Parasite Immunology, 2002, 24, 499 – 509

Cellular responses and cytokine profiles in Ascaris lumbricoides and

Immune
ORIGINALresponse
Blackwell to intestinal
Publishing
ARTICLE Ltd. helminthic co-infections

Trichuris trichiura infected patients

STEFAN M. GEIGER1,2, CRISTIANO L. MASSARA1, JEFFREY BETHONY1,3, PETER T. SOBOSLAY2, OMAR S. CARVALHO1
& RODRIGO CORRÊA-OLIVEIRA1

1
Fundação Oswaldo Cruz, Centro de Pesquisas René Rachou, Laboratório de Imunologia, Avenida Augusto de Lima 1715, 30190-002 Belo
Horizonte-MG, Brazil, 2Institute for Tropical Medicine, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany and 3Southwest
Foundation of Biomedical Research, San Antonio, Texas, USA

SUMMARY Keywords Ascaris lumbricoides, cytokines, immunity, intestinal

helminths, Trichuris trichiura
The impact of intestinal helminth infection, i.e. Ascaris lum-
bricoides and Trichuris trichiura, on cellular responsiveness and
cytokine production was investigated in young adults. Ascaris- INTRODUCTION
specific cellular responsiveness was higher in parasite-free
Intestinal nematode infections are among the most pre-
endemic controls than in patients infected with T. trichiura, or
valent diseases world-wide, affecting at least one-quarter of
A. lumbricoides, or patients co-infected with both parasites. Also,
the world’s population (1). The recent World Health Organ-
mitogen-induced tumour necrosis factor (TNF)-α, interleukin
ization (WHO) evaluation estimates are that 1·4 billion
(IL)-12 and interferon (IFN)-γ secretion by peripheral blood
individuals are infected with Ascaris lumbricoides, 1 billion
mononuclear cells (PBMC) was higher in negative endemic
with Trichuris trichiura and 1·3 billion with hookworms (2).
controls than in infected individuals. Ascaris antigen-specific
Although such chronic infections are normally not fatal, they
production of TNF-α, IL-12 and IFN-γ was low in singly
cause a high degree of morbidity (3). However, in heavily
Ascaris as well as in co-infected patients, whereas secretion of
infected individuals thousands of deaths per year occur
IL-10 and IL-13 was elevated and similarly high in all patient
due to intestinal obstruction, massive dysentery syndrome
groups. The detection of Trichuris-specific and Ascaris-specific
or severe iron-deficiency anaemia (2).
IgG4 revealed significantly higher serum antibody levels in
In experimental infections it was shown that intestinal
Trichuris or Ascaris patients when compared to endemic
helminth infections will skew host immune responses
controls (P < 0·05), whereas parasite-specific IgE antibody
towards a type 2 cytokine profile, which leads to IgE pro-
levels were similarly high in infected individuals and in endemic
duction, mast cell activation, mucus secretion and will
controls. In summary, chronically infected Ascaris and
ultimately lead to parasite elimination (4,5). In accordance
Trichuris patients with a high parasite load presented reduced
with these experimental infections, patients with an Ascaris
cellular reactivity and lower type 1 TNF-α, IFN-γ and IL-12
lumbricoides infection have highly polarized type 2 cytokine
responses when compared with endemic controls, whereas
responses (6) and children, putatively immune against A.
type 2 IL-10 and IL-13 productions were similar in all groups
lumbricoides, have significantly elevated IgE levels specific for
from the endemic area. The former may support parasite
a recombinant Ascaris allergen (7). Furthermore, Needham
persistence, whereas substantial type 2 cytokine release may
et al. (8) showed a protective role for IgA in immunity to
promote protective immunity, suggesting an adaptation of
T. trichiura in areas of intense transmission. However, apart
the host to control the parasite burden while minimizing
from these few reports, knowledge on the immune responses
immune-mediated host self-damage.
induced in humans infected with one or more intestinal
helminth species remains scarce.
In endemic tropical areas polyparasitism is commonly
Correspondence: Present address: Dr Stefan Geiger, Institute found, and such coexistence of several parasite species may
for Comparative Tropical Medicine and Parasitology, University
not only profoundly influence the host–parasite interaction
of Munich, Leopoldstraße 5, 80802 München, Germany
(e-mail: stefan.geiger@tropa.vetmed.uni-muenchen.de). (9,10), but also confound the development of acquired
Received: 15 February 2002 immunity against individual parasites. Experimental co-
Accepted for publication: 30 October 2002 infections with intestinal helminth parasites showed that a

