Entamoeba histolytica: localization Of the Gal/GalNAc adherence lectin in experimental amebic liver abscess.

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Archives of Medical Research 31 (2000) S242–S244
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Entamoeba histolytica
: Localization of the Gal/GalNAc Adherence Lectin in
Experimental Amebic Liver Abscess
Judith Pacheco-Yépez,* Rafael Campos-Rodríguez,*** Jesús Serrano-Luna,**
Martha Espinosa-Cantellano,* William A. Petri, Jr,**** Víctor Tsutsumi* and Mineko Shibayama*
*Departamento de Patología Experimental
**Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
***Departamento de Bioquímica, Escuela Superior de Medicina, Instituto Politécnico Nacional (IPN), Mexico City, Mexico
****Department of Medicine, University of Virginia, Charlottesville, VA, USA
Key Words: Entamoeba histolytica
, Gal/GalNAc lectin, Amebic liver abscess, Immunohistochemistry.
Introduction
Intestinal invasive amebiasis is characterized by ulceration
of the colonic epithelium, while amebic liver abscess, the
main extraintestinal form of the infection, produces large
necrotic areas that can be fatal unless properly treated. De-
spite great advances in the knowledge of the parasite, little
is known about the pathogenic mechanisms that participate
in the production of these lesions in the host.
In vitro
, the cytopathic effect of amebas involves adher-
ence, contact-dependent lysis, and phagocytosis. Several
molecules seem to play an important role in these events, in-
cluding lectins, pore-forming peptides, and proteases. One
of the best characterized molecules is the galactose/N-acetyl-
D
-galactosamine (Gal/GalNAc)-inhibitable lectin, which par-
ticipates in the
in vitro
adhesion to human erythrocytes, neu-
trophils, epithelial cells, and colonic mucins (1). In addition
to its role in amebic adherence, the lectin may also partici-
pate in the cytolytic event, because contact-dependent target
cell lysis is reduced in the presence of galactose, and a mon-
oclonal antibody against the heavy subunit is capable of in-
hibiting cytolysis without blocking adherence (2). Further-
more, the adhesin binds to purified C8 and C9 components
of complement and blocks the assembly of the membrane
attack complex on the amebic plasma membrane, suggest-
ing a role in mediating resistance to complement lysis
through components C5b–9 (3).
Because the Gal/GalNAc lectin plays an important role
in the
in vitro
cytopathic effect, the aim of this project was
to determine the participation of this molecule in the
experimental infection. We present here the localization of
in vivo
the Gal/GalNAc lectin in the amebic liver abscess at differ-
ent times of evolution.
Materials and Methods
Amebas. Entamoeba histolytica
1:IMSS were axenically cultured at 36
dium. Parasites were harvested after 72 h incubation by
chilling the culture tubes to 4
?
C and centrifuging at 160
for 5 min.
trophozoites strain HM-
?
C in BI-S-33 me-
?

g
Production of amebic liver abscess.
adult golden hamsters (
proximately 100 g were intraportally inoculated with 0.1
mL culture medium containing 5
phozoites. Groups of five animals were sacrificed at 6, 12,
24, and 48 h postinoculation. Lesions associated with nor-
mal hepatic tissue were fixed, embedded in paraffin, and
stained with hematoxilin and eosin to identify representa-
tive samples of each timepoint to be processed for immuno-
histochemistry.
Two-month-old male
) weighing ap-
Mesocricetus auratus
?
10
5

E. histolytica
tro-
Immunohistochemistry.
on glass slides covered with silane. After dewaxing with xy-
lene, endogenous peroxidase activity was blocked by incu-
bation with 0.3% H
2
O
2
in methanol for 1 h, and washed with
phosphate-buffered saline (PBS). Nonspecific reaction sites
were blocked for 1 h at room temperature with 10% fetal bo-
vine serum/3% rabbit serum in 0.05% PBS-Tween (PBS-T).
Slides were incubated overnight at 4
clonal anti-Gal/GalNAc lectin antibody or with a rabbit anti-
CD8 antibody (controls) diluted 1:100 in PBS-T. After sev-
eral washings in PBS-T, samples were incubated with a per-
oxidase-labeled goat antirabbit IgG antibody for 1 h at room
temperature. Peroxidase activity was detected by adding di-
Sections 6–7
?
m-thick were mounted
?
C with a rabbit poly-
Address reprint requests to: Mineko Shibayama, Departamento de
Patología Experimental, Cinvestav, Av. Instituto Politécnico Nacional #2508,
Col. San Pedro Zacatenco, 07369 México, D.F., México. Tel.: (
7107; FAX: (
?
525) 747-7107; E-mail: mineko@mail.cinvestav.mx
Presenting author: Judith Pacheco-Yépez.
?
525) 747-

