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Experimental Amebiasis: A Selected Review of Some In Vivo Models
Archives of Medical Research 37 (2006) 210–220
REVIEW ARTICLE
Experimental Amebiasis: A Selected Review of Some In Vivo Models Vı´ctor Tsutsumi and Mineko Shibayama Departamento de Patologı´a Experimental, CINVESTAV-IPN, Me´xico, D.F., Me´xico Received for publication September 22, 2005; accepted September 23, 2005 (ARCMED-D-05-00381).
The use of in vivo animal models in amebiasis has contributed significantly to the knowledge of this common human parasitic disease. Although there is no animal model that mimics the whole cycle of the human disease, the use of different susceptible and resistant laboratory animals and the availability for many years of techniques for the axenic culture of trophozoites of Entamoeba histolytica have allowed a better understanding of the parasite and the host–parasite relationship. The recent introduction of frontier methodologies in biology has increased our comprehension of this parasite. New information on the cellular and molecular biology and genetics of this organism has been extensively reported, and much of this has clearly required the more frequent use of animal models to verify specific facts. Based on experimental animals characterized previously, the introduction of new animal models with genetic or surgical modifications, especially in mice, has allowed a more adequate analysis of the mechanisms of pathogenesis. Multiple factors have been considered in the promotion of the invasiveness and virulence of E. histolytica. Additionally, the immunological and physiological responses of the host, depending on the environmental conditions, lead to the establishment or the rejection of the parasite. The role of inflammatory reaction to amebic infection constitutes one of the controversies that has been studied by several authors. In susceptible animals (hamsters and gerbils), inflammatory cell damage seems to be related to target cell lysis, while in resistant animals (mice), inflammatory cells appear to protect the host by lysing the parasite. Presently, the involvement of various substances in the development of lesions including lectins, proteases, amebapores, promoters of apoptosis, cytokines, nitric oxide, etc., is being examined using different in vivo models. Ó 2006 IMSS. Published by Elsevier Inc. Key Words: Amebiasis, Entamoeba histolytica, Animal models, Pathology.
Introduction Studies on the interaction of Entamoeba histolytica with different biological substrates have been aimed mainly at determining the pathogenic mechanisms participating in the production of amebic lesions. A better understanding of tissue destruction using animal models that reproduce intestinal or extraintestinal lesions similar to those seen in humans may contribute significantly in providing the basis for a more adequate treatment and prevention of the amebic disease.
Address reprint requests to: Vı´ctor Tsutsumi, Departamento de Patologı´a Experimental, CINVESTAV-IPN, Av. IPN No. 2508, CP. 07360, Me´xico, D.F., Me´xico; E-mail: [email protected]
Although the destructive or lytic capacity of amebas can be easily observed by light microscopy in all affected tissues, justifying the term histolytica, intrinsic mechanisms of the ameba and the host that play a role in infection are multifactorial and therefore constitute phenomena much more complex than a single in vitro host–parasite interaction. The commonly mentioned importance of the introduction of E. histolytica trophozoites cultured under axenic conditions for research in amebiasis (1) is evident from the large amount of knowledge that has been obtained regarding this etiologic agent. Most of the information on the physiopathology, fine structure, cell biology, biochemistry, immunology, genetics and molecular biology of E. histolytica has been acquired in the last 35 years using this technique of axenic culturing. Surprisingly, the great
0188-4409/06 $–see front matter. Copyright Ó 2006 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2005.09.011
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majority of these works has been achieved by using the same HM1-IMSS strain of trophozoites that was isolated close to 40 years ago. It should be emphasized that the virulence of this strain, which was originally very high, has been declining every year, requiring more frequent passages of amebas through the hamster liver. Especially when we consider virulence to be an important factor in experiments with E. histolytica, it should be pointed out that HM1-IMSS strain, which is one of the strains used in our laboratory and many others, cannot be regarded as the same strain isolated many years ago, at least with respect to virulence. Experimental amebiasis using in vivo models began when Lesch produced intestinal lesions in one of the dogs inoculated with feces from a patient suffering from dysentery (2). Subsequently, numerous studies have been performed using dogs, cats, rabbits and monkeys; however, their use is limited at present due to several factors including lack of uniformity, difficulty in handling and insufficient number of animals. Since the middle of the last century, easy access to many species of rodents for laboratory research, along with uniform conditions and acceptable susceptibility, has helped to increase our knowledge of the pathogenicity of amebiasis. These rodent models have also been used in many practical applications, including therapeutic and immunoprophylactic studies. However, an animal model that can reproduce the whole life cycle of E. histolytica as seen in humans is still not available. Thus, to date, the production of intestinal and extraintestinal amebic lesions in animals fed with E. histolytica cysts is not yet possible. Therefore, most of the in vivo studies of experimental amebiasis have been performed either with intestinal or hepatic models as separate entities, which have been produced by trophozoites injected directly into the target organ. Since relatively extensive reviews on different animal models in amebiasis have already been published (3–5), we will describe here briefly some previous basic works and then focus on recent data related mainly to the pathophysiology or mechanisms of amebic lesion production using intestinal and hepatic animal models.
Experimental Intestinal Amebiasis The production of typical intestinal amebic lesions in laboratory animals has been more complex and difficult than that of hepatic lesions. Many of the previous studies performed in different animals such as rats, guinea pigs and mice have yielded inconsistent and poor results, and in many cases this has been explained simply as due to a natural immune resistance of rodents to intestinal inoculation. Moreover, despite that experimental animals are inoculated with several million trophozoites of a known specific virulent strain, this inoculum mixes with a poorly characterized
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intestinal flora consisting of other protozoans, several strains of bacteria and other microorganisms as well. This mixture of intestinal organisms plus our lack of knowledge of the physicochemical characteristics of the intestinal milieu are important factors that have not been considered in the past and are likely responsible in great part for the heterogeneity of results. Differences in susceptibility have been reported by different groups, even when using similar laboratory animals under apparently identical conditions (4). In spite of these limitations, we believe that there are some useful laboratory animals for intestinal amebiasis, the choice depending on the specific objective sought. Therefore, interesting information regarding the early stages of intestinal infection, pathology and host immune response have been obtained, and much of the current knowledge about different aspects of the disease has come from the integration of data obtained using particular animal models. The current availability of various strains of rodents with different susceptibilities, especially in mice, has provided more information on the mechanisms of pathogenicity for intestinal amebiasis, although there are still many undetermined factors that are currently under study. At the molecular level, various research groups have been studying the genes or gene products of E. histolytica related to those of humans, through the evaluation of SCID mice (6) and other SCID modified mice (7), microarray analysis, and examination of several aspects of the mechanism of the immune response (8–11). Guinea pigs have also been used to study intestinal amebiasis, but there have been only a few reports concerning the successful production of amebic ulcers using amebas from axenic cultures. Diamond et al. (12) induced lesions in newborn guinea pigs inoculated intracecally; however, these results could not be reproduced consistently in a larger number of animals. Anaya et al. (13) described typical intestinal ulcers in guinea pigs and hamsters by producing an artificial cecal loop inoculated with axenic or monoxenic E. histolytica trophozoites (Figure 1). With this model, the authors showed the early stages of ameba invasion of the mucosal and the submucosal layers with a substantial inflammatory reaction (Figure 2). Electron microscopy analysis of the ulcer showed the possible role of leukocytes in the development of the amebic ulcer (Figure 3). Rats have been used in experimental amebiasis for many years; however, intestinal infections have been unclear. Jones (14) re-evaluated the use of rats in amebiasis studies and showed that weaning rats are more susceptible to amebic intestinal infection. This finding is interesting because it is known that rats rapidly develop resistance to infection when they begin to eat normal rat chow. A description of intestinal amebic lesions in this rodent was reported by Neal (15) who proposed the use of the Wistar strain and a scoring system of the amebic damage based on the macroscopic appearance of the intestinal wall and the
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Figure 1. Early intestinal lesions produced in the cecal loop model of guinea pig. At 48 h post-inoculation of axenic E. histolytica trophozoites, small whitish round lesions (arrows) surrounded by erythematous areas and congested blood vessels are shown.
