ARTICLE IN PRESS

Immunobiology 215 (2010) 101112

www.elsevier.de/imbio

Congenital exposure to Schistosoma mansoni infection: Impact on the

future immune response and the disease outcome
Ahmad A. Othmana,, Zeinab S. Shoheiba, Eman M. Saiedb, Rasha H. Solimanc
a

Department of Medical Parasitology, Faculty of Medicine, Tanta University, Tanta, Egypt

Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
c
Department of Medical Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
b

Received 19 February 2009; received in revised form 28 March 2009; accepted 1 April 2009

Abstract
Schistosomiasis mansoni is a widespread parasitic infection that may lead to several serious complications, such as
hepatic periportal brosis and portal hypertension, mainly due to deposition of schistosome eggs in the tissues.
However, people in endemic areas infrequently exhibit severe pathology and complications; this may be explained, in
part, by modulation of the disease in indigenous populations by in utero exposure to the parasite. This study
investigated the differences between mice born to Schistosoma mansoni-infected mothers and those born to noninfected ones in subsequent postnatal schistosomal infections. We found that the intensity of infection, evidenced by
hepatic egg load, was much reduced in mice born to infected mothers. No difference was found as regards total and
Schistosoma-specic immunoglobulin levels except for total IgG. The levels of gene expression of two regulatory
cytokines, namely interleukin-12 (IL-12) and transforming growth factor beta (TGF-b) were found to be signicantly
increased in prenatally exposed animals. Moreover, liver brosis was signicantly decreased in animals born to infected
mothers as revealed by histopathological and histochemical examination as well as by immunohistochemical
identication of activated hepatic stellate cells (HSCs) using antibody against glial brillary acidic protein (GFAP).
In conclusion, congenital exposure to S. mansoni seems to ameliorate the immunopathological changes in future
postnatal infections.
r 2009 Elsevier GmbH. All rights reserved.
Keywords: Congenital exposure; Cytokines; GFAP; Hepatic stellate cells; Immune response; Liver brosis; Schistosoma mansoni

Introduction
Abbreviations: BSA, bovine serum albumin; GFAP, glial brillary
acidic protein; HRP, horseradish peroxidase; HSCs, hepatic stellate
cells; IL-12, interleukin-12; MoAb, monoclonal antibody; p.i., postinfection; PBS, phosphate buffered saline; RT-PCR, reverse transcription polymerase chain reaction; TGF-b, transforming growth
factor beta; S. mansoni, Schistosoma mansoni.
Corresponding author at: Parasitology Department, Tanta Faculty
of Medicine, Tanta, Egypt. Fax: +20 2 40 3407734.
E-mail address: ahmed_ali44@hotmail.com (A.A. Othman).
0171-2985/$ - see front matter r 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.imbio.2009.04.004

Schistosomiasis is a water-borne parasitic disease that

plagues many tropical and subtropical regions all over
the world, aficting over 200 million people (Gryseels
et al. 2006). The principal events precipitating chronic
morbidity during infection with Schistosoma mansoni
(S. mansoni) develop as a result of schistosome eggs that
lodge in the liver, gut, and other organs causing
granulomatous inammation that ends in brosis and

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tissue damage. Although these egg-induced granulomas