© 2002 Blackwell Science Ltd 499

S. M. Geiger et al.
primary helminth-induced Th 2-type immune response
alters the outcome of a concurrent infection with a second
helminth parasite (11,12). In humans, however, the impact
of multiple parasite infections on expression of immunity is
controversial and remains poorly studied (13,14). In the
present study, we investigated the humoral and cellular
immune responses in children and young adults infected with
one or two of the common geohelminths, A. lumbricoides
and /or T. trichiura, and we observed that co-infection and a
high parasite load will not only exhaust parasite-specific
cellular responsiveness but will also reduce type 1 cytokine
responses.
MATERIALS AND METHODS
Study population
This study was conducted in two rural communities, Motas
and Pires, about 70 km south of Belo Horizonte, the capital
of the state of Minas Gerais, Brazil. Both communities
were situated close to each other and had the same socio-
economic conditions. The region around Motas and Pires
is characterized by small agricultural units and a big mining
area. Most of the households in the two communities have
no access to clean water supplies and appropriate sanita-
tion. In Motas a total of 378 subjects were examined for
gastrointestinal helminth infections, which covered over
95% of the population. In Pires 201 children from the local
primary and secondary school were examined. All Ascaris/
Trichuris positive subjects received a standard treatment of
6 × 100 mg mebendazole during three consecutive days.
Those individuals with Taenia sp. or with hookworm infec-
tion received a 6 × 200 mg mebendazole or a single 400 mg
dose of albendazole, respectively. Subjects with a Schisto-
soma mansoni infection received praziquantel treatment
from the local health authority. This study was approved by
the Ethical Committee of the Centro de Pesquisas René
Rachou. Participation in the study was dependent on written
consent, and in the case of children, written consent was
given by their parents or guardians.
Subject sampling and parasitology
Individuals were examined for intestinal helminth infections
using the Kato–Katz technique (15). Mean egg counts per
gram of faeces (epg) were calculated from at least two slides
per sample and up to 5000 eggs per slide were counted
under a microscope. Egg-negative endemic controls (EC)
were examined twice with two slides each time and within
1 week before enrolment in the study. Participants had not
received chemotherapy during the last 6 months before
enrolment into the study. Negative non-endemic controls
500
Parasite Immunology
(NEG) with no recent reports of helminth infections were
from the urban area of Belo Horizonte. Twenty millilitres of
blood were collected, from randomly selected individuals, in
a heparinized Vacutainer tube (Becton Dickinson, Franklin
Lakes, NJ). For differential blood counts and for a haemo-
gram, 5 mL were collected in a Vacutainer tube containing
EDTA (Becton Dickinson) and analysed in a Coulter counter
(Coulter Corporation, Miami, FL). For differential blood
counts, 5 µL of blood were put on a slide, dried, and stained
with a fast-staining solution (Laborclin, Pinhais-Paraná,
Brazil) and 100 white blood cells were counted under a light
microscope (400 × magnification) to determine eosinophilia
in the peripheral blood. Furthermore, blood was collected
in a non-heparinized tube and, after centrifugation, the serum
samples were stored at −70°C until further use.
Mitogen and antigen preparation
For polyclonal stimulation of PBMC phytohaemagglutinin-
L (PHA-L, Difco Laboratories, Detroit, MI) was used. Ascaris
lumbricoides (Ascaris antigen: ASC-Ag) and T. trichiura adult
worms (Trichuris antigen: TRIC-Ag) were collected from
treated individuals from the same endemic area. Worms
were extensively washed in cold phosphate buffered saline
(PBS), cut in pieces and transferred into a tissue grinder in
a small volume of PBS. The worms were homogenized on
ice and subsequently centrifuged at 20 000 g for 40 min at
4°C. Schistosoma mansoni parasitic stages were obtained
from Swiss mice experimentally infected at the animal facil-
ities of the Centro de Pesquisas René Rachou-FIOCRUZ in
Belo Horizonte. Schistosoma mansoni soluble adult worm
(SWAP) and egg antigens (SEA) were prepared as previ-
ously described (16). All PBS-soluble antigen fractions were
passed through a low protein binding 0·2 µm filter (Millipore,
Bedford, MA) and the protein concentration was determined
according to the method of Lowry et al. (17).
Peripheral blood mononuclear cell (PBMC) proliferation
assays
PBMC were isolated on a ficoll–diatriozoate cushion
(1·077 g/L, lymphocyte separation medium; Organon
Teknika, Charleston, SC) by centrifugation at 400 g for
40 min at room temperature (RT). PBMCs were collected,
washed three times with minimal essential medium (Gibco,
Grand Island, NY), and resuspended in RPMI 1640
medium (Gibco), containing 5% heat-inactivated normal
human AB serum, antibiotic/antimycotic solution (penicil-
lin: 100 U/mL, streptomycin: 100 µg/mL, amphotericin-B:
0·25 µg /mL; Sigma, St. Louis, MO), and 2 m -glutamine
(Winlab, Leicestershire, United Kingdom). The cell suspensions