characteristics of the luminal content. Although some authors have used rats for studying some aspects of intestinal amebiasis (effect of drugs, role of cholesterol in the ameba virulence and effect of different isolates), two unfavorable
conditions have to be considered when the study of pathogenesis is of interest: a) the use of weaning rats, although very susceptible to infection, have poorly developed immune systems and b) the requirement of continual bacterial association in the ameba inoculum can mask the real phenomena occurring during ulcer formation. The use of gerbils for experimental amebiasis was introduced by Diamond et al. (16). However, the application of this rodent in the study of intestinal amebiasis was not reported until 1985 by Chadee and Meerovitch (17) who analyzed the sequence of cecal infection from 1 to 10 days postinoculation. These latter authors noted that the trophozoites utilized were cultured under xenic or monoxenic conditions; however, there was no mention of inoculum size and the description of intestinal lesions was limited to erosion and edema of intestinal glands. Shibayama et al. (18), using axenic or monoxenic cultures, showed during the first hours of interaction an increase in mucus production mixed with trophozoites in the lumen. Microulcerative mucosal lesions appeared 24–72 h post-inoculation with inflammatory infiltrate and edema of the lamina propria associated with the necrotic ulcer. However, as in most ulcerative intestinal lesions observed in other rodents, lesions healed spontaneously after 96 h with complete absence of trophozoites. In the past, mice were not frequently used as models for amebiasis. The main reason was that these rodents were always considered as naturally resistant to E. histolytica infection because a substantial number of attempts to produce intestinal or hepatic lesions were practically unsuccessful. Further studies by Ghadirian and Kongshavn (19) using ten different strains of mice reported differences in
Figure 2. Light microscopy of an intestinal amebic ulcer produced in the previously mentioned cecal loop of guinea pig. A mucosal ulcer invading submucosa is composed by necrotic tissue, abundant trophozoites with fibrinous material and inflammatory infiltrate at the bottom of the ulcer. H&E stain. 370.
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Figure 3. Transmission electron microscopy of the necrotic zone of the amebic intestinal ulcer. Lesion was produced by using the cecal loop model in guinea pig. Various E. histolytica trophozoites (Eh) are surrounded by abundant lysed (Ly) vacuolar leukocytes. Some nuclear shadows of leukocytes are seen. Bar 5 10 mm.
susceptibility to the development of intestinal amebiasis after inoculation with trophozoites cultured axenically. The strains C3H/HeCr, BALB/c, NZB/B1N, B10.A, DBA/2 and C57Bl/6 were classified as susceptible models and the strains A/J, CE, DBA/1 and CD-1 were considered as resistant. The authors concluded that genetic factors may be involved in the differences of susceptibility to intestinal infection. In a similar study by Owen (20) using seven different strains of mice, a metastatic liver lesion was reported in the CBA/HN strain, whereas the athymic mouse of the same strain did not present liver invasion. The same author also reported intestinal lesions in C3H/HeJ strain with absence of liver metastasis, in contrast to a later report by Ghosh et al. (21). Among several factors involved in the intestine and hepatic invasion by E. histolytica trophozoites in rodents, Chauhan and Das (22) have mentioned a diet rich in cholesterol. Contrarily, factors associated with blocking the adhesion of and invasion by amebas are associated bacteria and the presence of other protozoans (23,24). Ghosh et al. (21) using C3H/HeJ mice reported the production of ulcerative inflammatory disease similar to that described in human amebiasis. In this study, E. histolytica trophozoites were inoculated intracecally and the animals sacrificed at different time-points, ranging from 5 to 30 days. On the 5th day there were tiny superficial erosions of the epithelium that progressed to deeper and more extensive lesions of the cecal wall. Flask-shaped ulcers and the presence of amebomas in the vicinity of ulcers were described in this model. Although ulcerative lesions were
found to be common and extensive, direct cell contact or tissue invasion by trophozoites was rarely observed. In addition to describing a relatively chronic intestinal lesion in this strain of mice, the authors suggested the possibility of distance damage to epithelial cells by toxic substances secreted by the amebas. Subsequently, an in vivo neutropenic model using BALB/c mice was reported (25). In this rodent, the role of neutrophils in the innate intestinal resistance to E. histolytica infection was studied. Animals treated with antineutrophil monoclonal antibody did not show any significant difference in the development of intestinal lesions when compared with control untreated mice. This work reports for the first time the production of granulomatous inflammatory reaction in the intestinal wall of mice when axenically cultured amebas were inoculated, and this type of inflammation was more common in treated animals. The importance of this chronic inflammatory reaction in an acute stage of amebic intestinal infection is still unknown. An interesting model of amebic colitis was developed in SCID mice when a fragment of human intestine was implanted into the subcapsular region (SCID-HU-INT) (7). This human intestinal xenograft was allowed to mature into a morphologically, mucin-secreting normal segment, which was then infected with E. histolytica trophozoites. Within 12 h of amebic inoculation into the lumen of this xenograft, the intestine developed focal ulcerations with invasion of the submucosa similar to that seen in humans with amebic colitis. Although there were differences between the model
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of human intestinal xenografts and the normal human intestine (e.g., the lack of normal flora, the absence of peristalsis and a closed loop), amebic infection in this model mimics a number of features observed in human amebic colitis, providing a system that allows the in vivo study of the interaction between E. histolytica and human intestine (8). In relation to the role of cysteine proteinase (ehcp5), the above authors suggested that this amebic enzyme produced damage of the xenografted human intestine that was consistent with invasion of mucosal tissue by trophozoites with a neutrophil-predominant inflammation (26). Further studies are required to clearly confirm the role of proteinases in the pathogenesis of amebic lesions. Experiments using C3H mice in the study of intestinal amebic infection were reported recently by Houpt et al. (27) who used C3H mice inoculated intracecally with E. histolytica trophozoites grown in a bacterial flora of the xenic strain CDC:0784. In this work, infection was followed for 18 months, and 60% of mice showed chronic cecal infection. Histopathologic changes were evident as early as 4 days after challenge, including crypt hyperplasia, epithelial ulceration, and submucosal infiltration. At that time, viable amebas were usually seen in areas of epithelial ulceration, and they were still seen in the lumen 3 weeks post-inoculation. Inflammation was severe and involved plasma cells, neutrophils, and mast cells. At 10 weeks, inflammation obscured the entire mucosa and morphologically resembled human colitis. Also, they determined the presence of different cytokines and a depletion of CD41 T cells. One of the interesting findings is that when CD41 cells were depleted, both parasite burden and inflammation diminished significantly correlating with a decrease in IL-4 and IL-13 production and loss of mast cell infiltration. This model revealed important immune factors that may influence susceptibility to infection and the physiopathology of intestinal amebiasis. Asgharpour et al. (28) used the same mice model and compared findings with those with C57BL/6 (a resistant mouse). They examined the role of neutrophils in the course of amebic colitis (C3H mouse) and found that neutrophil depletion using an anti-Gr-1 monoclonal antibody and dexamethasone treatment diminished the innate resistance in certain mouse strains (e.g., CBA). However, there was no effect on the high level of resistance of C57BL/6 mice, suggesting that the mechanisms of innate immunity to intestinal E. histolytica infection vary depending on the host genetic background.
