Clin.
exp.
Immunol.
(1982)
47,
65-73.
Schistosoma
mekongi
infection
in
man:
cellular
immune
responses
and
modulating
mechanisms
M.
BARRAL-NETTO,
M.
HOFSTETTER,
J.
GUSTAVO
DOS
SANTOS,*
A.
W.
CHEEVER
&
E.
A.
OTTESEN
Laboratory
ofParasitic
Diseases,
National
Institute
of
Allergy
and
Infectious
Diseases,
National
Institutes
ofHealth,
Bethesda,
Maryland,
and
*
Medical
College
of
Virginia,
Richmond,
Virginia,
USA
(Accepted
for
publication
3
July
1981)
SUMMARY
Cell-mediated
immune
responses
(CMI),
as
assessed
by
lymphocyte
proliferation
in
vitro,
were
evaluated
in
11
Laotian
patients
harbouring
asymptomatic
chronic
infections
by
Schistosoma
mekongi,
a
schistosome
closely
related
to
S.
Japonicum.
When
the
mononuc-
lear
cells
of
these
patients
were
cultured
in
autologous
plasma,
lymphocyte
responses
to
schistosome
antigens
were
essentially
nil,
not
differing
from
those
of
unexposed
North
American
controls.
Specific
lymphocyte
proliferation,
however,
was
seen
both
after
the
removal
of
mononuclear
cells
that
were
nylon-wool-adherent
and
after
substitution
of
the
autologous
serum
in
the
culture
with
normal
AB
serum.
Our
data
suggest
that
the
CMI
responses
of
humans
with
chronic
S.
mekongi
infections
are
'modulated'
by
adherent
suppressor
cells
and
serum-suppressive
factors,
and
that
modulation
of
CMI
supports
the
stable
host-parasite
relationship
in
a
similar
fashion
to
that
described
for
chronic
human
Schistosoma
mansoni
infection.
INTRODUCTION
Knowledge
of
the
human
immune
response
in
schistosomiasis
is
derived
largely
from
studies
of
patients
with
Schistosoma
mansoni
infection.
These
studies
have
shown
an
early
exuberant
cell-mediated
immune
response
(as
assessed
by
lymphocyte
transformation)
at
the
acute
stage
of
the
disease
with
subsequent
modulation
during
the
chronic
phase
of
infection.
Various
suppressive
mechanisms
have
been
implicated
in
this
modulation,
including
nylon-wool-adherent
peripheral
blood
mononuclear
cells
(Ottesen,
1979;
Todd,
Goodgame
&
Colley,
1979),
suppressor
T
cells
(Colley,
Lewis
&
Goodgame,
1978;
Ellner
et
al.,
1980),
and
poorly
characterized
suppressive
serum
factors
(Colley
et
al.,
1977;
Ottesen
&
Poindexter,
1980;
Rocklin
et
al.,
1980).
Little
is
known
about
the
human
immune
response
to
Schistosomajaponicum
(Lewert,
Yogore
&
Blas,
1979)
but
studies
in
animal
models
suggest
that
there
may
be
a
major
qualitative
difference
between
the
reaction
to
this
parasite
and
the
response
to
S.
mansoni;
namely,
that
the
prominent
T-cell
involvement
in
S.
mansoni
infection
may
be
less
important
in
the
response
to
S.
japonicum
(Coker
&
von
Lichtenberg,
1956;
Warren,
Domingo
&
Cowan,
1973;
Warren
et
al.,
1975;
Warren,
Grove
&
Pelley,
1978;
von
Lichtenberg,
Erickson
&
Sadun,
1973;
Byram
&
von
Lichtenberg,
1980;
von
Lichtenberg,
1978).
Abbreviations:
CMI
=
cell-mediated
immune
response(s),
ELIsA
=
enzyme-linked
immunoabsorbent
assay,
LT
=
lymphocyte
transformation
test,
PBMC
=
peripheral
blood
mononuclear
cells,
SK-SD
=
streptokinase-
streptodornase.
Correspondence:
Dr
E.
A.
Ottesen,
Laboratory
of
Parasitic
Diseases,
NIAID,
National
Institutes
of
Health,
Building
5,
Room
B1-04,
Bethesda,
Maryland
20205,
USA.
0009-9104/82/0100-0065$02.00
(0
1982
Blackwell
Scientific
Publications
65
We
recently
studied
11
Laotian
immigrants
infected
with
Schistosoma
mekongi,
a
species
closely
related to
S.
japonicum
(Voge,
Bruckner
&
Bruce,
1978).
While
infection
by
this
parasite
was
described
almost
a
quarter
of
a
century
ago
(Dupont
et
al.,
1957),
there
are
few
clinical
descriptions
of
the
infection
in
man
(Barbier,
1966;
Sornmani,
Vivatanasesth
&
Thirachantra,
1976)
and
little
is
known
of
the
immune
responses
elicited
by
this
parasite.
