect their protective activity in vivo (Miller et al., 2006).
Nguyen et al. (2003)
also showed increased expression of IL-12, IFN-g and TNF-a, but not IL-4 mRNAs in spleen cells after
infection with
T. gondii virulent RH and weakly virulent Beverly strains. High levels of circulating IL-12 and IFN-g were
detected in the serum of mice infected with strain RH, although TNF-
a levels remained low. In contrast, the same cytokines
were detected at only low levels in the serum of mice infected with the Beverly strain (
Nguyen et al., 2003). Konopka and
DzbenL ski (2001)
also found that splenic lymphocytes from T. gondii infected mice produced high levels of IFN-g in vitro. The
production of IFN-
g was increasing until 10th day of infection, reaching a level 1600-fold higher than that found in control
cultures, then it began to decrease. The level of IL-10 released by lymphocytes to the medium was also increasing up to the
10th day of infection, afterwards its content dropped down to the level 18-fold higher than that at the beginning of
experiments. There were no effects of immunization with antigens of killed parasites on the production of IFN-
g by splenic
cells in vitro (
Konopka & DzbenL ski, 2001). IFN-g overproduction and high level apoptosis were associated with high
virulence
T. gondii infection (Gavrilescu & Denkers, 2001), especially in an impaired production of endogenous IL-10
(
Gazzinelli et al., 1996b).
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
In congenital toxoplasmosis, the parasites first invade the umbilical vein endothelial cells and are then disseminated
throughout the fetus. Treatment of ovine umbilical vein endothelial cells with bovine recombinant IFN-
g blocked growth of
T. gondii
in a dose-dependent manner (Dimier & Bout, 1996). Maximum activation was achieved by incubating these cells
with 625 U/mL, and IFN-
g had no activity at 0.15 U/mL (Dimier & Bout, 1996).
It must be noted that spleen cells from male mice produced higher levels of IFN-
g in early stages of infection with T. gondii
than those from female mice (
Roberts, Cruickshank, & Alexander, 1995). In vitro examination of T. gondii-specific T-cell
proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice
than in their female counterparts. Thus, in male mice a rapid response to infection with this parasite with high levels of IFN-
g
and TNF-
a helps to control parasite multiplication, after which IL-10 production may be important in down regulating these
potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms T-cell proliferation and
IFN-
g production compared with their male counterparts may account for their poor survival rates and higher cyst burdens
(
Roberts et al., 1995). These observations may, at least in part, serve as an explanation for gender differences in behavioral/
personality profile changes in men and women induced by latent chronic toxoplasmosis (
Lindova et al., 2006), and a
correlation of duration of latent
T. gondii infection with personality changes in women (Flegr et al., 2000).
Finally, it must be emphasized that fetal high IFN-
g producing allele (IFN-g[+874T]) was associated with spontaneous
preterm delivery (odds ratio = 2.3 [1.2–4.4]) (
Speer et al., 2006). In addition, among preterm deliveries, maternal low TGF-b1
(codon 10C) producing genotypes correlated negatively with gestation, while fetal TNF-
a (308G) was significantly
associated with histologic chorioamnionitis.
7. Overproduction of nitric oxide in patients with ASD and its important role in control of
T. gondii infection
7.1. Patients with ASD
Zorog˘lu et al. (2003)
and Sweeten, Posey, and McDougle (2004) reported elevated plasma nitrite (a metabolite of NO)
levels in the autistic subjects, and
So¨gu¨ t et al. (2003) found increased NO levels in red blood cells of patients with autism. A
Table 9
Modulation of proinflammatory and anti-inflammatory cytokine levels involved in inflammation processes in autistic subjects and during
T. gondii
infection.