bacteria, either directly into the hepatic lobe or through the portal vein, which produced large amebic hepatic abscesses. Further studies using the same rodent but inoculated with axenic amebas confirmed high susceptibility and the uniformity of lesions (30) (Figure 4). Other data were obtained related to the pathogenesis of amebic damage, including aspects on the host immune response and many other factors involved in in vivo host–parasite interactions. A sequential morphological study of hepatic infection from the very early stages of post-inoculation with E. histolytica trophozoites in hamsters reported 20 years ago suggested the role of host inflammatory cells in the process of tissue damage (31) (Figure 5). In this work, which was confirmed with electron microscopic (32) (Figure 6) and immunocytochemical (33) (Figure 7) studies, the authors suggested that E. histolytica trophozoites do not produce amebic liver abscesses through direct lysis of hepatocytes. It was proposed that tissue damage is mediated instead by lysosomal enzymes released from disintegrating inflammatory cells that accumulate around the amebas and are killed by this parasite. This original finding has been the basis for numerous subsequent studies investigating inflammation as an important mechanism of host tissue destruction. Gerbils are another rodent highly susceptible to hepatic amebiasis (Figure 8). Lesions can be obtained similar to those produced in hamsters, although E. histolytica trophozoites apparently show a less virulent behavior in gerbils than in hamsters. Thus, regardless of the size of inoculums, progression of liver damage is slower and the eventual death of the infected animals takes a longer time. These characteristics allow performing similar sequential studies
Experimental Hepatic Amebiasis The first laboratory animal used successfully for hepatic amebiasis was the hamster, which was reported by Reinertson and Thompson (29). These authors injected trophozoites of E. histolytica cultured with three species of
Figure 4. Amebic hepatic lesion in hamster after 7 days post-inoculation. Large irregular whitish lesions are seen on the liver surface.
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Figure 5. Hamster liver at 3 h post-intraportal inoculation of E. histolytica trophozoites. The small amebic liver lesion is formed by a trophozoite (Eh) surrounded by normal (PMN) and lysed (Ly) polymorphonuclear leukocytes. Hepatocytes (H). Semithin section stained with toluidine blue stain. 3290.
of hepatic lesions (18). In addition to the association of inflammatory cells with trophozoites as shown in hamster liver, gerbils also showed trophozoites in direct contact with hepatocytes. The preceding data, plus the possibility of producing amebic intestinal lesions, have led some investigators to consider this rodent as being more similar to humans.
Gerbil has been useful in various fields of research in amebiasis. Studies on host immune response and the effect of various vaccine candidates purified from the parasite and administered parenterally or orally have been tested (34). More recently, gerbils have been used in molecular biology study of various proteins related to E. histolytica virulence (35).
Figure 6. Electron micrograph of an early liver lesion in hamster at 6 h post-intraportal inoculation of amebas. An E. histolytica (Eh) trophozoite is seen contacting undamaged (PMN) and lysed (Ly) polymorphonuclear leukocytes. Bar 5 10 mm.
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Figure 7. Immunohistochemistry of amebic liver lesion at 6 h post-intraportal inoculation. Trophozoites (Eh), inflammatory cells and peripheral hepatocytes (arrows) are positives to peroxidase labeled anti-amebic antibody. Hematoxylin counterstained. 3125.
In relation to the use of mice in hepatic amebiasis, the introduction of genetically modified animals, such as severe combined immunodeficient (SCID) mice, has provided interesting data. Cieslak et al. (6) using this strain of mouse developed liver abscesses when animals were challenged
intrahepatically with virulent E. histolytica trophozoites. In this study, only one of seven similarly challenged immunocompetent congenic C.B.-17 mouse (control) developed an abscess. They also used a polyclonal anti-E. histolytica antibody and reported protection in 7/12 SCID mice. These
Figure 8. Light microscopy of gerbil liver at 48 h after intrahepatic inoculation of amebas. (a) A group of centrally localized trophozoites (arrows) surrounded by abundant inflammatory cells. H&E. 3200. (b) Histochemistry of liver lesion in gerbil at 48 h post-inoculations. Gomori-Grocott stain shows trophozoites in black (arrows). 3200.
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authors concluded that SCID mouse constitutes a powerful model for studying the possible components of protective immunity in invasive amebiasis. In order to determine whether inflammatory cells play a similar role in mice as observed in hamster liver, BALB/c mice were treated with anti-neutrophil RB6-8C5 monoclonal antibodies and then challenged intrahepatically with E. histolytica trophozoites. Animals were sacrificed at different time-points and hepatic analysis showed that neutrophil-depleted mice showed abscesses larger than in untreated control animals. Based on previous data, the authors suggested that neutrophils may play a role in resistance mechanisms in mice, unlike in hamster or gerbil. However, it is important to mention that mice are in general resistant to amebic infection. In both RB6-8C5-treated and -untreated mice challenged similarly, animals survived to infection, although the treated animals required a longer time to heal completely from the hepatic lesion (36). In another study, an immunocytochemical analysis of the diverse cell population was performed in hepatic lesions produced in BALB/c and C3H/Hej strain mice (37). The authors suggested that mouse resistance to the development of liver abscesses depends on the activation of neutrophils, and this in turn is related to abundant nitric oxide production to kill the ameba. In addition to differences in the possible mechanisms of resistance or susceptibility due to inflammatory cells, contradictory roles of nitric oxide have also been mentioned. Previous in vitro (38) and in vivo (9) studies have suggested that nitric oxide has a lytic effect on trophozoites of E. histolytica. Another study in hamsters has shown that this molecule plays no role in arresting the development of amebic liver abscesses. The levels of nitric oxide measured by the presence of nitrites and nitrates in serum were directly proportional to the size of abscesses, suggesting that nitric oxide does not have a lytic effect on E. histolytica and is therefore incapable of providing protection against the hepatic lesion (39). In relation to apoptosis in hepatic damage, it has been shown that target cell killing may be caused in part by the triggering of this process by E. histolytica. Vela´zquez et al. (36) found areas of TUNEL-positive dead hepatocytes in normal and neutrophil-depleted mice. These apoptotic liver cells were found either close to the inflammatory infiltrate or at some distance free of ameba or inflammatory cells. The presence of TUNEL-positive hepatocytes could be the result of one or more possible mechanisms of tissue damage. It is known that inflammatory conditions may cause apoptosis or that amebas may release apoptosisinducing factors that affect hepatocytes at a distance (10). Induction of an ischemic status in liver parenchyma by trophozoites occluding blood vessels can also be considered as another factor (40,41). Studying the murine model of amebic liver abscess, Seydel and Stanley (42) showed that hepatocyte apoptosis requires activation of caspase but is independent from Fas and tumor necrosis factor (TNF) a
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receptor 1. Amebapores have been considered as necessary for both lytic and apoptotic pathways of cell death. Moreover, it is suggested that sublytic concentrations of pore-forming proteins can induce apoptosis in target cells (10). Other important molecules related to the pathogenesis of amebiasis are the proteinases. Previous studies, including those involving xenografted mice, have suggested the role of proteinases in tissue damage (8,11,26). In a recent study by Olivos-Garcı´a et al. (43), the authors concluded that cysteine proteinase plays either a minor or no role in the damage of liver parenchyma during the development of amebic liver abscess. These authors purified an amebic cysteine proteinase (EhCP2) and bound it to inert resin microspheres (22–24 mm in diameter), which were then injected into the portal vein of normal hamsters. A mild acute inflammation and occasional minimal necrosis of short duration was seen. Immunohistochemistry with the anti-EhCP2 antibody showed the presence of positive-labeled microspheres in tissues at 1 h after injection. Sections of experimental acute amebic liver abscesses presented strong labeling of trophozoites, although necrotic tissue and inflammatory infiltrates were negative, supporting the above notion. Another more recent study from the same group (44) reported that cysteine proteinase activity in trophozoites is required for survival of the trophozoites in experimental acute amebiasis in hamsters, whether or not this enzyme plays a role in the virulence of the parasite. Another recent study focused on the Gal/GalNAc lectin of E. histolytica trophozoites has corroborated morphologically the in vitro and in vivo adhesion roles of this lectin. Pacheco et al. (45) performed immunocytochemical studies using various target cells that included cultured human and hamster hepatocytes, hamster amebic liver abscesses and C3H mouse intestinal lesions. Lectin immunolabeling was positive in trophozoites, inflammatory cells and necrotic tissue. Damaged intestinal epithelium in the mouse model also showed labeling with this anti-lectin antibody. The authors concluded that Gal/GalNAc lectin is bound and captured by various target cells and these host cells containing the lectin showed different degrees of cell damage. However, the absence of lectin labeling in some damaged ameba-interacting cells suggests that the mechanisms of pathogenesis are multiple and other factors are also probably involved in cytotoxicity. Morphological changes produced by E. dispar trophozoites on hamster liver have been partially studied. Although early inflammatory foci around axenically cultured amebas were observed in the liver (Figure 9), including inflammatory cell damage (46) (Figure 10), lesions did not progress to abscess, and only small areas of inflammatory infiltrates were observed after 24 h. In another study using different strains of E. dispar cultured in xenic or monoxenic conditions, authors reported either absence or presence of hepatic lesions in hamsters, depending on the origin of
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Figure 9. Light microscopy of hamster liver inoculated with E. dispar (Ed) trophozoites. At 6 h post-intraportal inoculation of amebas, abundant inflammatory cells are seen associated with amebas. H&E stain. 3125.