Experimentally,
S.
japonicum
and
S.
mekongi
are
similar
(von
Lichtenberg,
1978)
in
that
both
are
extremely
pathogenic
for
susceptible
hosts.
Because
oriental
schistosomiasis
affects
a
large
human
population
and
may
evoke
immunopathogenic
mechanisms
qualitatively
different
from
those
involved
in
S.
mansoni
infection,
in
the
present
study
we
initiated
a
detailed
investigation
of
the
human
response
to
S.
mekongi
infection.
Cellular
immune
responses
were
evaluated
in
lymphocyte
proliferation
assays
and
potential
modulating
mechanisms
were
investigated
in
studies
similar
to
those
carried
out
with
S.
mansoni
infection
in
which
both
serum-suppressive
factors
and
adherent
suppressor
cells
were
sought.
Our
results
show
that,
despite
the
usual
heterogeneity
among
individuals,
CMI
to
chronic
S.
mekongi
infection
and
the
modulating
mechanisms
found
were
qualitatively
similar
to
those
described
previously
in
patients
with
chronic
S.
mansoni
infections.
MATERIALS
AND
METHODS
Patient
population.
Eleven
patients
(four
males
and
seven
females)
with
chronic
S.
mekongi
infections
were
studied
prior
to
treatment
at
the
Clinical
Center,
National
Institutes
of
Health.
Their
clinical
features
have
been
described
in
detail
elsewhere
(Hofstetter
et
al.,
1981)
but
are
summarized
in
Table
1.
Ages
ranged
between
4
and
47
years
and
stool
egg
counts
between
50
and
5,720
S.
mekongi
eggs/gram
faeces,
as
determined
by
the
Kato
technique
(Martin
&
Beaver,
1968).
Intestinal
parasites
included
Opisthorchis
(11
of
1
1),
Trichuris
(seven
of
1
1),
Trichostrongylus
(four
of
11),
Ascaris
(two
of
11)
and
Giardia
(one
of
11).
Table
1.
Clinical
characteristics
of
S.
mekongi-infected
patients
Cases
with
Intensity
of
hepatomegaly
No.
of
Age
range
infection
cases
(years)
(eggs/g
faeces)
No.
Age
I1
4-47
50-5,720
6
4-12
(875)*
*
Geometric
mean.
Five
healthy
North
American
volunteers
(three
males
and
two
females)
served
as
normal
controls.
Antigens
and
mitogens.
Schistosome
antigens
were
prepared
from
adult
worms
and
eggs
of
S.
japonicum.
S.
mekongi
parasites
were
not
available
in
adequate
numbers
to
be
used
as
a
source
of
antigen.
Techniques
for
the
preparation
of
the
saline
extracts
of
adult
worm
(Ottesen
et
al.,
1978)
and
soluble
egg
antigen
(Boros
&
Warren,
1970)
have
been
described.
Commercial
preparations
of
non-parasite
antigens
were
dialysed
and
used
in
vitro
in
previously
determined
optimal
concentrations.
Streptokinase-streptodornase
(SK-SD)
(Lederle
Laboratories,
Pearl
River,
New
York)
was
used
at
a
concentration
of
25
units
SK
and
6
3
units
SD/ml;
tetanus
toxoid
(Eli
Lilly
Company,
Indianapolis,
Indiana)
at
a
concentration
of
0-75
Lf/ml;
and
PPD
CT-68
(Connaught
Laboratories
Ltd,
Toronto,
Canada)
at
a
concentration
of
1
jug/ml.
Phytohaemagglutinin
(PHA;
Burroughs
Welcome,
Research
Triangle,
North
Carolina)
was
employed
in
the
cultures
at
three
concentrations
(10,
5
and
1
,ug/ml).
Pokeweed
mitogen
(GIBCO,
66
M.
Barral-Netto
et
al.
CMI
responses
in
human
S.
mekongi
infection
67
Grand
Island,
New
York)
was
used
at
0.10%
and
concanavalin
A
(Miles
Laboratories,
Elkhart,
Indiana)
at
10
and
5
,ug/ml.
Lymphocyte
cultures.
Peripheral
blood
mononuclear
cells
(PBMC)
used
for
the
lymphocyte
transformation
test
(LT)
were
obtained
from
venous
blood
layered
over
a
Hypaque-Ficoll
gradient
as
described
by
Ottesen
et
al.
(1978).
After
washing,
PBMC
were
resuspended
in
RPMI
1640
tissue
culture
medium
(GIBCO)
supplemented
with
penicillin
(124
pg/ml),
streptomycin
(70
pg/ml),
glutamine
(300
pg/ml)
and
10%
autologous,
heparinized
plasma
or
normal
human
AB
serum
(heat-inactivated
at
560C
for
30
min).
Cells
were
cultured
in
0
2
ml
of
supplemented
medium
at
a
density
of
100,000
cells
per
well
in
round-bottomed
tissue
culture
multi-well
plates
(Linbro
Division,
Flow
Laboratories
Inc.,
Hamden,
Connecticut).