effectively sequester toxic egg products, they lead to
severe hepatic brosis and portal hypertension (Wilson
et al. 2007).
Clinical observations in schistosomiasis-endemic
areas show that major complications of Schistosoma
infection develop in approximately 412% of the
population, while the majority of infected people remain
asymptomatic or exhibit mild non-specic symptoms
(Tachon and Borojevic 1978). Also, indigenous people
rarely manifest acute or toxaemic symptoms, whereas
individuals from non-endemic areas usually did (Caldas
et al. 2008). One possible reason for the presence or
absence of severe pathology is prenatal exposure to
schistosomiasis that modulates the future immune
response later in life (Malhotra et al. 1997). This notion
has been suggested by many authors and has been
explored in experimental models (Camus et al. 1976;
Montesano et al. 1999; King 2001).
Fibrosis is the ultimate sequel of chronic hepatic
schistosomiasis. A characteristic type of periportal
brosis occurs, which may lead to presinusoidal portal
hypertension and esophageal varices (Stavitsky 2004).
Apparently, hepatic stellate cells (HSCs) play a major
role in the process of brous tissue formation in the
liver. They are responsible for synthesis of components
of extra-cellular matrix and several types of collagen
(Parola and Robino 2001). These cells reside in the
Disses spaces of the liver sinusoids, and they constitute
a minor cell type, roughly 58% of the total liver cells
(Maubach et al. 2006). Following chronic injury, HSCs
differentiate into myobroblast-like cells, acquiring
contractile and brogenic properties (Zhang et al.
2006). For immunohistochemical identication of
HSCs, traditional antibodies against desmin (Yokoi
et al. 1984), vimentin, and a-smooth muscle actin
(Baroni et al. 1996) were used. More recently, glial
brillary acidic protein (GFAP), which is traditionally
used as a marker for astrocytes of the brain, was
established as a marker for HSCs (Lim et al. 2008).
Fibrosis is a complex process that is poorly understood,
but several immunological and non-immunological
determinants are suggested, and the role of cytokines
in this context is central (Alves-Oliveira et al. 2006).
Cytokines play a crucial role in the evolution and
regulation of Schistosoma-induced immunopathology.
In murine models, immune responses to schistosome
antigens manifest a striking shift from a moderate Th1
to a robust Th2-dominated response. Fibrosis and much
of the pathology is primarily mediated by Th2 while Th1
responses are presumed to be protective (Reiman et al.
2006). However, recent evidence suggests that maintaining a balanced and controlled Th1 or Th2 response is
critical in the case of schistosomiasis for protective
granuloma formation without excessive pathology
(Wilson et al. 2007). Transforming growth factor beta

(TGF-b) is secreted ubiquitously by many cell types,

including activated T cells and activated macrophages
(Miyazono et al. 1994). The bioactivity of TGF-b is
often bidirectional, depending on target cells or coexisting mediators (Tsutsui and Kamiyama 1999). However, its role in Schistosoma-induced inammation and
brosis remains controversial.
The aim of the present study was to evaluate the effect
of experimental congenital exposure to schistosomiasis
mansoni on the subsequent postnatal infection. We
assessed the intensity of infection, extent of brosis, as
well as several immunological parameters in mice born
to Schistosoma-infected mothers in comparison to those
born to non-infected ones.

Materials and methods

Parasite
Laboratory bred Biomphalaria alexandrina snails were
purchased from the Schistosome Biological Supply
Program, Theodore Bilharz Research Institute (Giza,
Egypt). After exposure to light for at least 4 h,
S. mansoni cercariae shed from the snails were used to
infect the experimental animals of the study.

Animals and experimental design

Laboratory bred and parasite free Swiss albino mice
(2025 g in weight) were used in this study. Mice were
purchased from Theodore Bilharz Research Institute
(Giza, Egypt) and were housed and infected in
accordance with the institutional guidelines. Eight weeks
old (n 10) female mice were infected with 200
cercariae of S. mansoni by subcutaneous injection as
described by Peters and Warren (1969). Each of 10
female mice was individually mated with a normal male
mouse 3 weeks post-infection (p.i.). Age-matched
uninfected female mice were also mated with male mice.
After delivery, offspring were divided into two main
groups: group I included 20 offspring born to noninfected mothers (infected control group), and group II
included 20 offspring born to S. mansoni-infected
mothers. Ten mice were left uninfected and served as
the non-infected control for immunological parameters
and immunohistochemical staining (group N). Groups I
and II were then infected subcutaneously with
S. mansoni cercariae 60710 cercariae/animal. Mice
of both groups were breast-fed, and were 8-week old at
the time of infection.
At 12 weeks p.i., animals of groups I, II, and N were
sacriced. Serum samples were collected from mice of
each group for detection of total and specic antiSchistosoma antibodies. The liver of each mouse was

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immediately removed and divided into three portions

for parasitological, immunological, histopathological,
and immunohistochemical studies.

Parasitological study
Liver egg load was estimated in groups I and II. One
gram from each liver was weighed, and then put in a test
tube containing 2 ml of 5% KOH and left overnight at
room temperature. The second day, all test tubes were
put in the incubator at 37 1C for 6 h. Each test tube was
shaken then 0.1 ml of the digest was examined microscopically for counting S. mansoni eggs. The total egg
count in 1 g liver tissue was then calculated (Cheever
1968).