E. dispar isolate. The effect of bacteria in the virulence of these ameba isolates was also mentioned and emphasized by the authors (47). Conclusions E. histolytica infects only humans and non-human primates; therefore, success in establishing reproducible animal
models of amebiasis has been limited. Nevertheless, knowledge obtained from different animal models used in the study of intestinal or hepatic amebiasis has allowed for a better understanding of the complex mechanisms operating in the target cell damage. As mentioned, we still do not have an animal model that mimics the complete cycle of E. histolytica as it occurs in the human disease; however, many studies conducted with various specific, normal, or
Figure 10. Transmission electron micrograph of hamster liver inoculated with E. dispar (Ed) trophozoites. After 6 h of intraportal inoculation, inflammatory focus shows normal polymorphonuclear (PMN) and lysed (Ly) leukocytes surrounding the ameba. Bar 5 10 mm.
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genetically or surgically altered laboratory animals have contributed to the better understanding of the pathogenesis and immunology of the amebic lesion. More recently, since the introduction of diverse strains of mice in research, our knowledge of the molecular pathology of amebiasis has increased. However, it is important to mention that in many cases intestinal models for amebic infections use artificial methodologies and in others, animals are greatly manipulated, introducing several different variables of the in vivo system unrelated to the human disease. A number of in vivo studies have suggested the role of the host bacterial flora in the pathogenesis of invasive amebiasis. Intestinal bacteria, motility and intestinal flow may all contribute to the protective role in amebic intestinal infection. Experimental studies related to the production of extraintestinal lesions have focused mainly on amebic liver abscess. Basically, there are two susceptible rodents, hamster and gerbil, and both have been used in multiple studies. These investigations involve the in vivo study of immunopathogenesis, pharmacology and effects of antiamebic drugs and studies on prophylaxis and vaccine testing. The role of inflammatory cells in hepatic damage in hamsters still constitutes an important controversial and interesting factor that is under investigation by several research groups. The special environment of the ameba in the liver and the complex interactions between parasite and host components have provided a better understanding of the role of some parasite products such as proteases, different lectins (170, 112, and 220 kDa) and several other components involved in the pathogenesis of experimental amebic liver. Mechanisms of hepatic damage related to ischemia produced by blood vessel occlusion by amebas cannot be discounted. Some liver lesions in either susceptible (hamster) or resistant animals (mouse) have shown histological characteristics of ischemic damage. The possible activation of the apoptotic process in hepatocytes by this ischemia or other factors has also been considered in different in vivo experimental models. Recent studies using the hepatic model in gerbils have provided some information on the virulence of E. histolytica. A molecular study has suggested that the survival of amebas within the hostile environment of the liver, and in particular during the development of liver abscess, is accomplished by a strong adaptive process requiring the specific regulation of a number of amebic proteins. Thus, this regulatory process seems to be an important factor for the general understanding of the mechanisms of E. histolytica pathogenesis. In reference to the ‘‘resistant animal models’’, their use has also provided valuable information about the cell types and the possible mechanisms that are involved in the natural immune response against E. histolytica infection. Rats, guinea pigs and different strains of mice have been useful in studies related to amebic resistance to intestinal or hepatic infection. Several studies have shown that
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polymorphonuclear cells, mainly neutrophils, are implicated in such resistance. Molecules such as nitric oxide complement proteins, and proinflammatory cytokines have been shown to be involved in the process of amebic rejection. Physical barriers such as epithelial cells and the secreted mucus are important blockers of the adhesion of E. histolytica trophozoites. Therefore, the mechanisms of innate protection include combinations of the host genetics, environmental factors such as intestinal bacterial flora in the intestine, and parasite intrinsic factors.
Acknowledgments We thank Silvia Galindo-Go´mez, Ange´lica Silva-Olivares and Mireya Sa´nchez-Palomera for their valuable technical assistance. Our gratitude to Dr. Ruy Pe´rez-Tamayo for his critical reading and helpful comments of the manuscript.