Triplicate
cultures
were
maintained
at
370C
in
an
atmosphere
of
95%
air
and
5%
CO2.
Cells
were
pulsed
with
I
pCi
of
3H-thymidine
(Amersham,
Arlington
Heights,
Illinois)
during
the
last
4
hr
of
culture
on
day
4
after
mitogen
stimulation
and
on
day
6
for
antigen-stimulated
cultures.
Cell
harvest
and
determination
of
3H-thymidine
incorporation
was
carried
out
as previously
described
(Ottesen,
1979).
Results
from
triplicate
cultures
were
averaged
and
expressed
as
counts
per
minute
of
stimulated
cultures
minus
the
value
of
the
unstimulated
control
cultures
(A
c.p.m.).
Techniques
for
the
removal
of
adherent
cells.
Cell
populations
deprived
of
adherent
cells
were
obtained
by
passage
of
PBMC
through
acid-washed
nylon-wool
columns
as
described
elsewhere
(Ottesen,
1979).
Briefly,
PBMC
were
incubated
for
45
min
at
370C
on
a
nylon-wool
column
wetted
with
RPMI
1640
medium
supplemented
with
antibiotics,
glutamine
and
heat-inactivated
normal
human
serum.
Non-adherent
cells
were
collected
by
washing
the
column
with
50
ml
of
supplemented
tissue
culture
medium.
After
repeated
washings,
the
cell
population
obtained
(usually
25
40%
of
the
cells
applied
to
the
column)
was
cultured
as
described
for
unfractionated
PBMC.
Antibody
determination.
An
enzyme-linked
immunoabsorbent
assay
(ELISA)
was
used
for
antibody
determination
as
described
previously
(Walls,
Bullock
&
Palmer,
Procedural
Guide
for
EIA
Microtitration
Test,
US
Public
Health
Center
for
Disease
Control,
Atlanta,
unpublished).
S.
japonicum
adult
worm
and
egg
antigens
were
diluted
in
0-06
M,
pH
9-5
carbonate
buffer
to
a
concentration
of
5
yg/ml
(defined
as
optimal
by
'box
titration'
of
positive
sera).
Microtitre
plates
(Dynatech
Laboratories,
Alexandria,
Virginia)
were
sensitized
with
0-
1
ml
of
antigen
solution
per
well,
incubated
at
37°C
for
3
hr
and
maintained
at
4°C
until
use.
Two-fold
dilutions
of
plasma
(beginning
at
1:8)
were
tested.
Peroxidase-conjugated
goat
anti-human
IgG
antibody
(Cappel
Laboratories,
Cochranville,
Pennsylvania)
was
used
in
a
dilution
of
1:200.
Ortho-
phenylenediamine
(Sigma,
St
Louis,
Missouri)
served
as
substrate.
After
30
min
at
room
temperature
in
the
dark,
0-05
ml
per
well
of
8
N
sulphuric
acid
was
used
to
stop
the
reaction.
Extinction
values
were
determined
using
densities
read
at
490
nm
on
an
ELISA
reader
(Clem
&
Yolken,
1978).
Statistical
treatment.
The
effects
of
replacing
autologous
serum,
removing
nylon-wool-adherent
cells
or
their
combination
on
the
lymphocyte
proliferation
response
were
analysed
by
the
paired-sample
Student's
t-test.
Comparison
of
mean
log
of
reciprocal
ELISA
antibody
titres
of
the
different
groups
was
made
by
the
independent-sample
Student's
t-test.
RESULTS
Lymphocyte
responses
to
S.
japonicum
antigens
All
patients'
PBMC
were
cultivated
in
autologous
serum
and
stimulated
with
adult
and
egg
antigens
at
concentrations
ranging
from
5
to
100
pg/ml.
Peak
responses
were
not
significantly
different
from
those
obtained
with
PBMC
from
uninfected
North
American
controls
(Fig.
1).
The
variability
of
individual
responses
is
noteworthy.
Although
these
data
are
compatible
with
a
deficiency
of
appropriately
sensitized
lymphocytes,
they
could
also
be
explained
by
suppression
of
otherwise
responsive
cells.
M.
Barral-Netto
et
al.
*
Adult
antigen
]r
§
S.
mekorgi
Norrnal
patients
controls
Fig.
1.
Comparison
of
lymphocyte
proliferation
responses
(unfractionated
cells
in
autologous
plasma)
of
S.
mekongi-infected
patients
and
uninfected
controls
to
adult
worm
and
egg
antigens.
Each
point
represents
the
mean
incorporation
of
3H-thymidine
from
triplicate
cultures
minus
incorporation
of
unstimulated
cultures
(A
c.p.m.)
from
each
patient.
Bar
represents
mean
in
each
group.
Identification
of
adherent
suppressor
cells
Lymphocyte
proliferative
responses
to
schistosome
antigens
were
greater
when
nylon-wool-adher-
ent
cells
were
removed
from
the
PBMC.