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S243
aminobenzidine and H
nally counterstained with hematoxilin.
2
O
2
as a substrate. Sections were fi-
Results and Discussion
Six hours after intraportal inoculation, the parasite was lo-
calized in the hepatic parenchyma surrounded by an acute
inflammatory infiltrate. Trophozoites were strongly labeled
with the peroxidase reaction, identifying the Gal/GalNAc
lectin on the amebas. Surprisingly, the lectin was also
present in inflammatory and sinusoidal cells and in the cyto-
plasm of neighboring hepatocytes, suggesting that
tolytica
releases the Gal/GalNAc lectin during the process
of liver invasion, and that the molecule is recognized by the
surrounding cells. Picnotic nuclei are observed in some cells
(Figure 1A). Labeling seems to be specific, as no reactivity
was observed in the control samples (data not shown). At 12
h the lesions increased in size, with a larger inflammatory
infiltrate around strongly labeled trophozoites (Figure 1B).
E. his-
Figure 1. Immunohistochemical localization of the Gal/GalNAc lectin in the hepatic parenchyma at 6 (A), 12 (B), 24 (C), and 48 (D) h postinoculation with
E. histolytica trophozoites. (A) After 6 h, an acute inflammatory infiltrate surrounds the trophozoites, which are strongly labeled with peroxidase (arrow).
Neighboring inflammatory, hepatic, and sinusoidal (arrowheads) cells are also labeled. Some hepatocytes show picnotic nuclei (n). 20?; (B) At 12 h, tropho-
zoites (arrows) are surrounded by multiple layers of labeled inflammatory cells. Hepatocytes close to the inflammatory focus are elongated and also labeled.
Most hepatocytes labeled with peroxidase show different degrees of damage, with the nucleus displaced to one pole of the cell, some with picnotic appear-
ance. Sinusoidal cells are also labeled (arrowheads). 20?; (C) Twenty-four h after inoculation, amebas (arrow) show strong peroxidase label and seem to have
ingested leukocytes in the cytoplasm. Inflammatory cells identified by the anti-Gal/GalNAc lectin antibody show signs of cell damage (vacuolization). Hepa-
tocytes present a lightly positive reaction. 40?, and (D) At 48 h, a newly formed granuloma characterized by the presence of epitheloid cells (ec) contains a
trophozoite (arrow) at the center and abundant chronic inflammatory cells. The anti-Gal/GalNAc lectin antibody recognized inflammatory cells and hepato-
cytes bordering the granuloma. Sinusoidal cells are also recognized (arrowheads). 20?.

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Pacheco-Yépez et al./Archives of Medical Research 31 (2000) S242–S244
Again, peroxidase label was identified in the surrounding
inflammatory, sinusoidal, and parenchymal cells, revealing
the presence of the Gal/GalNAc lectin. However, at this
stage most hepatocytes labeled with peroxidase showed dif-
ferent degrees of damage, with the nucleus displaced to one
pole of the cell and some with picnotic appearance. At 24 h
postinoculation, damage to the liver parenchyma was more
evident, the necrotic areas increasing in size, more hepato-
cytes showing signs of morphological damage, and abun-
dant lysed leukocytes (Figure 1C). The Gal/GalNAc lectin
was present in the amebas, inflammatory cells, and hepato-
cytes bordering the inflammatory infiltrates. A fine deposit
of peroxidase reaction was also identified in the necrotic
center, suggesting complete degradation of some cells that
bound the lectin at earlier timepoints. Analysis of the liver
lesions 2 days postinoculation showed characteristic granu-
lomas with a necrotic center surrounded by a large chronic
inflammatory infiltrate and limited by epithelioid cells (Fig-
ure 1D). The Gal/GalNAc lectin was still localized in the
amebas, parenchymal, sinusoidal, and inflammatory cells.
Our study demonstrates that in the hamster model of
amebic liver abscess
E. histolytica
Gal/GalNAc lectin during the process of invasion, which
can be detected in hepatocytes, sinusoidal, and inflamma-
tory cells since early time points of infection. These host
trophozoites release the
cells are more susceptible to cell damage, probably through
an apoptotic phenomenon (4). Whether the Gal/GalNAc
lectin is directly responsible for this effect, or the recogni-
tion of a foreign antigen on these cells renders them a target
for the host’s cellular immune system is unknown. Other
factors involved in hepatic damage remain to be elucidated.
Acknowledgments
We thank Ms. M. Miranda and Ms. C. Guadarrama for their valu-
able technical assistance.
References
1. Mann BJ, Lockhart LA. Molecular analysis of the Gal/GalNAc ad-
hesin of
Entamoeba histolytica.
J Eukaryot Microbiol 1998;45:13S.
2. Saffer LD, Petri WA Jr. Role of the galactose lectin of
tolytica
in adherence-dependent killing of mammalian cells. Infect Im-
mun 1991;59:4681.
3. Braga LL, Ninomiya H, McCoy JJ, Eacker S, Wiedmer T, Pham C,
Wood S, Sims PJ, Petri WA Jr. Inhibition of the complement mem-
brane attack complex by the galactose-specific adhesin of
histolytica.
J Clin Invest 1992;90:1131.
4. Ragland BD, Ashley LS, Vaux DL, Petri WA, Jr.
tica
: target cells killed by trophozoites undergo DNA fragmentation
which is not blocked by Bcl-2. Exp Parasitol 1994;79:460.
Entamoeba his-
Entamoeba
Entamoeba histoly-