Evaluation of humoral factors

Estimation of S. mansoni-specic IgE and IgG
Soluble antigens from S. mansoni adult worms
(SAWA) were prepared as described (El Ridi et al.
1993). In indirect ELISA, wells of polystyrene plates
(Costar, Cambridge, MA, USA) were coated with
500 ng soluble schistosome antigens, blocked with 1%
bovine serum albumin (BSA) in 0.05 M phosphate
buffered saline (PBS) pH 7.1, washed with PBS/0.05%
Tween 20 (washing buffer), and incubated with 100 ml
serum diluted in washing buffer. Specic, horseradish
peroxidase (HRP)-labelled rat monoclonal antibody
(MoAb) to mouse IgG and IgE were purchased from
Pharmingen (San Diego, CA, USA), and used diluted
1:1000 in washing buffer. HRP-labelled streptavidin
(Boehringer-Mannheim, Mannheim, Germany) was
used diluted 1:5000. Reactivity was estimated spectrophotometrically at 492 nm after adding o-phenylenediamine (Sigma Chemical Co., St. Louis, MO, USA)
substrate. Sera giving absorbance values higher than
the cut-off value ( mean absorbance of wells with
serum from control mice+3 SD) were considered
positive. Results were expressed in optical densities
(ODs).
Estimation of total IgE and IgG
Capture ELISA was used to determine the levels of
total IgG and IgE in mouse sera based on protocols
detailed by the manufacturer (Pharmingen). Wells of
polystyrene plates were coated with antimurine polyclonal IgG and antimurine IgE (1 chain-specic, clone
R35-72) rat MoAb. Following addition of the test
samples and standards and washing, HRP-conjugated
anti-mouse IgE (1 chain-specic, clone R35-118) rat
MoAb and biotin-labeled anti-mouse polyclonal IgG
were added to each well. The reaction was read
spectrophotometrically, and the serum levels of IgG

103

and IgE were calculated from a standard curve as

recommended by the manufacturer.

Estimation of levels of IL-12 and TGF-b mRNA

expression by semi-quantitative real-time PCR
Liver samples [10 randomly selected from groups I
and II, and 5 randomly selected from group N] were
taken and processed for RNA extraction (MagNA Pure
compact Nucleic Acid isolation kit I, Roche Diagnostics, GmbH, Mannheim, Germany) according to the
manufacturers recommendations. First strand cDNA
was synthesized from total RNA using Transcriptor
First Strand cDNA Synthesis Kit (Roche, Germany)
according to the manufacturers instructions.
Determination of IL-12 and TGF-b mRNA expression by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out using
b-globin as the internal gene control. PCR was carried
out using Roche LightCycler real-time PCR system.
Briey, in a 1.5 ml reaction tube on ice, the following
components were added in the same order they are
mentioned: 13 ml sterile H2O, PCR-grade, 2 ml LightCycler RT-PCR reaction mix SYBR Green I, 1.6 ml
MgCl2 stock solution (nal concentration 5 mM), 2 ml
cytokine primer mix (nal concentration 0.5 mM)
and 0.4 ml LightCycler RT-PCR enzyme mix, giving a
total volume of 19 ml. The components were mixed
gently then pipetted into pre-cooled LightCycler capillary. One microliter of the cytokine RNA template was
added giving a nal volume of 20 ml. The primer
sequences for IL-12 and TGF-b used in the study were
as follows:
IL-12p40 (sense 50 -GATGCTGGCCAGTACACC-30 ),
IL-12p40 (antisense 50 -TCCAGCACGACCTCAATG-30 ),
TGF-b (sense 50 -CTACTACGCCAAGGAGGTC-30 ),
TGF-b (antisense 50 -TGACCCGCAGAGAGGCTAT-30 ) (Techau et al. 2007). The optimal conditions for
PCR amplication were: an initial incubation at 95 1C
for 30 s, 45 cycles of denaturation at 95 1C, annealing at
55 1C (10 s), and extension at 72 1C (13 s). The gene
expression for measured cytokines and b-globin was
measured in copies/ml. For each cytokine, the amount
of RT-PCR product was normalized (as a ratio) to the
values obtained for b-globin, as an internal standard for
each sample.