Analyzed
biomarkers
Biological activity Autism findings
T. gondii infection findings
References References
IFN-
g Proinflammatory " (Jyonouchi et al., 2001;
Mills et al., 2007; Molloy
et al., 2006; Singh, 1996;
Stubbs, 1995
)
"
(Araujo & Slifer, 2003; Arsenijevic et al., 2001; Filisetti & Candolfi,
2004; Gavrilescu & Denkers, 2001; Gazzinelli et al., 1996a,b;
Hermes et al., 2008; Khan, Schwartzman, Matsuura, & Kasper,
1997; Mordue et al., 2001; Nguyen et al., 2003; Raymond et al., 1990
)a,b,c
NO Proinflammatory
" (Sweeten, Posey,
et al., 2004
)
"#
(Bohne, Heesemann, & Gross, 1994; Khan et al., 1997; Prada & Ngo-Tu, 2008)
IL-2 Proinflammatory
" (Molloy et al., 2006) " (Arsenijevic et al., 1997; Filisetti & Candolfi, 2004)
IL-12 Proinflammatory
" (Singh, 1996) " (Bliss, Marshall, Zhang, & Denkers, 1999; Filisetti & Candolfi, 2004;
Gazzinelli et al., 1996a,b; Nguyen et al., 2003
)
TNF-
a Proinflammatory " (Chez et al., 2007;
Jyonouchi et al., 2001
)
"
(Arsenijevic et al., 1998; Bliss et al., 1999; Filisetti & Candolfi, 2004;
Gazzinelli et al., 1996a,b; Khan et al., 1997; Prada & Ngo-Tu, 2008
)
IL-1
b Proinflammatory " (Jyonouchi et al., 2001) " (Arsenijevic et al., 1997)
IL-6
d Proinflammatory " (Stubbs, 1995) " (Arsenijevic et al., 1997; Filisetti & Candolfi, 2004; Prada & Ngo-Tu, 2008)
TGF-
be Pro- and
anti-inflammatory
"
TGF-b2 (Stubbs, 1995) " (Filisetti & Candolfi, 2004; Fischer, Nitzgen, Reichmann, & Hadding, 1997;
Gazzinelli et al., 1996a; Nagineni, Detrick, & Hooks, 2002
)
IL-4
f Anti-inflammatory " (Molloy et al., 2006) " (Arsenijevic et al., 1997; Filisetti & Candolfi, 2004)
IL-5
g Stimulation of
antibody production
by B cells
"
(Molloy et al., 2006) " (Arsenijevic et al., 1997; Filisetti & Candolfi, 2004)
IL-10 Anti-inflammatory
# (Mills et al., 2007)h " (Arsenijevic et al., 1997; Arsenijevic et al., 1998; Filisetti & Candolfi,
2004; Gazzinelli et al., 1996b
)
IL-13 Anti-inflammatory
" (Molloy et al., 2006)
"
, #, increased or decreased levels.
a
Human fetus is able to synthesize IFN-g as early as week 21 of pregnancy (Raymond et al., 1990).
b
It seems that IFN-g enhances, directly or indirectly, transplacental passage of T. gondii (Abou-Bacar et al., 2004). Increased levels of IL-6, TNF-a and NO
were often found to correspond with critical events during ocular toxoplasmosis, such as extended conjunctivitis, vitreous turbidity and/or temporary
blindness (
Prada & Ngo-Tu, 2008).
c
INF-a/b are also upregulated after T. gondii infection (Lang, Gros, & LuN der, 2007).
d
IL-6 was found to promote the intracellular multiplication of T. gondii in mice (Beaman, Hunter, & Remington, 1994).
e
TGF-b increased replication of the parasite on cultured retinal cells, suggesting that this cytokine may be involved in the immunopathogenesis of
retinochoroiditis (
Nagineni et al., 2002).
f
IL-4 may promote the passage of T. gondii through the placenta in mice (Alexander, Jebbari, Bluethmann, Brombacher, & Roberts, 1998; Thouvenin,
Candolfi, Villard, Klein, & Kien, 1997
).
g
IL-5 exerted a protective role during chronic T. gondii infection (Zhang & Denkers, 2001).
h
Mucosal lymphocyte IL-10 (Ashwood, Anthony, Torrente, & Wakefield, 2004).
