Studies on the natural immunity in hamsters using the intraperitoneal model of amebic liver abscess.

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Archives of Medical Research 31 (2000) S78–S80
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc.
PII S0188-4409(00)00187-9
Studies on the Natural Immunity in Hamsters Using the
Intraperitoneal Model of Amebic Liver Abscess
Mineko Shibayama,* Rafael Campos-Rodríguez,** Claudia Ramírez,**
Judith Pacheco-Yépez* and Víctor Tsutsumi*
*
Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
**
Departamento de Bioquímica, Escuela Superior de Medicina (ESM) del I.P.N., Mexico City, Mexico
Key Words: Entamoeba histolytica
, Peritoneal exudate, Natural immunity, Amebic liver abscess.
Introduction
In vivo
understand some of the mechanisms of pathogenesis in
amebiasis. Most of these studies have been performed in an-
imals susceptible to liver infection, such as hamsters and
gerbils. The susceptibility or resistance to amebic infection
(intestinal or hepatic) are related to multiple factors, which
include innate or natural immunity (1).
In a previous study, we reported the production of ame-
bic liver abscess (ALA) by inoculating
tica
trophozoites into the peritoneal cavity. This rapid and
efficient method to reproduce ALA in hamsters has been
used to study different aspects of the immune response in
amebiasis (2). Based on this and other experimental models,
we have suggested that polymorphonuclear (PMN) leuko-
cytes associated to amebas play an important role in the pro-
cess of host tissue invasion and destruction (3). However, in
these relatively acute conditions, the
peritoneal components, such as macrophages, lymphocytes,
and other chronic inflammatory cells and locally secreted
molecules, has not been determined. Moreover, whether a
previous stimulation of these peritoneal cells may have a
protective effect against amebic infection is unknown.
In this paper, we studied the effect of nonspecifically
stimulated peritoneal exudate on the production of amebic
liver abscess. An increase in peritoneal exudate was pro-
duced in hamsters by injecting different substances into the
peritoneum, and then animals were challenged with virulent
trophozoites of
E. histolytica
using the same route. Final ef-
fect was determined by analyzing the peritoneal exudate
content and measuring the liver lesions produced. Knowl-
edge of these host natural or innate factors may help us bet-
studies employing laboratory animals are useful to
Entamoeba histoly-
in vivo
role of other
ter understand the mechanisms of control of this parasitic
disease.
Materials and Methods
Amebic cultures.
1:IMSS were maintained axenically in BI-S33 medium (4).
This strain was subsequently cloned and passed three times
through hamster liver to increase virulence. The inoculum
was prepared from 72 h amebic cultures in logarithmic
growth phase. Each animal was injected intraperitoneally
(i.p.) with 2
?
10 amebas in 1 mL.
Trophozoites of
E. histolytica
strain HM-
6
Animals.
tus
), 2 months of age and weighing approximately 100 g,
were used throughout the experiments.
Male adult golden hamsters (
Mesocricetus aura-
Stimulation procedures.
control hamsters were not stimulated, but were challenged
with the same number of
E. histolytica
All animals were stimulated i.p.;
trophozoites.
Incomplete Freund’s adjuvant (IFA) and sterile mineral
oil.
Hamsters received a single dose of 1 mL IFA, or 3 mL
mineral oil (Sigma, St. Louis, MO, USA). Seven days later,
animals were challenged i.p. with 2
histolytica
, and sacrificed after 7 days.
?
10
6
trophozoites of
E.
BCG and complete Freund’s adjuvant (CFA).
group, animals were administered with a weekly dose of 0.5
mg for 3 weeks. CFA was given in a dose of 1 mL CFA (1
mg of
Mycobacterium
tuberculosis per mL) with saline
buffer for 3 weeks. At day 7 after the last inoculation, ani-
mals were challenged with
E. histolytica
sacrificed 1 week later.
In the BCG
trophozoites and
LPS treatment. Escherichia coli
applied i.p. to another group of hamsters 2 or 7 days before
LPS (100–200
?
g) was
Address reprint requests to: Víctor Tsutsumi, Departamento de Patología
Experimental, Cinvestav, Av. Instituto Politécnico Nacional #2508, Col. San
Pedro Zacatenco, 07360 México, D.F., México. Tel.: (
FAX: (
?
525) 747-7107; E-mail: vtsutsu@mail.cinvestav.mx
Presenting author: Mineko Shibayama.
?
525) 747-7107;