Histopathological, histochemical, and

immunohistochemical study
All the studied specimens were xed in 10% formalin
and subsequently embedded in parafn, then submitted
to

Haematoxylin and eosin (H&E) staining for histopathological examination. The composition of the

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granulomas was assessed and the granulomas were

classied into three types (brous, cellular, and
brocellular) according to the predominant component (Costa-Silva et al. 2002).
Massons trichrome staining to highlight brotic
changes and to conrm the type of granuloma. The
procedure of Massons trichrome stain was performed according to Jones (2002).
Immunohistochemistry was done for demonstration
of activated hepatic stellate cells using antibody
against glial brillary acidic protein. Immunohistochemical staining was performed on 35 mm sections
from randomly selected 10 parafn blocks from each
of the infected groups and 5 blocks from group N,
using the UltraVision Detection System (AntiPolyvalent, HRP/DAB Ready-to-Use, Cat. #TP-015HD, Lab Vision, USA). The procedure of immunostaining was conducted according to the manufacturers protocol. Briey, sections were deparafnized
with xylene and rehydrated with graded alcohol
series. Antigen retrieval was done by immersing the
sections in 10 mmol/l citrate buffer (pH 6.0) for
10 min at 100 1C in microwave. Endogenous peroxidase activity was blocked with hydrogen peroxide
block for 10 min. After thorough washing of the
sections with phosphate buffered saline, incubation
was done for 10 min with Ultra V block to prevent
non-specic background staining, followed by rinsing
the sections with PBS. Subsequently, an overnight
incubation of the sections with antibody against
GFAP (Ab-4 rabbit polyclonal antibody, Cat. #RB087-R7 Ready-to-Use, Lab Vision, USA) was done
at room temperature in a humidity chamber. The
sections were then washed with PBS and incubated
with biotinylated goat anti-polyvalent (secondary
antibody) for 10 min at room temperature followed
by washing with PBS, then incubated with streptavidin peroxidase solution for 10 min at room temperature, then rinsed with PBS. The reaction products
were visualized using 3-30 -diamino-benzidine-tetrahydrochloride (DAB), and the sections were then
counterstained with Mayers haematoxylin, dehydrated in alcohol and mounted in DPX. Sections
from the brain of a mouse were used as positive
controls, while negative controls were prepared by
omission of the primary antibody. Cells showing a
distinct brownish cytoplasmic reaction to GFAP
were considered positive. A semi-quantitative evaluation was performed in order to estimate the number
of GFAP-positive HSCs using scores from 0 to 4,
with 0 indicating the absence of positive staining; 1
indicating the presence of mild staining +; 2
indicating the presence of moderate staining ++;
3 indicating the presence of intense staining
+++; and 4 indicating the presence of very
intense staining ++++ (Gibelli et al. 2008).

Statistical analysis
Data were presented as means7standard deviation.
The probability of signicant differences among dual
means of groups was determined by Students t-test.
Fishers exact test was used for evaluating GFAP
expression. Differences were considered non-signicant
when (P40.05), signicant (Po0.05), and highly
signicant (Po0.001). The statistical analyses were
processed according to the conventional procedures
using Statistical Program of Social Sciences (SPSS)
software for windows, version 10.0.

Results
Hepatic egg load
Results of liver egg load are shown in Table 1. There
was highly signicant reduction in the mean numbers of
liver egg count in mice born to infected mothers (group
II) compared to infected control mice (group I). The
mean liver egg count in group I at 12 weeks p.i. was
(46807191.3) compared to group II (28307412.5),
Po0.001.

Antibody response
Results of antibody response 12 weeks p.i. are
demonstrated in Fig. 1. There were no signicant
differences between groups I and II regarding total
IgE, Schistosoma-IgE and Schistosoma-IgG. On the
other hand, highly signicant increase in total IgG level
was observed in group II compared to group I,
Po0.001.