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15. Neal RA. Some observations on the variation of virulence and response to chemotherapy of strains of Entamoeba histolytica in rats. Trans R Soc Trop Med Hyg 1951;44:439–452. 16. Diamond LS, Phillips BP, Bartgis IL. The clawed bird (Meriones unguiculatus) as an experimental animal for the study of hepatic amebiasis. Arch Invest Med (Mex) 1974;5:465–470. 17. Chadee K, Meerovitch E. The pathology of experimentally induced cecal amebiasis in gerbils (Meriones unguiculatus). Liver changes and amebic liver abscess formation. Am J Pathol 1985;119:485–494. 18. Shibayama-Salas M, Navarro-Garcı´a F, Lo´pez-Revilla R, Martı´nezPalomo A, Tsutsumi V. In vivo and in vitro intestinal amebiasis in gerbils. Parasitol Res 1997;83:170–176. 19. Ghadirian E, Kongshavn PAL. Genetic control of susceptibility of mice to infection with E. histolytica. Parasite Immunol 1984;6: 349–360. 20. Owen DG. Entamoeba histolytica in inbred mice: evidence of liver involvement. Trans R Soc Trop Med Hyg 1987;81:617–620. 21. Ghosh PK, Mancilla R, Ortı´z-Ortı´z L. Intestinal amebiasis: histopathologic features in experimentally infected mice. Arch Med Res 1994; 25:297–302. 22. Chauhan MS, Das SR. Simultaneous caecal and liver infections in hamster, mouse and guinea pigs by oral feeding of Entamoeba histolytica cysts. Curr Science 1987;56:1223–1224. 23. Ray DK, Chatterjee DK. Caecal amoebiasis in albino mice—an experimental model. Ann Trop Med Parasitol 1981;75:255–256. 24. Owen DG. Gnotobiotic, athymic mice: a possible system for the study of the role of bacteria in human amebiasis. Lab Anim 1990; 24:353–357. 25. Rivero-Nava L, Aguirre-Garcı´a J, Shibayama-Salas M, Herna´ndezPando R, Tsutsumi V, Caldero´n J. Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice. Exp Parasitol 2002;101:183–192. 26. Stanley SL, Zhang T, Rubin D, Li E. Role of Entamoeba histolytica proteinase in amebic liver abscess formation in severe combined immunodeficient mice. Infect Immun 1995;63:1587–1590. 27. Houpt ER, Glembocki DJ, Obrig TG, Moskaluk CA, Lockhart LA, Wright RL, Seaner RM, Keepers TR, Wilkins TD, Petri WA Jr. The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD41 T cells. J Immunol 2002;169:4496–4503. 28. Asgharpour A, Gilchrist C, Baba D, Hamano S, Houpt E. Resistance to intestinal Entamoeba histolytica infection is conferred by innate immunity and Gr-11 cells. Infect Immun 2005;73:4522–4529. 29. Reinertson JW, Thompson PE. Experimental amebic hepatitis in hamsters. Proc Soc Exp Biol Med 1951;76:518–520. 30. Tanimoto M, Sepu´lveda B, Va´zquez-Saavedra JA, Landa L. Lesiones producidas en el hı´gado del ha´mster por inoculacio´n de Entamoeba histolytica cultivada en medio axe´nico. Arch Invest Med (Mex) 1971;2: 275–284. 31. Tsutsumi V, Mena-Lo´pez R, Anaya-Vela´zquez F, Martı´nez-Palomo A. Cellular bases of experimental amebic liver abscess formation. Am J Pathol 1984;117:81–91. 32. Tsutsumi V, Martı´nez-Palomo A. Inflammatory reaction in experimental hepatic amebiasis. An ultrastructural study. Am J Pathol 1988;130: 112–119. 33. Ventura-Jua´rez J, Campos-Rodrı´guez R, Tsutsumi V. Early interactions of Entamoeba histolytica trophozoites with parenchymal and
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REVIEW ARTICLE
Experimental Amebiasis: A Selected Review of Some In Vivo Models Vı´ctor Tsutsumi and Mineko Shibayama Departamento de Patologı´a Experimental, CINVESTAV-IPN, Me´xico, D.F., Me´xico Received for publication September 22, 2005; accepted September 23, 2005 (ARCMED-D-05-00381).
The use of in vivo animal models in amebiasis has contributed significantly to the knowledge of this common human parasitic disease. Although there is no animal model that mimics the whole cycle of the human disease, the use of different susceptible and resistant laboratory animals and the availability for many years of techniques for the axenic culture of trophozoites of Entamoeba histolytica have allowed a better understanding of the parasite and the host–parasite relationship. The recent introduction of frontier methodologies in biology has increased our comprehension of this parasite. New information on the cellular and molecular biology and genetics of this organism has been extensively reported, and much of this has clearly required the more frequent use of animal models to verify specific facts. Based on experimental animals characterized previously, the introduction of new animal models with genetic or surgical modifications, especially in mice, has allowed a more adequate analysis of the mechanisms of pathogenesis. Multiple factors have been considered in the promotion of the invasiveness and virulence of E. histolytica. Additionally, the immunological and physiological responses of the host, depending on the environmental conditions, lead to the establishment or the rejection of the parasite. The role of inflammatory reaction to amebic infection constitutes one of the controversies that has been studied by several authors. In susceptible animals (hamsters and gerbils), inflammatory cell damage seems to be related to target cell lysis, while in resistant animals (mice), inflammatory cells appear to protect the host by lysing the parasite. Presently, the involvement of various substances in the development of lesions including lectins, proteases, amebapores, promoters of apoptosis, cytokines, nitric oxide, etc., is being examined using different in vivo models. Ó 2006 IMSS. Published by Elsevier Inc. Key Words: Amebiasis, Entamoeba histolytica, Animal models, Pathology.
Introduction Studies on the interaction of Entamoeba histolytica with different biological substrates have been aimed mainly at determining the pathogenic mechanisms participating in the production of amebic lesions. A better understanding of tissue destruction using animal models that reproduce intestinal or extraintestinal lesions similar to those seen in humans may contribute significantly in providing the basis for a more adequate treatment and prevention of the amebic disease.
Address reprint requests to: Vı´ctor Tsutsumi, Departamento de Patologı´a Experimental, CINVESTAV-IPN, Av. IPN No. 2508, CP. 07360, Me´xico, D.F., Me´xico; E-mail: [email protected]
Although the destructive or lytic capacity of amebas can be easily observed by light microscopy in all affected tissues, justifying the term histolytica, intrinsic mechanisms of the ameba and the host that play a role in infection are multifactorial and therefore constitute phenomena much more complex than a single in vitro host–parasite interaction. The commonly mentioned importance of the introduction of E. histolytica trophozoites cultured under axenic conditions for research in amebiasis (1) is evident from the large amount of knowledge that has been obtained regarding this etiologic agent. Most of the information on the physiopathology, fine structure, cell biology, biochemistry, immunology, genetics and molecular biology of E. histolytica has been acquired in the last 35 years using this technique of axenic culturing. Surprisingly, the great
0188-4409/06 $–see front matter. Copyright Ó 2006 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2005.09.011
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majority of these works has been achieved by using the same HM1-IMSS strain of trophozoites that was isolated close to 40 years ago. It should be emphasized that the virulence of this strain, which was originally very high, has been declining every year, requiring more frequent passages of amebas through the hamster liver. Especially when we consider virulence to be an important factor in experiments with E. histolytica, it should be pointed out that HM1-IMSS strain, which is one of the strains used in our laboratory and many others, cannot be regarded as the same strain isolated many years ago, at least with respect to virulence. Experimental amebiasis using in vivo models began when Lesch produced intestinal lesions in one of the dogs inoculated with feces from a patient suffering from dysentery (2). Subsequently, numerous studies have been performed using dogs, cats, rabbits and monkeys; however, their use is limited at present due to several factors including lack of uniformity, difficulty in handling and insufficient number of animals. Since the middle of the last century, easy access to many species of rodents for laboratory research, along with uniform conditions and acceptable susceptibility, has helped to increase our knowledge of the pathogenicity of amebiasis. These rodent models have also been used in many practical applications, including therapeutic and immunoprophylactic studies. However, an animal model that can reproduce the whole life cycle of E. histolytica as seen in humans is still not available. Thus, to date, the production of intestinal and extraintestinal amebic lesions in animals fed with E. histolytica cysts is not yet possible. Therefore, most of the in vivo studies of experimental amebiasis have been performed either with intestinal or hepatic models as separate entities, which have been produced by trophozoites injected directly into the target organ. Since relatively extensive reviews on different animal models in amebiasis have already been published (3–5), we will describe here briefly some previous basic works and then focus on recent data related mainly to the pathophysiology or mechanisms of amebic lesion production using intestinal and hepatic animal models.