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
positive correlation was found between nitrates and IFN-
g concentrations, indicating that elevated plasma NO may be
related to IFN-
g activity in ASD (Sweeten, Posey, et al., 2004). This is not surprising because the induction of iNOS is mediated
by some cytokines, namely IFN-
g, TNF-a, and IL-b1 (Nussler et al., 1992). In the cortex of autistic patients, the cholinergic
receptors known to be sensitive to NO toxicity were found to be decreased (
Perry et al., 2001). In addition, treatment with
cholinergic agonists improved behavioral abnormalities in autism (
Chauhan & Chauhan, 2006; Hardan & Handen, 2002). The
beneficial therapeutic effects may be explained by the finding that proinflammatory cytokine levels and excessive
inflammation can be regulated by specifically augmenting cholinergic signalling via the efferent vagus nerve and/or applying
selective cholinergic modalities targeting the
a7 subunit-containing nicotinic acetylcholine receptor (Gallowitsch-Puerta &
Pavlov, 2007; Jay, Kojima, & Gillespie, 1986; Pavlov, 2008; Yoshikawa et al., 2006
). Morever, it seems that because
proliferation of T. gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production
(
Shrestha et al., 2006), enhanced tobacco smoke exposure potentiation of superoxide anion generation by human
neutrophils (
Gillespie, Owasoyo, Kojima, & Jay, 1987) may be advantageous for the host infected with the parasite).
NO is known to affect the development and function of the central nervous system, such as neurite growth (
Hindley et al.,
1997
), synaptogenesis (Truman, De vente, & Ball, 1996), neurotransmitter release (Lonart, Wang, & Johnson, 1992), memory
processing and learning (learning was dose-dependently affected) (
HoN lscher & Rose, 1992; Myslivec, Hassmannova, Barcal,
S’afanda, & Z’alud, 1996
), and macrophage-mediated cytotoxicity (Hibbs, Taintor, Vavrin, & Rachlin, 1988). The expression of
iNOS and production of NO also affect inflammatory processes (
Wong & Billiar, 1995).
The role of NO in parasitic diseases is very important (
Liew, 1993). Normally, NO production is necessarily under tight
control but excessive NO can lead to development of immunopathology (diabetes, liver cirrhosis, rheumatoid arthritis). A
number of cytokines, including IL-4, IL-10 and TGF-
b, can down-regulate the induction of NO synthase in macrophages
(
Liew, 1993). Also NO can reduce the activity of NO synthase by feedback inhibition, and inhibits the production of IFN-g by
T
H1 cells with the regulatory pathways involving tyrosine kinase and protein kinase C (Liew, 1993). NO (and IFN-g) plays an
important role also in upregulation of VEGF gene expression (
Ramanathan, Giladi, & Leibovich, 2003), the factor known to be
markedly increased in the cerebrospinal fluid of patients with ASD (
Vargas et al., 2005).
NO is a cytotoxic effector molecule produced by macrophages that results in iron mobilization from tumor target cells,
which inhibits DNA synthesis and mitochondrial respiration. Mitochondria may contain a NO synthase and can produce
significant amounts of NO to regulate their own respiration. This function may therefore be important for physiological and
pathological (because of a known overproduction of NO in autism) regulation of energy metabolism (
Brown, 1999, 2001;
Brown & Borutaite, 1999
). NO-mediated iron mobilization is markedly potentiated by glutathione generated by the hexose
monophosphate shunt, and
Watts and Richardson (2002) reported that NO intercepted iron before incorporation into ferritin
and indirectly mobilized iron from ferritin in a glutathione-dependent manner.
7.2. T. gondii infection
Cytokines play an important role in the regulation of
T. gondii replication in the CNS (Hunter & Remington, 1994; Halonen
et al., 1998
). Studies indicate that IFN-g, TNF-a, IL-1, and IL-6 may control the growth of T. gondii in the brain via activation of
microglia (
Chao, Anderson, et al., 1993; Chao, Hu, et al., 1993; Chao, Gekker, Hu, & Peterson, 1994; Wang & Suzuki, 2007).