were adjusted to 1·25 × 106 cells /mL and 200 µL per well were
© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499–509
Volume 24, Number 11/12, November/December 2002
seeded in 96-well tissue culture plates (Costar, Corning, NY).
All cell culture experiments were performed in a humidified
incubator at 5% CO2 and at 37°C. Stimulation assays were
performed in triplicate and mitogen and antigens were
added at previously determined concentrations, known to
result in optimal proliferation, i.e. PHA (4 µg /mL), ASC-Ag
(20 µg /mL); TRIC-Ag (20 µg/mL), SEA (25 µg /mL), SWAP
(25 µg /mL). For mitogen stimulation, the cells were pulsed
after 48 h of culture with tritiated thymidine (Amersham
Pharmacia, Sao Paulo, Brazil; 0·5 µCi /well, specific activity
6·7 Ci/m) and harvested after further 18 h of culture.
Antigen-stimulated cell cultures were pulsed after 6 days
with tritiated thymidine and harvested after an additional 6 h
of culture. Incorporated activity was determined in a liquid
scintillation counter and the data expressed as stimulation
indices SI (SI = mean proliferation of stimulated culture /
mean proliferation of unstimulated culture).
Cytokine production in PBMC supernatants
PBMCs were isolated and adjusted as described above and
600 µL per well were seeded in 48-well tissue culture plates
(Costar), and then stimulated with mitogen or worm anti-
gens in the same concentrations as above. The cultures were
harvested at 48, 72, and 96 h, centrifuged, and the cell-free
supernatants stored at −70°C until cytokine quantification.
Cytokine-specific enzyme-linked immunosorbent assays
(ELISA) were performed according to the manufacturer’s
instructions (Opti-EIA Sets; Pharmingen, Heidelberg,
Germany) for the following cytokines: interleukin (IL)-10,
IL-12, IL-13, interferon-γ (IFN-γ), and tumour necrosis
factor-α (TNF-α). Briefly, the plates were sensitized with the
capture-antibody and incubated at 4°C overnight. After a
blocking step with PBS/10% foetal calf serum (FCS) (Biomast,
Rio de Janeiro, Brazil) for 1 h at room temperature (RT),
the plates were washed twice with PBS/ Tween-20 (0·05%)
and subsequently incubated for 2 h at RT with the cell
culture supernatants and the diluted cytokine standards.
After another washing step with PBS/ Tween-20, detection
antibody plus avidin–horseradish peroxidase were added to
the wells and incubated for 1 h at RT. The plates were
washed again (7×) and finally the substrate solution (tetram-
ethylbenzidine, TMB) was added. Colorimetric reaction was
stopped with 2  H2SO4 and absorbance measured at
450 nm in an automated ELISA reader. Cytokine concen-
trations in supernatants were measured in duplicate and
were calculated according to standard curves. Mean cytokine
concentrations are expressed in pg /mL and are shown as
net production from which background cytokine secretion
has been subtracted. The detection limits of the different
cytokine assays were below the standard curves as recom-
mended by the manufacturers for IL-10 (7·8 pg /mL), IL-13
© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499 –509
Immune response to intestinal helminthic co-infections
(3·1 pg /mL), IFN-γ (9·4 pg /mL), and TNF-α (15·6 pg/mL).
For IL-12, the detection limit was assessed to be at around
40 pg/mL.
Parasite-specific antibody reactivity in sera
Sera from infected patients, endemic controls and negative
controls were screened for Ascaris and Trichuris antigen-
specific antibodies in an indirect ELISA. Briefly, ELISA
plates (Nunc, Maxisorp, Naperville, IL) were coated over-
night at 4°C with 100 µL of ASC-Ag or TRIC-Ag (5 µg/mL)
diluted in carbonate buffer (15 m Na2CO3, 35 m NaHCO3,
0·02% NaN3, pH 9·6). The plates were washed twice with
PBS/0·05% Tween-20 and incubated with the individual
sera diluted in PBS/10% FCS (1 : 100 for IgG and IgA, and
1 : 50 for IgG1, IgG4, and IgE) at 4°C overnight. On the
following day, the plates were washed (5×) and 100 µL
per well of alkaline phosphatase-conjugated detection
antibody in PBS/10% FCS was added at the following
dilutions: IgG 1 : 1000 (Sigma-Aldrich, Steinheim, Germany);
IgG1, IgG4, IgA 1 : 1000 (Pharmingen); IgE 1 : 2000
(Pharmingen). The plates were incubated for 2 h at RT and
subsequently washed 7 times with PBS/0·05% Tween-20.
In a final step, 100 µL of p-nitrophenylphosphate substrate
(Sigma) diluted in substrate buffer (0·5 m MgCl2, 10%
diethanolamine, pH 9·8) were added to the plates and the
colorimetric reaction measured in an automated ELISA
reader at 405 nm. Serum antibody reactivity for individual
samples was performed in duplicate. Values from different
plates were compared and standardized according to standard
reference sera from Ascaris- and Trichuris-infected patients
and endemic control sera. Values are expressed as mean optical
densities and compared between the different patient groups.
Statistical analyses
All values were checked for normal distribution by the
Shapiro–Wilk W-test. Differences between the five patient
groups were checked for significance by using the nonpara-
metric Wilcoxon test. Bivariate analysis was done by perform-
ing the nonlinear Spearman’s rank test. Significance levels
were taken at the 5% level unless otherwise indicated. Due
to multiple testing, a Bonferroni–Holm test was performed