Experimental Intestinal Amebiasis The production of typical intestinal amebic lesions in laboratory animals has been more complex and difficult than that of hepatic lesions. Many of the previous studies performed in different animals such as rats, guinea pigs and mice have yielded inconsistent and poor results, and in many cases this has been explained simply as due to a natural immune resistance of rodents to intestinal inoculation. Moreover, despite that experimental animals are inoculated with several million trophozoites of a known specific virulent strain, this inoculum mixes with a poorly characterized
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intestinal flora consisting of other protozoans, several strains of bacteria and other microorganisms as well. This mixture of intestinal organisms plus our lack of knowledge of the physicochemical characteristics of the intestinal milieu are important factors that have not been considered in the past and are likely responsible in great part for the heterogeneity of results. Differences in susceptibility have been reported by different groups, even when using similar laboratory animals under apparently identical conditions (4). In spite of these limitations, we believe that there are some useful laboratory animals for intestinal amebiasis, the choice depending on the specific objective sought. Therefore, interesting information regarding the early stages of intestinal infection, pathology and host immune response have been obtained, and much of the current knowledge about different aspects of the disease has come from the integration of data obtained using particular animal models. The current availability of various strains of rodents with different susceptibilities, especially in mice, has provided more information on the mechanisms of pathogenicity for intestinal amebiasis, although there are still many undetermined factors that are currently under study. At the molecular level, various research groups have been studying the genes or gene products of E. histolytica related to those of humans, through the evaluation of SCID mice (6) and other SCID modified mice (7), microarray analysis, and examination of several aspects of the mechanism of the immune response (8–11). Guinea pigs have also been used to study intestinal amebiasis, but there have been only a few reports concerning the successful production of amebic ulcers using amebas from axenic cultures. Diamond et al. (12) induced lesions in newborn guinea pigs inoculated intracecally; however, these results could not be reproduced consistently in a larger number of animals. Anaya et al. (13) described typical intestinal ulcers in guinea pigs and hamsters by producing an artificial cecal loop inoculated with axenic or monoxenic E. histolytica trophozoites (Figure 1). With this model, the authors showed the early stages of ameba invasion of the mucosal and the submucosal layers with a substantial inflammatory reaction (Figure 2). Electron microscopy analysis of the ulcer showed the possible role of leukocytes in the development of the amebic ulcer (Figure 3). Rats have been used in experimental amebiasis for many years; however, intestinal infections have been unclear. Jones (14) re-evaluated the use of rats in amebiasis studies and showed that weaning rats are more susceptible to amebic intestinal infection. This finding is interesting because it is known that rats rapidly develop resistance to infection when they begin to eat normal rat chow. A description of intestinal amebic lesions in this rodent was reported by Neal (15) who proposed the use of the Wistar strain and a scoring system of the amebic damage based on the macroscopic appearance of the intestinal wall and the
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Figure 1. Early intestinal lesions produced in the cecal loop model of guinea pig. At 48 h post-inoculation of axenic E. histolytica trophozoites, small whitish round lesions (arrows) surrounded by erythematous areas and congested blood vessels are shown.
characteristics of the luminal content. Although some authors have used rats for studying some aspects of intestinal amebiasis (effect of drugs, role of cholesterol in the ameba virulence and effect of different isolates), two unfavorable
conditions have to be considered when the study of pathogenesis is of interest: a) the use of weaning rats, although very susceptible to infection, have poorly developed immune systems and b) the requirement of continual bacterial association in the ameba inoculum can mask the real phenomena occurring during ulcer formation. The use of gerbils for experimental amebiasis was introduced by Diamond et al. (16). However, the application of this rodent in the study of intestinal amebiasis was not reported until 1985 by Chadee and Meerovitch (17) who analyzed the sequence of cecal infection from 1 to 10 days postinoculation. These latter authors noted that the trophozoites utilized were cultured under xenic or monoxenic conditions; however, there was no mention of inoculum size and the description of intestinal lesions was limited to erosion and edema of intestinal glands. Shibayama et al. (18), using axenic or monoxenic cultures, showed during the first hours of interaction an increase in mucus production mixed with trophozoites in the lumen. Microulcerative mucosal lesions appeared 24–72 h post-inoculation with inflammatory infiltrate and edema of the lamina propria associated with the necrotic ulcer. However, as in most ulcerative intestinal lesions observed in other rodents, lesions healed spontaneously after 96 h with complete absence of trophozoites. In the past, mice were not frequently used as models for amebiasis. The main reason was that these rodents were always considered as naturally resistant to E. histolytica infection because a substantial number of attempts to produce intestinal or hepatic lesions were practically unsuccessful. Further studies by Ghadirian and Kongshavn (19) using ten different strains of mice reported differences in
Figure 2. Light microscopy of an intestinal amebic ulcer produced in the previously mentioned cecal loop of guinea pig. A mucosal ulcer invading submucosa is composed by necrotic tissue, abundant trophozoites with fibrinous material and inflammatory infiltrate at the bottom of the ulcer. H&E stain. 370.
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Figure 3. Transmission electron microscopy of the necrotic zone of the amebic intestinal ulcer. Lesion was produced by using the cecal loop model in guinea pig. Various E. histolytica trophozoites (Eh) are surrounded by abundant lysed (Ly) vacuolar leukocytes. Some nuclear shadows of leukocytes are seen. Bar 5 10 mm.
susceptibility to the development of intestinal amebiasis after inoculation with trophozoites cultured axenically. The strains C3H/HeCr, BALB/c, NZB/B1N, B10.A, DBA/2 and C57Bl/6 were classified as susceptible models and the strains A/J, CE, DBA/1 and CD-1 were considered as resistant. The authors concluded that genetic factors may be involved in the differences of susceptibility to intestinal infection. In a similar study by Owen (20) using seven different strains of mice, a metastatic liver lesion was reported in the CBA/HN strain, whereas the athymic mouse of the same strain did not present liver invasion. The same author also reported intestinal lesions in C3H/HeJ strain with absence of liver metastasis, in contrast to a later report by Ghosh et al. (21). Among several factors involved in the intestine and hepatic invasion by E. histolytica trophozoites in rodents, Chauhan and Das (22) have mentioned a diet rich in cholesterol. Contrarily, factors associated with blocking the adhesion of and invasion by amebas are associated bacteria and the presence of other protozoans (23,24). Ghosh et al. (21) using C3H/HeJ mice reported the production of ulcerative inflammatory disease similar to that described in human amebiasis. In this study, E. histolytica trophozoites were inoculated intracecally and the animals sacrificed at different time-points, ranging from 5 to 30 days. On the 5th day there were tiny superficial erosions of the epithelium that progressed to deeper and more extensive lesions of the cecal wall. Flask-shaped ulcers and the presence of amebomas in the vicinity of ulcers were described in this model. Although ulcerative lesions were
found to be common and extensive, direct cell contact or tissue invasion by trophozoites was rarely observed. In addition to describing a relatively chronic intestinal lesion in this strain of mice, the authors suggested the possibility of distance damage to epithelial cells by toxic substances secreted by the amebas. Subsequently, an in vivo neutropenic model using BALB/c mice was reported (25). In this rodent, the role of neutrophils in the innate intestinal resistance to E. histolytica infection was studied. Animals treated with antineutrophil monoclonal antibody did not show any significant difference in the development of intestinal lesions when compared with control untreated mice. This work reports for the first time the production of granulomatous inflammatory reaction in the intestinal wall of mice when axenically cultured amebas were inoculated, and this type of inflammation was more common in treated animals. The importance of this chronic inflammatory reaction in an acute stage of amebic intestinal infection is still unknown. An interesting model of amebic colitis was developed in SCID mice when a fragment of human intestine was implanted into the subcapsular region (SCID-HU-INT) (7). This human intestinal xenograft was allowed to mature into a morphologically, mucin-secreting normal segment, which was then infected with E. histolytica trophozoites. Within 12 h of amebic inoculation into the lumen of this xenograft, the intestine developed focal ulcerations with invasion of the submucosa similar to that seen in humans with amebic colitis. Although there were differences between the model
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of human intestinal xenografts and the normal human intestine (e.g., the lack of normal flora, the absence of peristalsis and a closed loop), amebic infection in this model mimics a number of features observed in human amebic colitis, providing a system that allows the in vivo study of the interaction between E. histolytica and human intestine (8). In relation to the role of cysteine proteinase (ehcp5), the above authors suggested that this amebic enzyme produced damage of the xenografted human intestine that was consistent with invasion of mucosal tissue by trophozoites with a neutrophil-predominant inflammation (26). Further studies are required to clearly confirm the role of proteinases in the pathogenesis of amebic lesions. Experiments using C3H mice in the study of intestinal amebic infection were reported recently by Houpt et al. (27) who used C3H mice inoculated intracecally with E. histolytica trophozoites grown in a bacterial flora of the xenic strain CDC:0784. In this work, infection was followed for 18 months, and 60% of mice showed chronic cecal infection. Histopathologic changes were evident as early as 4 days after challenge, including crypt hyperplasia, epithelial ulceration, and submucosal infiltration. At that time, viable amebas were usually seen in areas of epithelial ulceration, and they were still seen in the lumen 3 weeks post-inoculation. Inflammation was severe and involved plasma cells, neutrophils, and mast cells. At 10 weeks, inflammation obscured the entire mucosa and morphologically resembled human colitis. Also, they determined the presence of different cytokines and a depletion of CD41 T cells. One of the interesting findings is that when CD41 cells were depleted, both parasite burden and inflammation diminished significantly correlating with a decrease in IL-4 and IL-13 production and loss of mast cell infiltration. This model revealed important immune factors that may influence susceptibility to infection and the physiopathology of intestinal amebiasis. Asgharpour et al. (28) used the same mice model and compared findings with those with C57BL/6 (a resistant mouse). They examined the role of neutrophils in the course of amebic colitis (C3H mouse) and found that neutrophil depletion using an anti-Gr-1 monoclonal antibody and dexamethasone treatment diminished the innate resistance in certain mouse strains (e.g., CBA). However, there was no effect on the high level of resistance of C57BL/6 mice, suggesting that the mechanisms of innate immunity to intestinal E. histolytica infection vary depending on the host genetic background.