TNF-
a, IL-1 and IL-6 were up-regulated in the brains of mice with chronic toxoplasmosis (Deckert-Schluter, Albrecht, Hof,
Wiestler, & Schluter, 1995; Hunter, Roberts, Murray, & Alexander, 1992; Hunter, Litton, Remington, & Abrams, 1994
). IFN-g
has been shown to be the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain (
Suzuki, Conley, &
Remington, 1989; Suzuki, Orellana, Schreiber, & Remington, 1988
). Macrophages and microglia are phagocytic cells of
hemopoietic origin and are important IFN-
g-activated effector cells against T. gondii that exert potent anti-Toxoplasma
activity via the induction of iNOS and the production of NO (
Adams, Hibbs, & Kranhebuhl, 1990; Chao, Anderson, et al., 1993;
Chao, Hu, et al., 1993; Chao et al., 1994; Gazzinelli et al., 1993
). NO is believed to be directly toxoplasmacidal, resulting in
intracellular killing and/or stasis of parasites (
Halonen, Taylor, & Weiss, 2001). Activated macrophages control T. gondii
growth by NO production (
Seabra, de Souza, & Damatta, 2004; Minns et al., 2004). However, T. gondii active invasion inhibits
NO production, allowing parasite persistence. The mechanism used by
T. gondii to inhibit NO production persisting in
activated macrophages depends on phosphatidylserine exposure. TGF-
b1 led to iNOS degradation, actin filament (F-actin)
depolymerization, and lack of NF-
kB in the nucleus. All these features were reverted by TGF-b1 neutralizing antibody
treatment (
Minns et al., 2004; Seabra et al., 2004). It was also demonstrated that T. gondii tachyzoites inhibited
proinflammatory cytokines such as TNF-
a and IL-12 induction in infected macrophages by preventing NF-kB- and mitogenactivated
protein kinase-signalling cascades that may enable parasite survival within the host cells (
Butcher, Kim, Johnson, &
Denkers, 2001; Denkers, Butcher, Del Rio, & Kim, 2004
).
Cytokine-activated microglia are important host defense cells in CNS infections. Astrocytes are the predominant host cell
for
T. gondii in the brain and support prolific growth of the tachyzoite stage (Halonen, Lyman, & Chiu, 1996; Peterson, Gekker,
Hu, & Chao, 1993
). IFN-g has been demonstrated to inhibit parasite replication also in astrocytes (Halonen et al., 1998).
Although these authors (
Halonen et al., 1998) demonstrated that IFN-g-induced inhibition in murine astrocytes was found to
be affected via a nitric oxide- and tryptophan starvation-independent mechanism, in human astrocytes induction of NOS
activity by IL-1
b and IFN-g has been well documented (Lee, Dickson, Liu, & Brosnan, 1993). It was reported that human
astrocytes inhibit intracellular multiplication of Toxoplasma by NO-mediated mechanism (
Peterson, Gekker, Hu, & Chao,
1995
). Halonen et al. (2001) showed that IGTP, an IFN-g-regulated gene containing a GTP-binding sequence, localized to
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
endoplasmatic reticulum of cells (
Taylor et al., 1996) played a central role in the IFN-g-induced inhibition of T. gondii in
murine astrocytes through involvement in the processing or trafficking of antigens or cytokines (
Taylor et al., 1997). Recent
evidence indicates that cytokines can also activate astrocytes to inhibit growth of
T. gondii (Daubener et al., 1993, 1996;
Halonen et al., 1998; Pelloux et al., 1996; Peterson et al., 1995
), for example IFN-g has been shown to inhibit growth of T.
gondii
in the glioblastoma cell line 86HG39 (Daubener et al., 1993). The inhibition was shown to be via induction of
indoleamine 2,3-dioxygenase resulting in the degradation of intracellular tryptophan (
Daubener et al., 1996). Pelloux et al.
(1996)
found that in the astrocytoma cell line GHE, TNF-a inhibited, IL-1 stimulated, and IFN-g and IL-6 had no effect on
growth of
T. gondii.
Finally, in primary human astrocytes, IFN-
g and IL-1 in combination have been shown to inhibit growth of T. gondii via
production of NO (
Taylor et al., 1996, 1997). Studies of Halonen et al. (1998) showed that pretreatment of astrocytes with
IFN-
g resulted in 65% inhibition of T. gondii growth and that neither TNF-a, IL-1, nor IL-6 alone had any effect on T. gondii
growth, while IFN-
g in combination with either TNF-a, IL-1, or IL-6 caused a 75–80% inhibition of growth.