in order to correct significance values in each experiment,
and adjusted P-values are indicated in the figures and tables.
RESULTS
Study group characteristics
The prevalence of A. lumbricoides in Motas and Pires was
43·7% and 53·7%, respectively. For T. trichiura infection the
501
S. M. Geiger et al.
Table 1 Study characteristics in patients infected with Ascaris lumbricoides or Trichuris trichiura, and in uninfected control groups
ASC + TRIC
n = 16
Sex: M/F 10/6
Mean age, years 11·5
(7–19)
Ascaris lumbricoides, epg 10 944
(12–120 000)
Trichuris trichiura, epg 227
(12–972)
Eosinophilia percentage 5·7
Eggs per gram stool (epg) are indicated as geometric mean. Numbers in parentheses indicate the range. A. lumbricoides eggs are counted for
a maximum of 5000 eggs per slide and set as 120 000 eggs per gram stool. M, male; F, female. Study groups: ASC + TRIC, A. lumbricoides
and T. trichiura co-infected; ASC, A. lumbricoides mono-infected; TRIC, T. trichiura mono-infected; EC, parasite-free endemic controls; NEG,
parasite-free non-endemic controls.
prevalence was 20·6% (Motas) and 37·8% (Pires). Individu-
als with S. mansoni infection (prevalence: Motas: 0·5%;
Pires 1·5%) or hookworm infection (prevalence: Motas:
0·8%; Pires 0·5%) had migrated into the study area and sup-
posedly got this infection in other localities where these
helminth parasites are endemic. Demographic and parasito-
logical data for the study groups are shown in Table 1. The
patient groups from the endemic area had no statistically
significant differences in their age distribution. The mean
infection intensity for A. lumbricoides in the mono- and co-
infected groups was moderate (A. lumbricoides mono-
infected: 12 454 epg; co-infected: 10 944), whereas patients
with T. trichiura infection had light infections (T. trichiura
mono-infected: 171 epg; co-infected: 227 epg). Analysis of
variance () did not reveal significant differences in
intensities of infection between the mono- and co-infected
groups for both parasite species. Ascaris lumbricoides and T.
trichiura patients presented low to moderate eosinophilia
(Table 1), as determined by differential blood counts, and
none of the individuals showed signs of severe anaemia or
malnutrition. For parasite-specific ELISA, the number of
patients enrolled in each study group was increased as indi-
cated below ( Table 3a,b).
Lymphocyte proliferation and cytokine production
The proliferation of PBMC after stimulation with parasite
antigens is indicated as stimulation indices and presented in
Figure 1. In response to Ascaris antigen, endemic controls
showed the highest cellular responses, whereas cellular reac-
tivity in egg-positive Ascaris patients was in the range of
non-endemic negative controls. After stimulation with
Trichuris antigens, PBMC from T. trichiura mono-infected
patients and endemic controls showed the highest prolifera-
tion when compared with the other groups, but the differences
502
Parasite Immunology
ASC TRIC EC NEG
n = 14 n = 14 n=9 n=8
5/9 7/7 3/6 3/5
14·6 13·9 13·6 29·4
(8–27) (9–25) (11–17) (25–32)
12 454 0 0 0
(12–120 000)
0 171 0 0
(24–1044)
6·7 4·2 3·7 1·1
were not statistically significant. The response to S. mansoni
soluble egg antigens (SEA) was low in all groups. Interest-
ingly, an elevated cellular reactivity was observed in PBMC
from endemic and non-endemic controls after stimulation
with SWAP. Here, mono-infected Ascaris and co-infected
patients showed the lowest proliferative responses, and dif-
ferences between co-infected patients and non-endemic
controls were statistically significant (P < 0·05) (Figure 1). The
reactivity of PBMC in response to the mitogen PHA did not
differ between the patient groups (data not shown).
The in vitro production of cytokines by mitogen- and
antigen-stimulated PBMC is shown in Table 2a for PHA
and in Table 2b for Ascaris antigen stimulation. After
mitogen stimulation with PHA, PBMC from endemic and
non-endemic controls showed an elevated production of
TNF-α. The differences in TNF-α secretion between
endemic controls and non-endemic controls and mono-
infected Ascaris patients were statistically significant
(P < 0·05). The PHA-induced IL-12 production was highest
in endemic controls and mono-infected Trichuris patients;
those groups also presented significantly higher levels of
IL-12 production than mono-infected Ascaris patients
(P < 0·05). Similarly, IFN-γ production was elevated in
mitogen-stimulated PBMC from endemic controls, and was
significantly higher (P < 0·05) than that of mono-infected
Ascaris, mono-infected Trichuris patients or negative
non-endemic controls (Table 2a). For PHA-induced IL-10
production, no significant differences between the patient
groups were observed (Table 2a). The PHA-induced IL-
13 production of PBMC was lowest in non-endemic con-
trols when compared to the groups from the endemic area,
but these differences were not statistically significant
(Table 2a).
For the cytokine production after antigenic stimulation,
all patients responded most strongly to the A. lumbricoides
© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499–509
Volume 24, Number 11/12, November/December 2002 Immune response to intestinal helminthic co-infections