bacteria, either directly into the hepatic lobe or through the portal vein, which produced large amebic hepatic abscesses. Further studies using the same rodent but inoculated with axenic amebas confirmed high susceptibility and the uniformity of lesions (30) (Figure 4). Other data were obtained related to the pathogenesis of amebic damage, including aspects on the host immune response and many other factors involved in in vivo host–parasite interactions. A sequential morphological study of hepatic infection from the very early stages of post-inoculation with E. histolytica trophozoites in hamsters reported 20 years ago suggested the role of host inflammatory cells in the process of tissue damage (31) (Figure 5). In this work, which was confirmed with electron microscopic (32) (Figure 6) and immunocytochemical (33) (Figure 7) studies, the authors suggested that E. histolytica trophozoites do not produce amebic liver abscesses through direct lysis of hepatocytes. It was proposed that tissue damage is mediated instead by lysosomal enzymes released from disintegrating inflammatory cells that accumulate around the amebas and are killed by this parasite. This original finding has been the basis for numerous subsequent studies investigating inflammation as an important mechanism of host tissue destruction. Gerbils are another rodent highly susceptible to hepatic amebiasis (Figure 8). Lesions can be obtained similar to those produced in hamsters, although E. histolytica trophozoites apparently show a less virulent behavior in gerbils than in hamsters. Thus, regardless of the size of inoculums, progression of liver damage is slower and the eventual death of the infected animals takes a longer time. These characteristics allow performing similar sequential studies
Experimental Hepatic Amebiasis The first laboratory animal used successfully for hepatic amebiasis was the hamster, which was reported by Reinertson and Thompson (29). These authors injected trophozoites of E. histolytica cultured with three species of
Figure 4. Amebic hepatic lesion in hamster after 7 days post-inoculation. Large irregular whitish lesions are seen on the liver surface.
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Figure 5. Hamster liver at 3 h post-intraportal inoculation of E. histolytica trophozoites. The small amebic liver lesion is formed by a trophozoite (Eh) surrounded by normal (PMN) and lysed (Ly) polymorphonuclear leukocytes. Hepatocytes (H). Semithin section stained with toluidine blue stain. 3290.
of hepatic lesions (18). In addition to the association of inflammatory cells with trophozoites as shown in hamster liver, gerbils also showed trophozoites in direct contact with hepatocytes. The preceding data, plus the possibility of producing amebic intestinal lesions, have led some investigators to consider this rodent as being more similar to humans.
Gerbil has been useful in various fields of research in amebiasis. Studies on host immune response and the effect of various vaccine candidates purified from the parasite and administered parenterally or orally have been tested (34). More recently, gerbils have been used in molecular biology study of various proteins related to E. histolytica virulence (35).
Figure 6. Electron micrograph of an early liver lesion in hamster at 6 h post-intraportal inoculation of amebas. An E. histolytica (Eh) trophozoite is seen contacting undamaged (PMN) and lysed (Ly) polymorphonuclear leukocytes. Bar 5 10 mm.
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Figure 7. Immunohistochemistry of amebic liver lesion at 6 h post-intraportal inoculation. Trophozoites (Eh), inflammatory cells and peripheral hepatocytes (arrows) are positives to peroxidase labeled anti-amebic antibody. Hematoxylin counterstained. 3125.
In relation to the use of mice in hepatic amebiasis, the introduction of genetically modified animals, such as severe combined immunodeficient (SCID) mice, has provided interesting data. Cieslak et al. (6) using this strain of mouse developed liver abscesses when animals were challenged
intrahepatically with virulent E. histolytica trophozoites. In this study, only one of seven similarly challenged immunocompetent congenic C.B.-17 mouse (control) developed an abscess. They also used a polyclonal anti-E. histolytica antibody and reported protection in 7/12 SCID mice. These
Figure 8. Light microscopy of gerbil liver at 48 h after intrahepatic inoculation of amebas. (a) A group of centrally localized trophozoites (arrows) surrounded by abundant inflammatory cells. H&E. 3200. (b) Histochemistry of liver lesion in gerbil at 48 h post-inoculations. Gomori-Grocott stain shows trophozoites in black (arrows). 3200.