In
analysing
this
phenomenon,
cells
were
cultured
with
normal
serum
to
eliminate
the
effect
of
potential
serum-suppressive
factors.
Responses
to
both
adult
and
egg
antigen
increased
in
eight
of
the
11
patients
after
the
removal
of
adherent
cells
from
the
PBMC
population
(Fig.
2).
Interestingly,
the
same
three
patients
who
lacked
adherent
Adult
antigen
Egg
antigen
12
_
\
/
15
k
SK-SD
12
9
6
3
15
5
UNF
N-ADH
Phytohoemagglutinin
UNF
N-ADH
Fig.
2.
Effect
of
removing
adherent
cells
on
3H-thymidine
incorporation
of
S.
mekongi-infected
patients'
lymphocytes
in
response
to
adult
worm
and
egg
antigens,
SK-SD
and
PHA-P.
UNF=unfractionated
cells,
N-ADH
=
nylon-wool-non-adherent
cells.
Mean
response
of
triplicate
cultures
minus
unstimulated
controls
(A
c.p.m.).
Cells
were
cultured
in
medium
supplemented
with
normal
human
AB
plasma.
68
?-
10
ci
(3
T1
Egg
antigen
0
0
S.
mekxiggi
patients
controls
CMI
responses
in
human
S.
mekongi
infection
69
suppressor
cells
to
adult
antigen
also
had
no
suppressor
cells
to
egg
antigen.
The
increase
in
response
to
schistosome
antigens
occurred
despite
the
fact
that
the
unfractionated
PBMC
responded
better
than
the
adherent-cell-depleted
population
to
mitogen
PHA.
Both
populations
had
similar
responses
to
SK-SD.
These
findings
suggest
that
circulating
suppressor
cells
modulated
lymphocyte
proliferative
responses
to
parasite
antigens.
Identification
of
serum-suppressive
factors
Inhibitory
factors
in
the
sera
of
these
patients
were
investigated
by
comparing
lymphocyte
responses
in
autologous
plasma
to
those
in
normal
human
AB
serum.
Use
of
only
the
non-adherent
mononuclear
cell
population
for
this
comparison
avoided
the
possible
confusing
effects
of
adherent
suppressor
cells
in
the
cultures.
It
is
clear
from
Fig.
3
that
the
use
of
normal
serum
increased
responses
to
adult
antigen
in
six
of
the
10
patients
thus
examined
and
to
egg
antigen
in
nine
of
the
10
patients
(P<0-01)
despite
higher
lymphocyte
responses
to
PHA
in
the
autologous
plasma.
The
serum
change
did
not
significantly
affect
the
responses
to
SK-SD.
These
data
indicate
the
existence
of
specific
inhibitory
elements
in
the
autologous
plasma
but
again
emphasize
the
heterogeneity
of
individual
responses.
Effect
of
combined
removal
of
adherent
suppressor
cells
and
autologous
plasma
Our
findings
imply
a
combined
immunoregulatory
role
for
serum
suppressive
factors
and
adherent
suppressor
cells
on
the
patients'
lymphocyte
responses
to
parasite
antigens.
Of
interest
then
was
the
result
of
combined
removal
of
adherent
suppressor
cells
and
serum
inhibitory
factors
in
these
patients
(Fig.
4).
In
the
egg-antigen-stimulated
cultures
every
individual
showed
enhanced
proliferation
after
this
procedure.
In
the
cultures
stimulated
with
adult
antigen,
nine
of
the
11
patients
showed
increased
proliferative
responses.
This
enhancement
in
the
absence
of
serum
and
adherent
cell
suppression
was
statistically
significant
(P
<0-01
for
egg
antigen;
P
<
0-05
for
adult
15
Adult
antigen
Egg
antigen
12
9
6-
3-
Ci
55
ro
SK-SD
Phytohaemagglutinin
45-
12
35-
9
25
6-
15-
3-
5-
AUTOL
AB
AUTOL
AB
Fig.
3.
Influence
of
autologous
(AUTOL)
or
normal
human
(AB)
serum
on
patients'
lymphocyte
proliferation
in
response
to
adult
worm
and
egg
antigens
SK-SD
and
PHA-P.
Mean
incorporation
of
3H1-thymidine
from
triplicate
cultures
minus
incorporation
of
unstimulated
cultures
(A
c.p.m.)
from
each
patient.
All
cultures
contained
only
rylon-wool-non-adherent
cells.
M.
Barral-Netto
et
al.
15
k
Adult
antigen
55'
15
k
SK-SD
45
35
25
15
UNF.
N-ADH
AUTOL
AB
Egg
antigen
_
\
Phytohoemagglutinin
X
UNF.
N-ADH
AUTOL
AB
Fig.
4.
Effect
of
combined
removal
of
nylon-wool-adherent
cells
and
autologous
serum
on
3H-thymidine
incorporation
of
patients'
lymphocytes
in
response
to
adult
worm
and
egg
antigens
SK-SD
and
PHA-P.
UNF.