Figure 1 In vitro proliferation of peripheral blood mononuclear cells (PBMC), shown as stimulation indices (SI), in different patient groups
after stimulation with several helminth antigens. PBMC were stimulated with A. lumbricoides adult worm antigens (ASC-Ag, 20 µg /mL),
T. trichiura adult worm antigens (TRIC-Ag, 20 µg /mL), S. mansoni egg (SEA, 25 µg /mL) and adult worm antigens (SWAP, 25 µg /mL). Data
are shown as mean values ± standard error of the mean (SEM). Because of multiple testing a Bonferroni adjustment was performed and
P-values with P < 0·05 were considered significant and are indicated with an asterisk. Patient groups: ASC + TRIC, Ascaris and Trichuris
co-infected; ASC, Ascaris mono-infected; TRIC, Trichuris mono-infected; EC, endemic controls; NEG, non-endemic controls.

antigen preparation (Table 2b), whereas individually secreted
cytokine levels were low for the T. trichiura, SEA, or SWAP
antigens, and frequently were at or below the detection
limits of the cytokine assays (data not shown). Following
A. lumbricoides antigenic stimulation, an elevated production
of TNF-α and IL-12 was detected in culture supernatants
from mono-infected Trichuris patients and from endemic
and non-endemic controls, whereas their concentrations
were lowest in mono-infected Ascaris and co-infected
patients (Table 2b), albeit these differences were not statistically
significant. Also, antigen-induced IFN-γ production was
highest in endemic controls (Table 2b). For type 2 cytokines,
PBMC from non-endemic controls produced the lowest
amounts of IL-10 and IL-13 in response to Ascaris antigens
when compared to infected patients and endemic controls
(Table 2b). Furthermore, mitogen- and Ascaris antigen-
induced IL-10 production was positively correlated with
Ascaris egg counts both in mono-infected Ascaris (rho =
0·4967 and rho = 0·3458) and in co-infected Ascaris and
Trichuris patients (rho = 0·6752 and rho = 0·2089). For the
co-infected patients the correlation between mitogen-induced
IL-10 secretion and Ascaris egg counts reached statistical
significance (P < 0·01).
All patients from the endemic region were tested for any
correlation with antigen- or mitogen-induced cytokine
production and age. Here, PHA-induced IL-13 secretion
resulted in a significantly negative correlation with age (rho =
−0·3170; P = 0·0207). The negative correlation of PHA-
induced IL-13 production and age was even stronger when
only infected patients were considered (rho = −0·3803; P =
0·0109). For the other cytokines no correlation with age
was found.
When mitogen- and Ascaris antigen-induced cytokine
secretions in endemic individuals were checked for sex dif-
ferences, no statistically significant differences were found
for IL-10, IL-12, IL-13, and IFN-γ. However, when TNF-α
secretion was checked for sex differences in all individuals
from the endemic area, the mean TNF-α secretion after
stimulation with Ascaris antigen was significantly lower
(P < 0·01) in female (107 ± 25 pg/mL) than in male individ-
uals (230 ± 39 pg/mL). If TNF-α secretion was checked
only in infected patients, again, the mean TNF-α production

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S. M. Geiger et al. Parasite Immunology

Table 2a–b Mean cytokine production by PBMC cultures from patients with intestinal helminth infections and from negative controls.
(a) Cultures stimulated by PHA. (b) Cultures stimulated by Ascaris antigen

(a) PHA stimulation

ASC + TRIC ASC TRIC EC NEG

Cytokine Mean Mean Mean Mean Mean
pg/mL (± SEM) (± SEM) (± SEM) (± SEM) (± SEM)

TNF-α 407 211a*,b* 378 610a 592b

(± 68) (± 54) (± 57) (± 77) (± 134)
IL-12 52 22c*,d* 78c 100d 50
(± 12) (± 8) (± 14) (± 17) (± 12)
IFN-γ 2528 1015e* 1594e* 6511e 1741e*
(± 659) (± 184) (± 611) (± 1278) (± 183)
IL-10 325 452 296 302 513
(± 38) (± 99) (± 38) (± 60) (± 45)
IL-13 1097 960 1024 1054 826
(± 38) (± 75) (± 60) (± 77) (± 109)

(b) Ascaris-AG

ASC + TRIC ASC TRIC EC NEG

Cytokine Mean Mean Mean Mean Mean
pg/mL (± SEM) (± SEM) (± SEM) (± SEM) (± SEM)

TNF-α 119 123 253 176 192

(± 37) (± 37) (± 58) (± 43) (± 78)
IL-12 43 52 60 88 60
(± 16) (± 25) (± 14) (± 41) (± 21)
IFN-γ 52 17 81 95 69
(± 26) (± 6) (± 41) (± 35) (± 54)
IL-10 86 168 115 96 63
(± 15) (± 32) (± 29) (± 28) (± 13)
IL-13 75 73 111 125 50
(± 12) (± 14) (± 20) (± 36) (± 18)

Significant differences between the patient groups are indicated by letters a– e. P-values are corrected for multiple testing (*P < 0·05). Patient
groups: ASC + TRIC, Ascaris and Trichuris co-infected; ASC, Ascaris mono-infected; TRIC, Trichuris mono-infected; EC, endemic controls;
NEG, non-endemic controls.