7.3. Depressed metabolism of endogenous and exogenous substances in the patients with ASD probably is due to a significantly
diminished activity of several enzymes by hypercytokinemia and overproduction of reactive oxygen species (ROS) during
neuroinflammation caused by T. gondii
Proinflammatory cytokines play an important role in depression of cytochrome P450-dependent and UDP glucuronosyl
transferase-dependent drug biotransformation in mammals during infection and inflammation, and NO markedly prevents
the inhibition of glucuronidation induced by cytokines (
Monshouwer, Witkamp, Nujmeijer, Van Amsterdam, & Van Miert,
1996
). CYP450 superfamily of enzymes are distributed primarily in the liver and adrenal glands, but they also may be found
in intestines, skin, lungs and brain (
De Wildt, Kearns, Leeder, & van den Anker, 1999; Norris, Hardwick, & Emson, 1996).
CYP450 enzymes in the brain are approximately 0.5–3% of the content in the liver and should not significantly contribute to
overall drug elimination, but may alter local actions or concentrations of endogenous and exogenous substances (
Majewska,
Harrison, Schwartz, Barker, & Paul, 1986; Nicholson & Renton, 2002
). It was reported that CYP450 in the brain have
homeostatic functions because its isoforms have been shown to participate in cerebral blood vessel tone and also in the
synthesis of neuroactive steroids (
Harder, Lange, Gebremdhin, Birks, & Roman, 1997; Walther, Ghersi-Egea, Minn, & Siest,
1987; Warner, Wyss, Yoshida, & Gustafsson, 1994
). Current evidence shows that CYP1A1/2, CYP2B1, CYP2E1, CYP2D1, novel
forms from CYP3A and CYP4F families, and CYP7B exist in the brain and are regionally located in both neuronal and glial cells
(
Miksys, Hoffmann, & Tyndale, 2000; Strobel et al., 1995).
In response to an immune stimulus, glial cells, specifically astrocytes and microglia became activated in the process
termed gliosis (
Andersson, Perry, & Gordon, 1992), and stimulated the acute phase response characterized by the release of
cytokines, proteases, prostaglandins, NO via the increased expression of iNOS, and stimulation of the arachidonic acid
cascade with increased production of ROS (
Gottschall, Komaki, & Arimura, 1992; Jersmann, Rathjen, & Ferrante, 1998; Lopez-
Figueroa et al., 2000; Matyszak, 1998; Montero-Menei et al., 1996; Nicholson & Renton, 2002; Rivest et al., 2000
).
In the brain, even localized inflammatory responses cause a concomitant down-regulation of cytochrome P450 and drugmetabolizing
activity in the liver and the brain (
Renton, 2000; Renton & Nicholson, 2000). To explain the pivotal role of viral/
bacterial/parasite infections in the pathomechanism of ASD, it must be noted that many infectious agents and drugs
stimulate/depress interferon and other cytokine and NO production, which inactivates cytochromes P450 enzymes (phase I
biotransformation enzymes) involved in the metabolism of several endogenous lipophilic substances (e.g. steroids, lipidsoluble
vitamins, prostaglandins, leukotriens, thromboxanes) and exogenous substances (drugs and/or their metabolites,
pesticides, and other environmental agents) (
Armstrong & Renton, 1994; Arnold, Hill, & Sansom, 1981; Barkin, Schwer, &
Barkin, 1999; Chang, Bell, Lauer, & Chai, 1978; De Wildt et al., 1999; Franko, Powell, & Nahata, 1982; Kearns, 1995; Leeder &
Kearns, 1997; Prandota, 2000, 2001, 2004a, 2004b, 2005; Renton & Nicholson, 2000
). There was a considerable interindividual
variability in the hepatic expression of P450 enzymes (
Shimada, Yamazaki, Mimura, Inui, & Guengerich, 1994),
and for a given individual, the pathway and rate of a compound’s metabolic clearance is a function of that individual’s unique
phenotype with respect to the forms and amounts of P450 species expressed (
Leeder & Kearns, 1997; Wrighton & Stevens,
1992
). Cytochromes P450 known to be quantitatively important for human drug metabolism are found in the CYP1, CYP2,
and CYP3 gene families. It is important to note that, for example CYP 3A4/5 demonstrates the greatest intersubject variability
for the plasma clearance of many CYP3A substrates in adults (
Gonzales & Idle, 1994), with more than a 10-fold variation in
the amount of hepatic CYP3A4 messenger RNA content present in humans, and up to 50–60-fold differences in constitutive
CYP3A activity found between individuals, depending on the phenotyping method used (
Gonzales & Idle, 1994; Kearns,
1995; Schuetz, Beach, & Guzelian, 1994
). Moreover, CYP1A2 expression shows considerable variability even up to 100-fold or
more for some activities (
Butler, Iwasaki, Guengerich, & Kadlubar, 1989). All this may suggest that in some genetically
predisposed individuals even administration of therapeutic doses of a drug may result in serious clinical adverse effects, if an
important concomitant risk factor, such as acute viral infection, is involved.