AUTOL
=
cultures
of
unfractionated
cells
in
autologous
plasma,
N-ADH
AB
=
cultures
of
nylon-wool-non-
adherent
cells
with
normal
human
AB
plasma.
Mean
response
of
triplicate
cultures
minus
unstimulated
controls
(A
c.p.m.)
for
each
patient's
response.
Adult
worm
antigen
(P<O00)
1,024
D
256
,.-
64
D
16
4
;F
4-
S.
maodgi
Normol
patients
controls
Egg
antigen
(P<&OI)
~Lii
(9
mx
128
32
_
8
S.
me*ongi
Normal
patients
controls
Patients
Patients
without
senu
with
evidlone
suppessi
factos
serum
effect
Fig.
5
Fig.6
Fig.
5.
IgG
ELISA
antibody
titres
to
adult
worm
and
egg
antigens
in
S.
mekongi-infected
patients
and
normal
controls.
Bar
represents
geometric
mean
in
each
group.
The
P
value
from
Student's
t-test
between
mean
titres
for
patient
and
control
population
for
each
antigen.
Fig.
6.
IgG
ELISA
titres
to
adult
worm
antigen
in
patients
with
or
without
evidence
of
serum
suppressive
factor(s)
on
lymphocyte
responses
to
this
antigen.
Bar
represents
geometric
mean
in
each
group.
70
121
9
6
3
0
i)
a
12
9
6
3
.
I
CMI
responses
in
human
S.
mekongi
infection
antigen).
Again,
the
changes
in
the
responses
to
parasite
antigens
were
markedly
different
from
those
stimulated
by
PHA
or
streptococcal
antigen
(SK-SD).
There
was
no
correlation
of
lymphocyte
responsiveness
and
egg
output
in
the
patients'
faeces.
Antibody
titres
All
11
patients
had
positive
ELISA
antibody
titres
against
egg
antigen
and
all
but
two
against
adult
antigen
(Fig.
5).
The
mean
log
of
patients'
reciprocal
antibody
titres
was
higher
than
that
of
controls
(P
<
0-01)
to
both
adult
and
egg
antigens.
We
investigated
the
potential
relationship
between
the
presence
of
serum-suppressive
factors
and
specific
antibody
titres
by
dividing
the
patients
into
two
groups
according
to
presence
or
absence
of
serum
suppression
of
the
LT
response
to
adult
antigen.
As
seen
in
Fig.
6,
the
antibody
titres
were
very
similar
(P
>
0-8).
A
similar
analysis
could
not
be
done
with
egg
antigen
responses
since
only
one
patient
lacked
serum-suppressive
factors
in
the
LT
response
to
this
antigen.
DISCUSSION
The
present
study
demonstrated
a
sensitized
and
responsive
cellular
arm
of
the
human
immune
system
in
chronic
S.
mekongi
infections.
Lymphocyte
reactivity
to
parasite
antigens
was,
however,
inapparent
in
cultures
of
PBMC
in
autologous
plasma
because
of
the
presence
of
active
immunoregulatory
mechanisms.
These
mechanisms
were
shown
to
include
adherent
mononuclear
suppressor
cells
and
serum-suppressive
factors.
In
all
patients
at
least
one
suppressive
mechanism
was
found,
and
in
some
patients
both
effects
were
present.
On
the
other
hand,
in
a
few
patients
the
serum-suppressive
mechanism,
although
present
in
the
response
elicited
by
egg
antigen,
was
lacking
entirely
in
the
response
to
adult
antigen.
Cellular
and
humoral
mechanisms
were
capable
of
independent
action,
since
autologous
plasma
suppressed
the
responses
of
non-adherent
PBMC
to
schistosome
antigens
and
the
suppression
mediated
by
adherent
cells
was
also
maintained
in
cultures
with
normal
plasma.
The
combined
action
of
these
two
mechanisms,
apparent
in
almost
all
of
our
patients,
has
also
been
reported
with
chronic
human
S.
mansoni
infections
(Ottesen,
1979;
Todd
et
al.,
1979).
In
all
of
these
studies
both
the
lymphocyte
responses
to
schistosome
antigens
and
their
modulating
mechanisms
showed
marked
heterogeneity
in
different
individuals.
The
nature
of
the
serum-suppressive
factors
present
in
chronic
human
S.
mekongi
infections
was
not
determined.
Specific
inhibition
of
LT
responses
to
schistosome
antigens
may
involve
immune
complexes
with
parasite
antigens,
as
suggested
for
both
human
and
experimental
infections
with
S.
mansoni
(Rocklin
et
al.,
1980;
Cottrell,
Sturrock
&
Vanhoegaerden,
1980).
As
the
anti-adult
worm
antibody
titres
were
very
similar
in
patients
with
and
without
the
serum-suppressive
factor,
the
idea
of
sequestration
of
the
antigen
by
anti-parasite
antibodies
in
the
autologous
plasma
is
not
supported by
these
data.