Cytokine mRNA expression

Fig. 2 shows levels of mRNA expression of the
measured cytokines. In the liver homogenates, levels of
IL-12 and TGF-b mRNA expression were signicantly
increased in mice born to infected mothers (group II)
compared to mice born to non-infected mothers (group I),
and non-infected animals (group N), Po0.001.
Table 1. Liver egg count (mean7SD) recovered from mice
born to non-infected mothers (group I) and mice born to
infected mothers (group II).
Group

Egg count

Reduction (%)a

P-value

Group I (n 20)
Group II (n 20)

46807191.3
28307412.5

39.5

o0.001

a
Percent reduction was calculated according to the formula [(group I
meangroup II mean)/group I mean]  100.
 Po0.001 (signicant reduction).

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Total IgG

IL-12
4

14
12
mg/dl

10

Group N

3.5

Group I
Group II

Group N
Group I
Group II

2.5

8
6

1.5

0.5
0

Total IgE
5

IU/ml

105

TGF-
5

Group N
Group I
Group II

Group N

Group I
Group II

absorbance (492nm)

0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

2
Group I

Group II

Sch- IgG

Sch- IgE

Fig. 1. Values of total IgG, total IgE, Schistosoma-IgG (SchIgG), and Schistosoma-IgE (Sch-IgE) in infected control mice
(group I) compared to mice born to infected mothers (group
II) expressed in means7SD. Signicant elevation in total IgG
level is observed in group II compared to group I (n 20).
Po0.001.

Histopathological and histochemical ndings

Histopathological examination revealed the presence
of granulomatous reactions within the portal tracts.
Each granuloma was formed around a Schistosoma
ovum and was composed of cellular collection of
eosinophils, histiocytes, lymphocytes, and plasma cells.
Peripheral broblasts with variable amounts of collagen
deposition were seen in brocellular and brous types.
Percentages of the different types of granulomas in the
studied groups are illustrated in Fig. 3. In group I, the
brous granulomas were the most prevalent type
(Fig. 4A), whereas in group II, the brocellular type
predominated (Fig. 4B). The brotic changes within the
granulomas were highlighted by the Massons trichrome
stain (Fig. 4C and D).

Fig. 2. Cytokine mRNA expression levels in liver tissues 12

weeks p.i. Vertical bars represent the mean (7SD) of these
results for each group (group N, n 5, groups I and II,
n 10). Signicant increase in levels of IL-12 and TGF-b
mRNA was found in mice born to infected mothers (group II),
Po0.001.

Immunohistochemical evaluation of GFAP

expression
In the normal control group (group N), very few
scattered HSCs showing faint positivity for GFAP were
detected. In both infected groups, GFAP-positive HSCs
appeared in two locations: (a) sinusoidal (parenchymal)
HSCs, which appeared as thin irregular positive bands
along the sinusoids with a dislocated nucleus and (b)
mesenchymal HSCs located in the hepatic lobule close
to the portal tracts, within the portal tracts, and within
the granulomas. Immunohistochemical localization of
GFAP in both groups revealed that group I showed
higher GFAP expression in comparison to group II both
in sinusoidal (Fig. 5A and B) and mesenchymal (Fig. 5C
and D) HSCs, indicating a more active and more
extensive brotic process in group I compared to group II.
GFAP expression in the infected groups is illustrated in
Table 2. The predominant grades of GFAP expression
in group I were grades 3 and 4, while in group II the
predominant grades were grades 1 and 2. The difference
between both groups was statistically signicant
(Po0.05).

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Group I (infected control group)

Group II (born to infected mothers)

8.20%

27.30%

32.30%

29.40%

62.40%
40.40%

Cellular

fibrocellular

fibrous

Cellular

fibrocellular

fibrous

Fig. 3. Comparison between group I (infected control) and group II (born to infected mothers) regarding the percentages of the
different types of granulomas. Group I granulomas are mainly of the brous type while in group II brocellular granulomas
predominate.

Fig. 4. Photomicrographs of schistosomal hepatic granulomas: (A) a large number of granulomas formed around Schistosoma ova,
most of them are of the brous variety arrows in infected control group I (H&E  100), (B) fewer granulomas arrows in
prenatally exposed mice group II. The granulomas are brocellular (H&E  100), (C) extensive brotic changes within the
granuloma in group I (Massons trichrome  400), and (D) little brotic changes within the granuloma in group II (Massons
trichrome  400).