In clinical practice, after the intramuscular administration of recombinant human interferon alpha A there was a
significant increase in the elimination half-life, area under the curve and mean residence time of theophylline in association
with a decrease in plasma clearance of theophylline/antipyrine (
Jonkman et al., 1989; Williams & Farrell, 1986; Williams,
Baird-Lambert, & Farrell, 1987
). These pharmacokinetic parameters of theophylline changed markedly also during acute
respiratory viral illness and/or asthma in children and adults (
Arnold et al., 1981; Chang et al., 1978; Franko et al., 1982). Even
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associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid
immunization with influenza virus and rubella virus vaccines resulted in an increased 2–5A synthetase activity, and IFN-
a as
well as IFN-
g levels rose selectively depressing the oxidative metabolism of various drugs in humans (Meredith et al., 1985;
Penn &Williams, 1984; Renton & Mannering, 1976; Williams & Farrell, 1986; Williams et al., 1987
). Interferon-b treatment
of patients with chronic hepatitis C also caused a marked decrease of total body clearance of theophylline and an increase in
its elimination half-life, but the magnitude of the decreases in analyzed liver enzymes activities varied widely in individual
patients and did not correlate with the dose of interferon administered (
Okuno et al., 1993).
Another clinical example, which emphasized importance of depression of metabolism of endogenous and exogenous
substances during viral/bacterial infections and/or chronic inflammation states, was the down-regulation of phosphoenolpyruvate
carboxykinase activity by TNF, IL-1, IL-6, bacterial endotoxin, adenovirus E1A, oxidative stress (H
2O2), and
vitamin A deficiency, in the pathomechanism of sudden infant death syndrome (SIDS) (
Alm et al., 2003; Christ, Nath,
Heinrich, & Jungerman, 1994; Christ, Yazici, & Nath, 2000; Ghoshal, Pasham, Odom, Furr, & McGrane, 2003; Hill & McCallum,
1991, 1992; Kalvakolanu, Liu, Hanson, Harter, & Sen, 1992; Prandota, 2004a, 2004b; Wang, Deutschman, Clemens, & De
Maio, 1995; Yamauchi et al., 2001
). The repression of PEPCK activity resulted in a significant impairment of gluconeogenesis
and glyceroneogenesis processes in the liver, kidney, and adipocytes of SIDS victims, and overproduction and accumulation
of non-esterified fatty acids, the biochemical disturbances characteristic for the early phase of type 2 diabetes (
Beale,
Antoine, & Forest, 2003; Beale, Hammer, Antoine, & Forest, 2004; Forest et al., 2003; McGary, 2002; Prandota, 2004a, 2004b;
Sasaki et al., 1984; Unger, 2003
).
Table 10
(Paucha Smith, 2008; with own modification) and Table 11 (Mannering, Renton, el Azhary, & Deloria, 1980; with
own modification) summarized effects of several interferon-inducing agents and vaccinations that also increased
concentrations of many other cytokines.