Other
serum
factors
capable
of
modulating
immune
responses
(Mota-Santos
et
al.,
1977;
Dessaint
et
al.,
1977)
cannot
now
be
excluded.
Lack
of
suppression
of
the
LT
responses
elicited
with
SK-SD
points
to
antigen
specificity
of
the
suppressive
mechanisms;
however,
in
those
patients
in
whom
either
PPD
or
tetanus
antigens
were
also
used,
serum-suppressive
factors
and
nylon-wool-adherent
suppressor
cells
could
often
be
demonstrated.
Interpretation
of
these
findings
is
difficult;
however,
all
patients
were
immunized
for
tetanus
3
months
before
the
study
and
all
had
positive
PPD
skin
tests.
Adherent
suppressor
cells
have
already
been
described
in
tuberculosis
(Ellner,
1978)
and
it
is
well
recognized
that
both
suppressor
and
effector
mechanisms
develop
in
the
post-immunization
period.
The
adherent
suppressor
cell
removed
by
nylon-wool
column
fractionation
was
not
character-
ized,
but
the
technique
used
primarily
removes
the
C3-receptor-bearing
cells
and
the
esterase-stain-
ing
population
(Ottesen,
1979;
Graves
&
Grown,
1974;
Yu
et
al.,
1977).
Of
these
cells,
the
monocyte-macrophage
lines
are
more
likely
to
mediate
suppression
as
described
for
S.
mansoni
(Ottesen,
1979;
Todd
et
al.,
1979)
and
tuberculosis
(Ellner,
1978),
but
suppression
mediated
by
adherent
B
lymphocytes
(Asherson
&
Zembala,
1975)
has
also
been
described.
The
possibility
that
71
72
M.
Barral-Netto
et
al.
the
suppressor
population
is
a
T-cell
population
that
constitutes
a
minute
proportion
of
the
cells
retained
on
the
nylon-wool
column
must
also
be
considered.
Many
studies
using
injection
of
purified
S.
mansoni
eggs
into
mouse
lung
have
shown
that
the
granulomatous
reaction
to
these
eggs
is
T-lymphocyte-dependent
(Coker
&
von
Lichtenberg,
1956;
Warren
et
al.,
1973).
Use
of
S.
japonicum
eggs
with
the
same
model,
however,
has
led
to
the
suggestion
that
the
granulomas
in
this
case
are
B-cell-mediated
(Warren
et
al.,
1975,
1978).
It
is
uncertain
whether
purified
S.
japonicum
eggs
as
used
in
the
above
model
preserve
all
of
their
important
antigenic
characteristics,
as
S.
japonicum
eggs
injected
into
S.
japonicum-infected
mice
elicit
little
reaction
at
the
time
that
eggs
laid
by
the
worms
are
surrounded
by
large
granulomas
(Warren
etal.,
1975).
However,
other
studies
in
the
rodent
model
of
schistosomiasis
seem
to
support
the
idea
that
T
cells
are
less
important
for
the
reaction
to
S.
japonicum
eggs
than
for
the
reaction
to
S.
mansoni
eggs.
Hamsters
infected
with
S.
japonicum
show
histiogranulocytic
granulomas
characterized
by
central
accumulation
of
granulocytes
around
the
eggs,
dense
plasma
cell
infiltration
and
presence
of
Hoeppli
phenomena,
findings
which
suggest
a
role
for
humoral
immune
reactions
in
their
pathogenesis
(von
Lichtenberg
et
al.,
1973).
Furthermore,
granulomas
in
S.
mansoni-infected
nude
mice
are
much
smaller
than
granulomas
in
immunologically
normal
heterozygotes,
while
circumoval
granulomas
in
S.
japonicum-infected
nude
mice
are
more
similar
to
those
in
intact
mice
(von
Lichtenberg,
1978,
personal
communication).
Unfortunately,
the
pathology
of
human
S.
mekongi
infection
is
not
known,
but
in
this
study
the
CMI
to
parasite
antigen
of
humans
infected
with
S.
mekongi
were
low
due
to
modulation
by
serum
factors
and
adherent
suppressor
cells
instead
of
lack
of
appropriately
sensitized
lymphocytes.
Evidence
for
similar
CMI
findings
was
also
obtained
in
two
S.
japonicum-infected
patients
we
had
the
opportunity
to
study
(unpublished
data).
Together
these
findings
support
the
idea
that
the
CMI
responses
to
schistosomes
of
the
S.
japonicum
complex
are
prominent
in
the
human
host
and
similar
to
those
described
in
the
immune
responses
to
infection
with
S.
mansoni.
We
greatly
appreciate
the
invaluable
contribution
of
Dr
T.
E.
Nash
to
the
clinical
care
and
evaluation
of
these
patients.
The
statistical
assistance
of
Dr
David
Alling
is
gratefully
acknowledged.
Excellent
editorial
assistance
was
provided
by
Ms
Linda
Sacchetti.