was significantly lower in females than in males, both
after PHA stimulation (females: 254 ± 50 pg /mL; males:
416 ± 53 pg/mL; P < 0·05) and after stimulation with Ascaris
antigen (females: 93 ± 27 pg /mL; males: 232 ± 44 pg /mL;
P < 0·05).
Parasite-specific antibody responses
Parasite-specific antibody reactivities against A. lumbri-
coides and T. trichiura adult worm antigens are presented
in Table 3a and Table 3b, respectively. For A. lumbricoides-
specific reactivity, total IgG was significantly elevated
(P < 0·05) in all infected patient groups when compared to
the non-endemic negative controls (Table 3a). Higher levels
of parasite-specific total IgG were also detected in sera from
endemic controls when compared to the non-endemic con-
trols (Table 3a). No significant differences were observed
between the patient groups for the IgG1 reactivity against
A. lumbricoides adult worm antigens (Table 3a). Compared
to endemic and non-endemic controls, significantly
increased levels of Ascaris-specific IgG4 were detected in
co-infected patients (P < 0·05); furthermore, mono-infected
Ascaris patients showed significantly (P < 0·05) higher IgG4
levels than endemic controls (Table 3a). Specific IgE was
elevated in sera of all patient groups from the endemic area
when compared to the non-endemic controls. For mono-
infected Trichuris patients and co-infected patients these
differences were statistically significant (P < 0·05) (Table 3a).
With respect to parasite-specific IgA, no significant differ-
ences were detected between the groups, although reactivity
was higher in the infected than in the negative patient
groups (data not shown).
The T. trichiura-specific reactivity of total IgG in the
patient groups is shown in Table 3b. In mono-infected T.

504 © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499–509

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Table 3a A. lumbricoides-specific IgG, IgG1, IgG4 and IgE reactivity in sera from patients with intestinal helminth infections and from
negative controls as determined by ELISA

Ascaris-Antigen

ASC + TRIC ASC TRIC EC NEG

Ig Mean OD Mean OD Mean OD Mean OD Mean OD
isotype (± SEM) (± SEM) (± SEM) (± SEM) (± SEM)

IgG 0·71a* 0·77a* 0·72a* 0·56 0·29a

(± 0·09) (± 0·10) (± 0·16) (± 0·07) (± 0·04)
IgG1 0·33 0·26 0·37 0·27 0·2
(± 0·04) (± 0·03) (± 0·11) (± 0·05) (± 0·06)
IgG4 0·81b*,c* 0·4b* 0·4 0·13b 0·13c
(± 0·16) (± 0·11) (± 0·14) (± 0·04) (± 0·06)
IgE 0·34d* 0·13 0·34d* 0·3 0·05d
(± 0·08) (± 0·04) (± 0·09) (± 0·13) (± 0·02)

Table 3b T. trichiura-specific IgG, IgG1, IgG4, and IgE reactivity in sera from patients with intestinal helminth infections and from negative
controls as determined by ELISA

Trichuris-Antigen

ASC + TRIC ASC TRIC EC NEG

Ig Mean OD Mean OD Mean OD Mean OD Mean OD
isotype (± SEM) (± SEM) (± SEM) (± SEM) (± SEM)

IgG 1·25a 0·49a*,b* 1·21b 0·55a*,b* 0·36a*,b*

(± 0·16) (± 0·06) (± 0·18) (± 0·11) (± 0·06)
IgG1 0·57c 0·21c*,d* 0·54d 0·25c*,d* 0·25c*
(± 0·08) (± 0·02) (± 0·08) (± 0·06) (± 0·08)
IgG4 1·27e 0·18e*,f* 1·05f 0·18e*,f* 0·17e*,f*
(± 0·19) (± 0·05) (± 0·20) (± 0·07) (± 0·10)
IgE 0·47 0·23 0·49 0·54 0·20
(± 0·11) (± 0·05) (± 0·11) (± 0·18) (± 0·05)

Shown are the mean optical density (OD) values, with SEM values in parentheses. Significant differences (*P < 0·05) between the patient
groups are indicated by letters a–f. P-values are corrected for multiple testing. Patient groups: ASC + TRIC (n = 23), Ascaris and Trichuris
co-infected; ASC (n = 27), Ascaris mono-infected; TRIC (n = 19), Trichuris mono-infected; EC (n = 17), negative endemic controls; NEG
(n = 9), negative non-endemic controls.

trichiura patients or co-infected patients, significantly ele-
vated (P < 0·05) total IgG levels were found when compared
with mono-infected Ascaris patients or with endemic or
non-endemic controls (Table 3b). In the mono-infected
Trichuris patients, parasite-specific IgG1 reactivity was
higher when compared to mono-infected Ascaris patients
or endemic controls (both P-values < 0·05). Co-infected
patients had significantly higher IgG1 levels than mono-
infected Ascaris patients, endemic controls, or non-endemic
controls (P < 0·05) (Table 3b). Similarly, Trichuris patients
and Ascaris/Trichuris co-infected individuals showed stronger
IgG4 reactivity than mono-infected Ascaris patients, endemic
controls or non-endemic controls (P < 0·05) (Table 3b). For
Trichuris-specific IgE (Table 3b) as well as IgA (data not
shown), serological reactivity was elevated in mono- and
co-infected Trichuris patients as well as in endemic controls,
however, these differences were not statistically significant
when compared to the other groups.
Pearson product correlation between egg counts and
antibody isotype levels revealed a significant positive correla-
tion between T. trichiura egg counts and IgG4 antibody
levels against T. trichiura antigen in co-infected patients
(rho = 0·419; P < 0·01).
DISCUSSION
Intestinal helminth parasites are among the most prevalent
causes of tropical diseases in developing countries, and con-
current infections with multiple helminth species are fre-
quently found in endemic populations. Up to date, however,

© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499 –509 505
S. M. Geiger et al.
only few studies have investigated the effects of multiple
and chronic intestinal helminth infections on the cellular
immune response in infected humans. Normally, these
parasites cause chronic and non-fatal infections (3), even
though it was recently shown that these helminth infections
cause T cell alterations and changes in the T cell activation
profiles and, as such, exert a profound impact on the host’s
immune response (18). Furthermore, intestinal helminth
infections will not only induce parasite-specific immune
responses, but also broadly activate immune responses,
which may then interfere with other diseases and, as previ-
ously shown, may impede (13,19) or improve (20) the capa-
bility of the host to defend itself against pathogens, or may
even impair the success of vaccination (21,22). Hence, it
remains important to understand the impact that common
intestinal helminth infections exert on the host’s immune
response and to reveal to which extent parasite co-infections
may activate, bias or exhaust the immune response of
infected individuals, especially children.
In this study, we investigated the cellular immune response
and cytokine production of patients singly and co-infected with
A. lumbricoides and T. trichiura. The proliferation of PBMC
in response to Ascaris and Trichuris antigens was distinctly
different in the various patient groups. Those mono-infected
with Ascaris or co-infected with Ascaris and Trichuris
showed the lowest parasite-specific cellular reactivity, which
was in the range of that observed for non-endemic controls.
In contrast, responses to Ascaris antigen were highest in
endemic controls. Previously, Cooper et al. (6) reported that
cellular reactivity to Ascaris adult worm and larval stage
antigens was higher in Ascaris-infected patients when com-
pared to non-endemic controls. However, this may be attrib-
uted to the lower infection intensities, i.e. lower mean A.
lumbricoides egg counts (6728 epg), than those found in the
present study population. Such specifically suppressed cellu-
lar reactivity in Ascaris and Trichuris co-infected as well as
mono-infected Ascaris patients may be caused by the high
intensity of infection found in the patients of this study.
Furthermore, a pronounced cellular responsiveness was
observed in non-infected controls to S. mansoni SWAP,
whereas this reactivity remained low in helminth-infected
patients. This may reflect not only possible cross-reactivities
between antigenic epitopes, but also, that higher parasite
loads will influence negatively cellular responsiveness in
patients exposed to related parasite antigens, which is the
case with concurrent helminth infections.
In the present study, the secretion of cytokines by antigen-
and mitogen-stimulated PBMC was analysed in vitro. Al-
though the peripheral blood immune response may not
reflect the local responses in the intestinal wall or the drain-
ing lymph nodes, in the mouse experimental model it was
recently shown, that in a T. muris infection (23) the peri-
506
Parasite Immunology
pheral cytokine responses corresponded to those occurring
at the site of the infection. Therefore, peripheral cytokine
responses may provide valuable information on the parasite-
induced cytokine environment. After stimulation with
mitogen, our results disclosed a dominant type 1 cytokine
production in endemic controls, represented by elevated
levels of TNF-α, IL-12, and IFN-γ, whereas in patently
infected patients those type 1 and pro-inflammatory
cytokines were detected at lower amounts in their PBMC
cultures. In addition, after stimulation with Ascaris antigen,
less TNF-α, IL-12, and IFN-γ were detected in culture
supernatants from singly Ascaris or Ascaris and Trichuris
co-infected patients when compared with endemic controls,
although this did not reach statistical significance. Previ-
ously, no differences in the IL-2, IL-10, or IFN-γ secretion
after mitogen or antigen stimulation were reported between
Ascaris-infected individuals and negative individuals from a
low-transmission area (6). Furthermore, Ascaris-infected
patients showed a markedly elevated IL-5 secretion after
stimulation with mitogen or specific Ascaris antigens (6). In
the present study, we examined parasite-free controls from
the same highly endemic area. These negative endemic con-
trols, in all likelihood, had previous contact with the two
intestinal helminthic species and, apart from possible pro-
tective immune responses, chemotherapy in the past, use of
plant extracts as therapeutic agents or very low-levels of
infections cannot be excluded. In order to investigate the
immune response in an endemic area, we consider it appro-
priate to include such endemic controls, because those
individuals represent an indigenous group who are exposed
but potentially not yet patently infected or that may have
acquired protective immunity to the infection. As such,
negative endemic controls were also used in studies where
the effect of intestinal helminth infections on vaccination
efficiency was investigated (22).
The lower TNF-α production in females, both after
mitogen and antigen stimulation, when compared with male
individuals from the endemic area is interesting. Since we
were not able to detect a sex-related difference in intensity of
infection, which might explain the lower TNF-α production
in females, it may be explained by the different hormonal
regulation in pre-adulthood influencing the patients’ immune
response.
In several animal models it has been shown that Th2-type
cytokines such as IL-4, IL-5, and IL-13 play an important
role in the elimination of gastrointestinal nematodes (4,5).
In our study groups, we investigated type 2 cytokine
responses, i.e. IL-13 and IL-10, in in vitro stimulated PBMC
cultures, but we did not detect significant differences
between patients and controls, both after mitogen and
parasite antigen-specific stimulation. It is noteworthy that
PBMC from non-endemic controls showed the lowest
© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499–509