Discussion
Schistosomiasis remains a major cause of morbidity in
tropical and subtropical countries (Engels et al. 2002). It
is quite intriguing that in endemic areas few people

develop severe pathology and sequelae, while the

majority show asymptomatic or mild pathology. Intensity and duration of the infection are major
determinants, but other factors are also involved. These
include genetic background of the host, nutritional

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107

Fig. 5. Hepatic immunohistochemical staining for GFAP: (A) section from infected control group I showing grade 4 GFAP
expression located in sinusoidal HSCs arrow (immunoperoxidase GFAP  400), (B) section from prenatally exposed mice group
II showing grade 2 GFAP expression located in sinusoidal HSCs arrow (immunoperoxidase GFAP  400), (C) section from
group I showing grade 3 GFAP expression located in both mesenchymal arrow and sinusoidal HSCs (immunoperoxidase GFAP
 400), and (D) section from group II showing grade 2 GFAP expression located in mesenchymal HSCs arrow
(immunoperoxidase GFAP  400).

Table 2.

GFAP expression in the infected groups.

Groups

Group I (n 10)
Group II (n 10)

GFAP expression
Grade 0
GFAP staining

Grade 1
GFAP staining (+)

Grade 2
GFAP staining (++)

Grade 3
GFAP staining
(+++)

Grade 4
GFAP staining
(++++)

0
0

0
0

1
5

10
50

2
4

20
40

4
1

40
10

3
0

30
0

There is a signicant difference between both groups, Po0.05.

status, parasite strain differences, and frequency of

infection. Maternal infection status in that offspring of
the infected mothers may be primed to mount a
modulated type of response at rst infection has been
proposed by many authors in order to explain the
individual differences (Butterworth and Thomas 1999).

Epidemiology provides some evidence: individuals who

move from non-endemic to endemic areas, such as
migrants, travelers, or military personnel, often express
more severe acute and chronic schistosomiasis compared to those who grew up within endemic areas (Nash
et al. 1982).

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In the present study, mice born to infected mothers

showed signicant reduction in liver egg count compared to infected control animals. Reduction in adult
worm count may provide an explanation for these
ndings. Attallah et al. (2006) demonstrated that adult
worm count was signicantly reduced in the offspring of
Schistosoma-infected mothers compared to mice born to
non-infected ones, but no explanation was offered. We
assume that the situation is perhaps similar to that of
concomitant immunity in which continuous antigenic
stimulation (by the presence of some adult worms)
stimulates protective immune response against further
infections. Early stimulation of the immune system by in
utero exposure to schistosomiasis may, therefore,
provide quite strong protective immunity against initial
postnatal infections. In sum, congenital exposure to
Schistosoma infection seems to reduce the intensity of
infection in subsequent postnatal exposure.
As regards the humoral immune response, no
signicant differences were noted in the levels of
Schistosoma-specic IgG and IgE, as well as total IgE
between mice born to infected mothers and infected
control animals. In contrast, total IgG level was
signicantly increased in prenatally exposed mice.
Attallah et al. (2006) found that Schistosoma-specic
IgG levels were lower in prenatally exposed mice
compared to control. However, IgG levels were measured earlier in the course of infection than in our study.
The increase in total IgG levels in our study should be
interpreted with caution, for different classes of
IgG serve different functions: whereas IgG2 class is
considered to confer immunity (Dunne et al. 1995;
Noya et al. 1995), IgG4 are blocking antibodies
that may block IgE-mediated effects (Noya et al. 1995;
King 2001), and may play a role in immunological
tolerance in prenatally sensitized individuals (Caldas
et al. 2008). Although IgE levels are correlated with
resistance to infection especially after treatment (Satti
et al. 1996), no difference was noted in our study
between both infected groups as regards total and
specic IgE levels.
Interleukin-12 (IL-12) is a heterodimer composed of
two disulde-linked polypeptide chains that are products of two distinct genes. It plays a pivotal role in
promoting cell-mediated immune response. It promotes
the differentiation of uncommitted T helper cells to the
Th1 subset (Trinchieri and Scott 1999). A major action
of IL-12 is its ability to induce the production of IFN-g,
doing so synergistically in combination with IL-2. The
major producers of IL-12 are the dendritic cells and
macrophages (Gately et al. 1998).
In our study, expression levels of IL-12 were
signicantly increased in mice born to infected mothers
compared to those born to non-infected ones. Few data
are available regarding the role of IL-12 in schistosomiasis, but all point out a benecial effect (Wilson et al.