We
are
grateful
to
Dr
Yung-san
Liang
for
the
ready
availability
of
S.
japonicum-infected
snails,
which
he
furnished
through
National
Institute
of
Allergy
and
Infectious
Diseases
(National
Institutes
of
Health)
supply
contract
No.
Al
72724.
Dr
Barral-Netto
is
a
recipient
of
a
W.
K.
Kellogg
Foundation
Fellowship.
REFERENCES
ASHERSON,
G.L.
&
ZEMBALA,
M.
(1975)
Suppressor
T
cells
in
cell-mediated
immunity.
Br.
Med.
J.
ii,
476.
BARBIER,
M.
(1966)
Determination
d'un
foyer
de
bilharziose
arterio-veineuse
au
Sud-Laos
(Province
de
Sithndone).
Bull.
Soc.
Pathol.
Exot.
59,
974.
BOROS,
D.L.
&
WARREN,
K.S.
(1970)
Delayed
hyper-
sensitivity-type
granuloma
formation
and
dermal
reaction
induced
and
elicited
by
a
soluble
factor
isolated
from
Schistosoma
mansoni
eggs.
J.
exp.
Med.
132,
488.
BYRAM,
J.E.
&
VON
LICHTENBERG,
F.
(1980)
Experi-
mental
infection
with
Schistosoma
mekongi
in
laboratory
animals:
parasitological
and
pathologi-
cal
findings.
In
The
Mekong
Schistosome
(ed.
by
J.
I.
Bruce,
S.
Sornmani,
H.
L.
Asch
and
K.
A.
Craw-
ford),
Suppl.
2,
p.
125.
Malacological
Review,
Whitmore
Lake,
Michigan.
CLEM,
T.T.
&
YOLKEN,
R.H.
(1978)
Practical
colori-
meter
for
direct
measurement
of
microplates
in
enzyme
immunoassay
systems.
J.
clin.
Microbiol.
7,
55.
COKER,
C.M.
&
VON
LICHTENBERG,
F.
(1956)
A
revised
method
for
isolation
of
S.
mansoni
eggs
for
biological
experimentation.
Proc.
Soc.
exp.
Biol.
Med.
92,
780.
COLLEY,
D.G.,
COOK,
J.A.,
FREEMAN,
G.L.,
JR,
BARTHOLOMEW,
R.K.
&
JORDAN,
P.
(1977)
Immune
responses
during
human
schistosomiasis
mansoni.
III.
Regulatory
effect
of
patient
sera
on
human
lymphocyte
blastogenic
responses
to
schistosome
antigen
preparations.
Am.
J.
Trop.
Med.
Hyg.
26,
917.
COLLEY,
D.G.,
LEWIS,
F.A.
&
GOODGAME,
R.W.
(1
978)
Immune
responses
during
human
schistoso-
miasis
mansoni.
IV.
Induction
of
suppressor
cell
activity
by
schistosome
antigen
preparations
and
concanavalin
A.
J.
Immunol.
120,
1225.
COTTRELL,
B.J.,
STURROCK,
R.F.
&
VANHOEGAERDEN,
M.
(1980)
An
immunosuppressive
factor
in
the
serum
of
baboons
(Papio
anubis)
infected
with
Schistosoma
mansoni.
Immunology,
39,
589.
DESSAINT,
J.P.,
CAMUS,
D.,
FISCHER,
E.
&
CAPRON,
A.
(1977)
Inhibition
of
lymphocyte
proliferation
by
factor(s)
produced
by
Schistosoma
mansoni.
Eur.
J.
Immunol.
7,
624.
DUPONT,
V.,
BERNARD,
E.,
SOUBRANE,
J.,
HALLU,
B.
&
CMI
responses
in
human
S.
mekongi
infection
73
RICHIR,
C.
(1957)
Bilharziose
a
Schistosomajaponi-
cum
a
forme
hepat6-splnique
revelee
par
une
grande
hematemese.
Bull.
MeWm.
Soc.
Mid.
H6p.
Paris,
73,
933.
ELLNER,
J.J.
(1978)
Suppressor
adherent
cells
in
human
tuberculosis.
J.
Immunol.
121,
2573.
ELLNER,
J.J.,
OLDS,
G.R.,
KAMEL,
R.,
OSMAN,
G.S.,
ELKHOLY,
A.
&
MAHMOUD,
A.A.F.
(1980)
Sup-
pressor
splenic
T
lymphocytes
in
human
hepato-
splenic
schistosomiasis
mansoni.
J.
Immunol.
125,
308.
GRAVES,
M.F.
&
GROWN,
G.
(1974)
Purification
of
T
and
B
lymphocytes.
J.
Immunol.
112,
420.
HoFsTETTER,
M.,
NASH,
T.E.,
CHEEVER,
A.W.,
GusTAvo
DOS
SANTOS,
J.
&
OTrESEN,
E.A.
(1981)
Schistosomiasis
mekongi
in
Southeast
asian
refu-
gees.
J.
Infect.
Dis.
(In
press).
LEWERT,
R.M.,
YoGoRE,
M.G.,
JR
&
BLAS,
B.L.