Table 12 (Prandota, 2005; with modification) presented down-regulating effects of
several cytokines, NO, growth factors and other agents on the metabolism of endogenous and exogenous substances.
Activation of systemic host defense mechanisms resulted in down-regulation of various induced and constitutive isoforms of
cytochrome P450 also in response to other than IFNs cytokines, which are released from activated immune cells in patients
with infections and/or disease states that have an inflammatory component (
Renton, 2000; Renton & Nicholson, 2000).
Several cytokins, such as IL-1, IL-1
b, IL-4, IL-6, TNF-a, interferon-a, aA/D, -b and -g, TGF-b1, human hepatocyte growth
factor, and lymphotoxin, down-regulated gene expression of major cytochrome P450 enzymes with the specific effects on
messenger RNA levels, protein expression, and enzyme activity observed with a given cytokine varying for each P450 studied
(
Abdel-Razzak et al., 1993; Abdel-Razzak, Corcos, Fautrel, Campion, & Guillouzo, 1994; Abdel-Razzak, Corcos, Fautrel, &
Guillouzo, 1995; Bertini, Bianchi, Villa, & Ghezzi, 1988; Chen, Strom, Gustafsson, & Morgan, 1995; Delaporte & Renton, 1997;
Donato, Gomez-Lechon, Jover, Nakamura, & Castell, 1998; Leeder & Kearns, 1997; Muntane-Relat, Ourlin, Domergue, &
Maurel, 1995; Okuno et al., 1993; Paton & Renton, 1998; Tapner, Liddle, Goodwin, George, & Farrel, 1996
). In addition, the
combinations of IL-1 and IL-6 (CYP2C11) (
Echizen et al., 1990), and INF-g and TNF (ethoxycoumarin deethylase activity)
(
Bertini et al., 1988) had an additive effect in depressing liver cytochrome P450-dependent drug metabolism. Moreover, it
appeared that IL-1
b even antagonized phenobarbital induction of several major cytochromes P450 (Abdel-Razzak et al.,
1995
). One must remember, however, that e.g. IL-4 also inhibited expression of TNF-a and -b, IL-1b, IL-6, and IFN-g, thus
being an important regulator of intensity of the inflammatory immune responses (
Lee, Rhoades, & Economou, 1995; Loyer
et al., 1993
), as well as up-regulated CYP2E1 mRNA, and glutathione S-transferases (phase II enzymes) involved in drug
detoxication and in protection against lipid peroxidation (
Delaporte & Renton, 1997; Langouet et al., 1995). Also during
bacterial infections, e.g.
Listeria monocytogenes infection, the activation of host defense mechanisms has been shown to cause
a depression in hepatic cytochrome P450-mediated metabolism in humans, and it appeared that hemolysin was an essential
component of the mechanism responsible for the down-regulation of cytochrome P450 (
Armstrong & Renton, 1994). The
above-presented down-regulatory functions of inflammatory cytokines which play a key role through activation and
coordinating the immune responses are understandable in the light of demonstration that, e.g. TNF-
a and IL-1, were found to
be involved in the human papillomavirus type 16 (HPV) gene regulation, thus contributing to the hosts’s defense against HPV
infection (
Kyo et al., 1994). Also local inflamed tissue imbalance in the ratio of different cytokines, e.g. IL-1b, IL-6 and TNF-a,
Table 10
Effects of some interferon-inducing agents administered on different exposure gestation day to rats and mice that induced maternal immune activation
causing increases of cytokine levels in the fetal brain.
Triggering factors Increased cytokine levels in the fetal brain References
LPS 4 mg/kg, i.p.; E18 rat TNF-
a, IL-1b (Cai, Pan, Pang, Evans, & Rhodes, 2000)a
LPS 2.5 mg/kg, i.p.; E16 rat TNF-
a (Urakubo, Jarskog, Lieberman, & Gilmore, 2001)
LPS 1 mg/kg, i.p. TNF-
a, IL-1b, iNOS (Paintlia, Paintlia, Barbosa, Singh, & Singh, 2004)a
LPS 0.05 mg/kg, i.p.; E18 rat No change in TNF-a,
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