2007). Vaccination of mice with parasite eggs and IL-12

inhibits the Th1 to Th2 shift and results in amelioration
of hepatosplenic pathology following infection (Wynn
et al. 1995). Similarly, mice lacking IL-12 develop a
vigorous Th2 response that is detrimental during the
chronic phase of infection and display signicant
mortality by 1215 weeks post-infection (Hoffmann
et al. 2000). The high levels of IL-12 may account in part
for the ameliorated liver pathology and brosis noted in
our experiment.
Transforming growth factor-b (TGF-b) is a pleiotropic cytokine that is involved in a number of
biological processes. It induces collagen and bronectin
production by broblasts. Further, it inhibits many
products of lymphocyte function including T-cell proliferation, and plays a critical role in T-cell homeostasis
(Gorelik and Flavell 2000). It is produced by special
subset of CD4+ T helper cells, sometimes called Th3
(Chatila 2005). Its role in Schistosoma-induced pathology and brosis is unclear especially in humans. Some
authors believe that during hepatic brogenesis, TGF-b
has a pivotal role in initiation and progression of
differentiation of HSCs into myobroblasts (Gressner
and Weiskirchen 2006; Chu et al. 2007). On the other
hand, de Jesus et al. (2004) did not nd differences in
TGF-b levels in patients with different degrees of
hepatic brosis. Others suggested that TGF-b is a
regulatory cytokine which provides an effective mechanism of control of the progression of brosis in
association with IL-10 (Kitani et al. 2003; Hesse et al.
2004). Moreover, an experimental study has demonstrated that brosis and much of the pathology of
schistosomiasis is mediated by Th2 cytokines especially
IL-13, and not by TGF-b (Kaviratne et al. 2004).
We found that TGF-b mRNA expression levels were
signicantly higher in mice born to infected mothers
compared to infected control. This may account for
reduced pathology and brosis observed in our study,
and this is probably related to its inhibitory effect on
T-cell proliferation. The brogenic activity of TGF-b
might be offset by its immunoregulatory function.
Furthermore, IFN-g suppresses the brogenic activity
of TGF-b by down-regulating the expression of its
receptors (Zhang et al. 2004). Thus, increased IL-12 may
have attenuated the brogenesis stimulated by TGF-b.
Presumably, other regulatory mechanisms may be
involved, such as CD4+CD25+ T regulatory cells,
since these cells increase in experimental Schistosomainduced hepatic granulomas from 12% at 8 weeks to
88% at 16 weeks during the course of infection, and they
ameliorate liver pathology in experimentally infected
mice (Singh et al. 2005). However, this assumption
remains to be tested.
Fibrosis is the main culprit in Schistosoma-induced
pathology. The hepatic stellate cells (HSCs) play a
major role in this process, and there has recently been

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unprecedented interest in this cell (and its activated

form) as a prognostic indicator of progression of liver
brosis, as well as a potential target for therapeutic
intervention (Moreira 2007). In addition, multiple
studies have demonstrated a positive correlation between the severity of hepatic brosis and the number of
activated stellate cells present in the liver (Russo et al.
2005). However, the different stimuli that initiate and
perpetuate HSC activation in chronic liver diseases are
poorly understood. Remarkably, HSCs affect adversely
the hepatic microcirculation. When activated, they
transform into myobroblasts that contract around
the hepatic sinusoids, increasing the vascular resistance
and contributing to portal hypertension (Friedman
2000).
In our study, histopathological and histochemical
examination revealed that most granulomas were
brocellular in prenatally exposed offspring, whereas
most were brous in infected control group. In addition,
cellular granulomas were more abundant in prenatally
exposed mice. Evident reduction in the activity of HSCs
was observed in mice born to infected mothers
compared to infected control group as demonstrated
by immunohistochemical staining for GFAP. Previous
animal studies did demonstrate smaller-sized granulomas in offspring of infected mothers compared to
control (Hang et al. 1974; Montesano et al. 1999;
Attallah et al. 2006), but this is the rst time, to the best
of our knowledge, to demonstrate the direct effect of
maternal infection on HSC activity in schistosomiasis.
The reduced size of granulomas observed by many
authors and in our experiment (data not shown), and the
reduction in brosis in congenitally exposed mice may
be attributed to attenuated Th2 response. The latter may
be due to, at least in part, an increase in counterregulatory cytokines such as IL-12 and TGF-b.
Furthermore, recent research stresses the role of free
oxygen radicals as activators of HSCs (Loguercio and
Federico 2003). As the overall liver inammation was
expected to decrease in congenitally exposed mice
due to decreased egg deposition and granuloma formation, less free oxygen radicals would be produced with
attenuated stimulation of HSCs compared to infected
control mice.
Perinatal immunological sensitization may occur by
transplacental and/or transmammary passage of schistosome antigens (Carlier et al. 1980), anti-idiotypic
antibodies (Montesano et al. 1999), or immune complexes (Uhr et al. 1957). Attallah et al. (2006) have
already detected Schistosoma antigens in the tissues of
offspring born to infected mothers particularly in the
liver and kidneys. In addition, several authors suggested
that cellular hypersensitivity to S. mansoni antigen could
in part be transmitted in milk of infected mothers to
their infants (Eissa et al. 1989; Santoro et al. 1977).
Presumably, exposure to parasite antigen very early in