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authors concluded that SCID mouse constitutes a powerful model for studying the possible components of protective immunity in invasive amebiasis. In order to determine whether inflammatory cells play a similar role in mice as observed in hamster liver, BALB/c mice were treated with anti-neutrophil RB6-8C5 monoclonal antibodies and then challenged intrahepatically with E. histolytica trophozoites. Animals were sacrificed at different time-points and hepatic analysis showed that neutrophil-depleted mice showed abscesses larger than in untreated control animals. Based on previous data, the authors suggested that neutrophils may play a role in resistance mechanisms in mice, unlike in hamster or gerbil. However, it is important to mention that mice are in general resistant to amebic infection. In both RB6-8C5-treated and -untreated mice challenged similarly, animals survived to infection, although the treated animals required a longer time to heal completely from the hepatic lesion (36). In another study, an immunocytochemical analysis of the diverse cell population was performed in hepatic lesions produced in BALB/c and C3H/Hej strain mice (37). The authors suggested that mouse resistance to the development of liver abscesses depends on the activation of neutrophils, and this in turn is related to abundant nitric oxide production to kill the ameba. In addition to differences in the possible mechanisms of resistance or susceptibility due to inflammatory cells, contradictory roles of nitric oxide have also been mentioned. Previous in vitro (38) and in vivo (9) studies have suggested that nitric oxide has a lytic effect on trophozoites of E. histolytica. Another study in hamsters has shown that this molecule plays no role in arresting the development of amebic liver abscesses. The levels of nitric oxide measured by the presence of nitrites and nitrates in serum were directly proportional to the size of abscesses, suggesting that nitric oxide does not have a lytic effect on E. histolytica and is therefore incapable of providing protection against the hepatic lesion (39). In relation to apoptosis in hepatic damage, it has been shown that target cell killing may be caused in part by the triggering of this process by E. histolytica. Vela´zquez et al. (36) found areas of TUNEL-positive dead hepatocytes in normal and neutrophil-depleted mice. These apoptotic liver cells were found either close to the inflammatory infiltrate or at some distance free of ameba or inflammatory cells. The presence of TUNEL-positive hepatocytes could be the result of one or more possible mechanisms of tissue damage. It is known that inflammatory conditions may cause apoptosis or that amebas may release apoptosisinducing factors that affect hepatocytes at a distance (10). Induction of an ischemic status in liver parenchyma by trophozoites occluding blood vessels can also be considered as another factor (40,41). Studying the murine model of amebic liver abscess, Seydel and Stanley (42) showed that hepatocyte apoptosis requires activation of caspase but is independent from Fas and tumor necrosis factor (TNF) a
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receptor 1. Amebapores have been considered as necessary for both lytic and apoptotic pathways of cell death. Moreover, it is suggested that sublytic concentrations of pore-forming proteins can induce apoptosis in target cells (10). Other important molecules related to the pathogenesis of amebiasis are the proteinases. Previous studies, including those involving xenografted mice, have suggested the role of proteinases in tissue damage (8,11,26). In a recent study by Olivos-Garcı´a et al. (43), the authors concluded that cysteine proteinase plays either a minor or no role in the damage of liver parenchyma during the development of amebic liver abscess. These authors purified an amebic cysteine proteinase (EhCP2) and bound it to inert resin microspheres (22–24 mm in diameter), which were then injected into the portal vein of normal hamsters. A mild acute inflammation and occasional minimal necrosis of short duration was seen. Immunohistochemistry with the anti-EhCP2 antibody showed the presence of positive-labeled microspheres in tissues at 1 h after injection. Sections of experimental acute amebic liver abscesses presented strong labeling of trophozoites, although necrotic tissue and inflammatory infiltrates were negative, supporting the above notion. Another more recent study from the same group (44) reported that cysteine proteinase activity in trophozoites is required for survival of the trophozoites in experimental acute amebiasis in hamsters, whether or not this enzyme plays a role in the virulence of the parasite. Another recent study focused on the Gal/GalNAc lectin of E. histolytica trophozoites has corroborated morphologically the in vitro and in vivo adhesion roles of this lectin. Pacheco et al. (45) performed immunocytochemical studies using various target cells that included cultured human and hamster hepatocytes, hamster amebic liver abscesses and C3H mouse intestinal lesions. Lectin immunolabeling was positive in trophozoites, inflammatory cells and necrotic tissue. Damaged intestinal epithelium in the mouse model also showed labeling with this anti-lectin antibody. The authors concluded that Gal/GalNAc lectin is bound and captured by various target cells and these host cells containing the lectin showed different degrees of cell damage. However, the absence of lectin labeling in some damaged ameba-interacting cells suggests that the mechanisms of pathogenesis are multiple and other factors are also probably involved in cytotoxicity. Morphological changes produced by E. dispar trophozoites on hamster liver have been partially studied. Although early inflammatory foci around axenically cultured amebas were observed in the liver (Figure 9), including inflammatory cell damage (46) (Figure 10), lesions did not progress to abscess, and only small areas of inflammatory infiltrates were observed after 24 h. In another study using different strains of E. dispar cultured in xenic or monoxenic conditions, authors reported either absence or presence of hepatic lesions in hamsters, depending on the origin of
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Figure 9. Light microscopy of hamster liver inoculated with E. dispar (Ed) trophozoites. At 6 h post-intraportal inoculation of amebas, abundant inflammatory cells are seen associated with amebas. H&E stain. 3125.
E. dispar isolate. The effect of bacteria in the virulence of these ameba isolates was also mentioned and emphasized by the authors (47). Conclusions E. histolytica infects only humans and non-human primates; therefore, success in establishing reproducible animal
models of amebiasis has been limited. Nevertheless, knowledge obtained from different animal models used in the study of intestinal or hepatic amebiasis has allowed for a better understanding of the complex mechanisms operating in the target cell damage. As mentioned, we still do not have an animal model that mimics the complete cycle of E. histolytica as it occurs in the human disease; however, many studies conducted with various specific, normal, or
Figure 10. Transmission electron micrograph of hamster liver inoculated with E. dispar (Ed) trophozoites. After 6 h of intraportal inoculation, inflammatory focus shows normal polymorphonuclear (PMN) and lysed (Ly) leukocytes surrounding the ameba. Bar 5 10 mm.
In Vivo Models in Amebiasis
genetically or surgically altered laboratory animals have contributed to the better understanding of the pathogenesis and immunology of the amebic lesion. More recently, since the introduction of diverse strains of mice in research, our knowledge of the molecular pathology of amebiasis has increased. However, it is important to mention that in many cases intestinal models for amebic infections use artificial methodologies and in others, animals are greatly manipulated, introducing several different variables of the in vivo system unrelated to the human disease. A number of in vivo studies have suggested the role of the host bacterial flora in the pathogenesis of invasive amebiasis. Intestinal bacteria, motility and intestinal flow may all contribute to the protective role in amebic intestinal infection. Experimental studies related to the production of extraintestinal lesions have focused mainly on amebic liver abscess. Basically, there are two susceptible rodents, hamster and gerbil, and both have been used in multiple studies. These investigations involve the in vivo study of immunopathogenesis, pharmacology and effects of antiamebic drugs and studies on prophylaxis and vaccine testing. The role of inflammatory cells in hepatic damage in hamsters still constitutes an important controversial and interesting factor that is under investigation by several research groups. The special environment of the ameba in the liver and the complex interactions between parasite and host components have provided a better understanding of the role of some parasite products such as proteases, different lectins (170, 112, and 220 kDa) and several other components involved in the pathogenesis of experimental amebic liver. Mechanisms of hepatic damage related to ischemia produced by blood vessel occlusion by amebas cannot be discounted. Some liver lesions in either susceptible (hamster) or resistant animals (mouse) have shown histological characteristics of ischemic damage. The possible activation of the apoptotic process in hepatocytes by this ischemia or other factors has also been considered in different in vivo experimental models. Recent studies using the hepatic model in gerbils have provided some information on the virulence of E. histolytica. A molecular study has suggested that the survival of amebas within the hostile environment of the liver, and in particular during the development of liver abscess, is accomplished by a strong adaptive process requiring the specific regulation of a number of amebic proteins. Thus, this regulatory process seems to be an important factor for the general understanding of the mechanisms of E. histolytica pathogenesis. In reference to the ‘‘resistant animal models’’, their use has also provided valuable information about the cell types and the possible mechanisms that are involved in the natural immune response against E. histolytica infection. Rats, guinea pigs and different strains of mice have been useful in studies related to amebic resistance to intestinal or hepatic infection. Several studies have shown that
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polymorphonuclear cells, mainly neutrophils, are implicated in such resistance. Molecules such as nitric oxide complement proteins, and proinflammatory cytokines have been shown to be involved in the process of amebic rejection. Physical barriers such as epithelial cells and the secreted mucus are important blockers of the adhesion of E. histolytica trophozoites. Therefore, the mechanisms of innate protection include combinations of the host genetics, environmental factors such as intestinal bacterial flora in the intestine, and parasite intrinsic factors.
Acknowledgments We thank Silvia Galindo-Go´mez, Ange´lica Silva-Olivares and Mireya Sa´nchez-Palomera for their valuable technical assistance. Our gratitude to Dr. Ruy Pe´rez-Tamayo for his critical reading and helpful comments of the manuscript.
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