(1979)
Lymphocyte
responsiveness
to
phyto-
hemagglutinin
and
to
worm
and
egg
antigens
in
human
schistosomiasisjaponica.
Am.
J.
Trop.
Med.
Hyg.
28,
92.
MARTIN,
L.K.
&
BEAVER,
P.C.
(1968)
Evaluation
of
Kato
thick-smear
technique
for
quantitative
diag-
nosis
of
helminth
infections.
Am.
J.
Trop.
Med.
Hyg.
17,
382.
MOTA-SANTOS,
T.A.,
TAVARES,
C.A.P.,
GAZZINELLI,
G.
&
PELLEGRINO,
J.
(1977)
Immunosuppression
mediated
by
adult
worms
in
chronic
schistoso-
miasis
mansoni.
Am.
J.
Trop.
Med.
Hyg.
26,
727.
OTTESEN,
E.A.
(1979)
Modulation
of
host
response
in
human
schistosomiasis.
I.
Adherent
suppressor
cells
that
inhibit
lymphocyte
proliferative
responses
to
parasite
antigens.
J.
Immunol.
123,
1639.
OTTESEN,
E.A.,
HIATT,
R.A.,
CHEEVER,
A.W.,
ZOTO-
MAYOR,
Z.R.
&
NEVA,
F.A.
(1978)
The
acquisition
and
loss
of
antigen-specific
cellular
immune
respon-
siveness
in
acute
and
chronic
schistosomiasis
in
man.
Clin.
exp.
Immunol.
33,
38.
OTrEsEN,
E.A.
&
POINDEXTER,
R.W.
(1980)
Modula-
tion
of
the
host
response
in
human
schistosomiasis.
II.
Humoral
factors
which
inhibit
lymphocyte
proliferative
responses
to
parasite
antigens.
Am.
J.
Trop.
Med.
Hyg.
29,
592.
ROCKLIN,
R.E.,
BROWN,
A.P.,
WARREN,
K.S.,
PELLEY,
R.P.,
HOUBA,
V.,
SIONGOK,
T.K.A.,
OUMA,
J.,
STURROCK,
R.F.
&
BUTTERWORTH,
A.E.
(1980)
Factors
that
modify
the
cellular-immune
response
in
patients
infected
by
Schistosoma
mansoni.
J.
Immunol.
125,
1916.
SORNMANI,
S.,
VIVATANASESTH,
P.
&
THIRACHANTRA,
S.
(1976)
Clinical
study
of
Mekong
schistosomiasis
at
Khong
Island,
Southern
Laos.
Southeast
Asian
J.
Trop.
Med.
Public
Health,
7,
270.
TODD,
C.W.,
GOOODGAME,
R.W.
&
COLLEY,
D.G.
(1979)
Immune
responses
during
human
schistoso-
miasis
mansoni.
IV.
Suppression
of
schistosome
antigen-specific
lymphocyte
blastogenesis
by
adherent/phagocytic
cells.
J.
Immunol.
122,
1440.
VOGE,
M.,
BRUCKNER,
D.
&
BRUCE,
J.I.
(1978)
Schistosoma
mekongi
sp.
n.
from
man
and
animals,
compared
with
four
geographic
strains
of
Schisto-
soma
japonicum.
J.
Parasitol.
64,
577.
VON
LICHTENBERG,
F.
(1978)
Mechanisms
in
parasitic
infection
with
emphasis
on
schistosomiasis.
South-
east
Asian
J.
Trop.
Med.
Public
Health,
9,
186.
VON
LICHTENBERG,
F.,
ERICKSON,
D.G.
&
SADUN,
E.G.
(1973)
Comparative
histopathology
of
schis-
tosome
granulomas
in
the
hamster.
Am.
J.
Pathol.
72,
149.
WARREN,
K.S.,
BOROS,
D.L.,
HANG,
L.M.
&
MAH-
MOUD,
A.A.F.
(1975)
The
Schistosoma
japonicum
egg
granuloma.
Am.
J.
Pathol.
80,
279.
WARREN,
K.S.,
DOMINGO,
E.O.
&
COWAN,
R.B.T.
(1973)
Granuloma
formation
around
schistosome
eggs
as
a
manifestation
of
delayed
hypersensitivity.
Am.
J.
Pathol.
51,
735.
WARREN,
K.S.,
GROVE,
D.I.
&
PELLEY,
R.P.
(1978)
The
Schistosoma
japonicum
egg
granuloma.
II.
Cellular
composition,
granuloma
size
and
immuno-
logic
concomitants.
Am.
J.
Trop.
Med.
Hyg.
27,
271.
Yu,
A.H.,
WATTS,
H.,
JAFFE,
N.
&
PARKMAN,
R.
(1977)
Concomitant
presence
of
tumor
specific
cytotoxic
and
inhibitor
lymphocytes
in
patients
with
osteogenic
sarcoma.
N.
Engl.
J.
Med.
297,
12
1.