109

the development of the immune system results in

immunological tolerance. The antigens can be no longer
recognized as totally foreign. On initial postnatal
exposure, the primed immune system may be more
prone to develop less striking shift from Th1 to Th2
responses as seen in truly immunologically nave
individuals (Caldas et al. 2008). A more balanced
T-cell response may be responsible for improved
immunopathological processes and perhaps better host
parasite interaction in indigenous populations.
These results can be relevant to humans since many
children are indeed born to infected mothers in endemic
areas. Unfortunately, no human studies have directly
shown that in utero exposure to schistosomiasis affects
the subsequent outcome of the disease. However, vast
amount of empirical evidence exists. For example,
children in Schistosoma-endemic areas rarely manifest
acute manifestations such as acute dermatitis or
katayama fever. These children are born to mothers
who had schistosomiasis during their pregnancy, and are
probably already primed for many anti-schistosome
responses at the time of their rst exposure to cercariae
(Caldas et al. 2008). Interestingly, congenital transmission of Schistosoma japonicum to the offspring when
the mother acquires the infection during pregnancy
was conrmed in pigs (Willingham et al. 1999), and in
humans (Kikuchi 1957). If this proves to be true for
human schistosomiasis mansoni, this will add to the
complexity of the situation, and might provide a
stronger background for immunomodulation in subsequent infections.
Not surprisingly, the inuence of in utero exposure to
infections has been suggested in other chronic parasitic
diseases. For example, in laria-endemic areas fetuses
are exposed to substantial amount of larial antigen.
Exposure to antigens at this stage of development
promotes tolerance by the immune system (Hightower
et al. 1993). Thus, a child born to a microlaremic
mother is 2.9 times more likely to become microlaremic
than a child born to an amicrolaremic mother.
Likewise, many individuals in endemic areas are
asymptomatic microlaremics (Kumar 1997). The same
pattern is found in malaria-endemic areas where
non-endemic individuals react differently than indigenous people (Rasheed et al. 1995). Moreover, transplacental transfer of Opisthorchis felineus antigens in the
hyperendemic foci of infection does not prevent superinfection, but does prevent the acute phase of disease
and signicantly mitigates the organic lesions in the
chronic phase in spite of a very high intensity of
infection (Ozeretskovskaia 2000). One recent study
demonstrated that experimental maternal infection
with Opisthorchis viverrini affected worm fecundity
and worm load in acquired postnatal infections of
the offspring with the same parasite (Intapan and
Maleewong 2006).

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In conclusion, prenatal exposure to S. mansoni

infection affects the immune response and the disease
outcome in the future postnatal infections; the intensity
of the infection as well as the immunopathological
changes are moderated in congenitally exposed hosts.
These ndings warrant further investigations regarding
the underlying mechanisms of immunomodulation here
and in other parasitic infections, and they may also be
applicable to other Th2-dominated diseases, such as
allergy and bronchial asthma. Finally, these results may
give us insight for prevention and control strategies in
schistosomiasis, and perhaps in other chronic infectious
diseases as well.

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