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XIV Seminar on Amebiasis, Mexico City/Archives of Medical Research 31 (2000) S78–S80
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challenge with
route, and sacrificed at day 7.
E. histolytica
trophozoites using the same
Recovery of the peritoneal exudate.
was injected with 20 mL of RPMI-1640 culture medium
supplemented with 5% heat-inactivated fetal calf serum con-
taining heparin and antibiotics. The peritoneal exudate was
aspirated and washed three times by centrifugation. The
number of cells was counted in a hemocytometer and viabil-
ity was determined by trypan blue exclusion in a light micro-
scope. To identify peritoneal cell population, the exudate
was fixed with 2.5% glutaraldehyde in sodium cacodylate
The peritoneal cavity
buffer, pH 7.2, for 2 h at room temperature, postfixed for 2 h
with 1% osmium tetroxide in the same buffer, and embedded
in Epon resin. Thin sections of representative regions were
stained with uranyl acetate and lead citrate. The samples
were examined with a Zeiss EM-10 electron microscope.
Results and Discussion
Hamsters administered with nonbiologic and nonspecific
peritoneal stimulants such as IFA or mineral oil by the peri-
toneal route and challenged 1 week later with
E. histolytica
Figure 1. Effect of peritoneal stimulants on amebic liver lesion development in hamsters. (A) Effect of a single i.p. dose of 1 mL incomplete Freund’s adjuvant
(IFA) or 3 mL mineral oil. Untreated control animals showed ca. 40% of hepatic lesion, whereas no lesions were developed in the stimulated groups. (B) Simul-
taneous administration of peritoneal stimulants and E. histolytica trophozoites. C: control; TIOG: thioglycolate; Oil: mineral oil; IFA: incomplete Freund’s
adjuvant; CFA: complete Freund’s adjuvant. Liver lesions were similar in all cases, suggesting that stimulants do not have a direct effect on amebas. (C) Effect
of three weekly doses of CFA or BCG before challenge with 2 ? 106 amebas. Control group presented 45% of lesion; complete protection was achieved with
CFA, while only 5% of lesion was produced with BCG. (D) Correlation between liver abscess size and presence of immune cells in the peritoneal exudate. Ani-
mals treated with LPS, BCG, or mineral oil 7 days before challenge with E. histolytica trophozoites show abundant macrophages/monocytes, which correlates
with significantly smaller liver lesions when compared with the control group (p ?0.01). All values represent the mean ? SD of five hamsters in each group.

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Shibayama et al./Archives of Medical Research 31 (2000) S78–S80
trophozoites did not show significant liver damage at day 7
after challenge. In contrast, nonstimulated control hamsters
presented ALA of
ca.
40% of the total hepatic weight (Fig-
ure 1A). Analysis of the peritoneal exudate after stimulation
showed that mononuclear cells (macrophages/monocytes)
were the most abundant cells, suggesting that these were the
main effector cells affecting amebic viability, and that min-
eral oil and IFA strongly stimulates their production and/or
recruitment.
To test whether these substances (mineral oil, IFA, CFA,
and thioglycolate), which have been used as peritoneal stim-
ulants in different experiments, are directly damaging the
amebas, we performed experiments in which both the stim-
ulant and the amebas were given simultaneously to the same
animal using the same route. The results showed that ALAs
developed under these conditions were similar to controls
(Figure 1B), indicating that these compounds were not dam-
aging the trophozoites.
Hamsters inoculated i.p. with three doses of 0.5 mg per
week (total 1.5 mg) of BCG 7 days before challenge devel-
oped only small ALAs (Figure 1C). Also, animals immu-
nized weekly with 1 mL of CFA per week for 3 weeks did
not develop ALAs ( Figure 1C). These data suggest that bio-
logic, but nonspecific, proteins are also capable of inducing
a peritoneal protective response. Protection with BCG is
only observed when amebas are inoculated through the
same route. This suggests that the protective effect must be
local (peritoneal). Although BCG injected into the perito-
neum enters the systemic circulatory system and can also
stimulate hepatic Kupffer cells, this appears to be insuffi-
cient for blocking amebic invasion when the liver is chal-
lenged by direct injection of trophozoites.
Another nonspecific stimulant of the peritoneum is LPS,
which is extracted from
E. coli.
against amebic infection was only observed 7 days after in-
jecting LPS into the peritoneal cavity, when the peritoneal
exudate contained large amounts of macrophages and
monocytes. Two days after LPS stimulation, the peritoneal
exudate was mainly composed of PMN neutrophils, and
macrophages were scarce (Figure 1D).
In conclusion, in the model of ALA produced by i.p. in-
oculation of
E. histolytica
trophozoites, the nonspecific
stimulation of peritoneal cells, especially mononuclear in-
flammatory cells, appears to be sufficient to eliminate the
parasite. Therefore, a specific immune response does not
seem to be important for controlling the infection. Studies
conducted to elucidate the mechanisms involved in this pro-
tection, including passive transfer of activated cells, are
presently being carried out in the laboratory.
The protective effect
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