Volume 24, Number 11/12, November/December 2002
production of IL-10 and IL-13 after stimulation with Ascaris
antigen. This supports the idea that our non-endemic
controls may truly represent negative controls. On the
other hand, recent history of exposure as well as of infection
has imprinted immunological memory and upon in vitro
stimulation with parasite-specific antigens activated a
mixed Th1- and Th2-type cytokine production, as observed
in our endemic controls. The predominance of either type 1
or 2 T cell responses occurs only in a chronic helminth
infection in immunopathological reactions or in acquired
immunity (24).
Interestingly, IL-13 secretion, which shares some of the
important properties with IL-4, was induced in PBMC cul-
tures of patients and endemic controls, and this indicated
the generation of potentially protective Th2-type cytokine
responses as required for the expulsion of experimental
gastrointestinal nematodes (5). In Ascaris-infected and co-
infected patients with a moderate to high intensity of infec-
tion, cellular unresponsiveness was accompanied by a reduced
secretion of pro-inflammatory TNF-α and other type 1
cytokines. Such a down-modulation of type 1 cytokines
and inflammatory responses may favour parasite persistence
but at the same time may serve to reduce intestinal path-
ology, as proposed for other intestinal helminth infections
(25,26). As such, elevated local and systemic TNF-α levels
were detected in children with a high intensity of T. trichiura
infection and Trichuris dysentery syndrome (TDS), and
were associated with intestinal pathology (27), whereas
elevated TNF-α levels were not detected in low-intensity
trichiuriasis (28).
The lack of substantial cytokine secretion after stimula-
tion with T. trichiura crude adult worm antigen was recently
reported by different research groups (6,29), whereas ele-
vated secretion of IL-10 and TNF-α were detected in super-
natants with whole blood cultures from Trichuris patients
after stimulation with excretory–secretory antigens from
T. trichiura or T. muris (29).
For the parasite-specific humoral immune responses, high
levels of Ascaris- and Trichuris-specific IgG4 antibodies
were detected in the sera of infected individuals, whereas
they remained at low levels in both control groups. In co-
infected Ascaris and Trichuris patients the levels of Trichuris-
specific IgG4 antibodies correlated significantly with the
T. trichiura egg output. Although such a positive corre-
lation was already found in an epidemiological study (30),
the same study showed a negative correlation between
Trichuris-specific IgA antibodies and intensity of infection
in adults. We were not able to detect such correlations for
parasite-specific IgA antibodies, which might be due to the
lower mean age in our patients. In contrast to Cooper et al.
(6), we were able to detect considerable levels of Trichuris-
and Ascaris-specific IgE antibodies in sera of our endemic
© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 499 –509
Immune response to intestinal helminthic co-infections
controls, which again is indicative for previous and repeated
parasite exposure. Therefore, specific IgE may be important
for the development of acquired immunity to A. lumbri-
coides infection, as proposed by others (7,31), and should
be further investigated, especially in those individuals con-
stantly exposed, although without signs of infection. Such
IgE-mediated protective immune responses were also reported
in reinfection studies with S. haematobium or S. mansoni
(32,33). In addition, specific IgG1 or IgG4 antibodies
against Trichuris crude adult worm antigens were con-
firmed to be useful tools to detect a patent T. trichiura
infection with low cross-reactivity to A. lumbricoides antigens,
as already shown by Lillywhite et al. (34). Interestingly,
in patients infected with T. trichiura and A. lumbricoides,
elevated parasite-specific reactivities of both IgG4 and IgE
were detected. In S. haematobium infections, elevated IgG4
antibody levels were postulated to be involved in the blockage
of the IgE protective response (32); however, the question
to what extent IgG4 may block acquisition of protective
immunity in ascariasis and trichiuriasis has to be determined
in further studies.
In summary, we observed elevated levels of TNF-α, IL-
12, and IFN-γ production by PBMC from parasite-free
endemic controls, whereas these cytokines were suppressed
in the patient groups with high parasite loads, e.g. mono-
infected Ascaris and co-infected Ascaris and Trichuris patients.
Whether these elevated levels of Th1-type cytokines
play an essential role in parasite expulsion and protective
immunity, or whether they may just reflect the parasite-
free state of infection, has to be further investigated. In
order to better determine those immune mechanisms that
protect against infection, longitudinal studies remain
necessary to identify and characterize those individuals
who remain resistant to infection over extended periods of
time. In filariasis and schistosomiasis, it has recently been
shown that resistance in endemic normal individuals is asso-
ciated with a mixed rather than with a clearly polarized type
1 or type 2 immune response (35,36). In A. lumbricoides
infection, natural immunity has been associated with a high
specific IgE response, favouring a protective type 2 immune
response (7,31). However, further detailed studies on cellu-
lar responses and cytokine production profiles in a well-
defined group of resistant individuals are required to better
define those parameters necessary for acquisition of protec-
tive immunity.
ACKNOWLEDGEMENTS
This work was supported by grants from FAPEMIG and
FIOCRUZ. S. M. Geiger received postdoctoral research
fellowships at institutions of higher education in the
Federal Republic of Germany from the German Academic
507
S. M. Geiger et al.
Exchange Service (DAAD) and from Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq). J.
Bethony is supported by an International Scientists Research
Development Award, from the John C. Fogarty Center,
NIH.
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