Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis. Received: 10 August 2000 / Accepted: 5 September 2000     

Entamoeba histolytica Induces Host Cell Death in Amebic Liver Abscess by a Non-Fas-Dependent, Non-Tumor Necrosis Factor Alpha-Dependent Pathway of Apoptosis

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999     

Entamoeba histolytica : production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N ^G -nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators. Received: 16 February 2000 / Accepted: 19 June 2000     

Trophozoites of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> express a CD59-like molecule in human colon

In vitro studies have proved the presence of epitopes of CD59 in the surface of trophozoites of Entamoeba histolytica (E. histolytica). However, it has not been proved if CD59 molecules are expressed in the surface during the trophozoites’ tissue invasion. The aim of the present study was to determine whether the complement-regulatory protein CD59 is present on trophozoites of E. histolytica in human colon. Eleven specimens of amoebic colitis were studied by immunohistochemistry and electron microscopy techniques with a monoclonal antibody against human CD59 molecule. Our results show that a CD59-like molecule is expressed in trophozoites of E. histolytica found in colonic amebic lesions. Also, a CD59-like molecule was detected by western blot analysis in whole lysate of E. histolytica as well as on the plasma membrane by immunocytochemistry. These results suggest that E. histolytica can use CD59-like protein against the lytic action of membrane attack complex.     

Effect of zinc on Entamoeba histolytica pathogenicity

The present study analyzes the effects of zinc on Entamoeba histolytica activity and on its pathogenicity. Metal activity was evaluated in vitro with regard to the parasite's viability, replication, and adhesion to epithelial cells and in vivo with regard to its pathogenicity. The results obtained in vitro show that zinc at 1.0 mM concentration does not affect amebic viability; however, it does decrease amebic replication and adhesion (P < 0.001). In vivo studies performed on a model of experimental liver abscess in the hamster indicate that the intraperitoneal administration of a single dose of zinc at 48 h after the intrahepatic inoculation of amebic trophozoites significantly inhibits (P < 0.001) abscess development. The results indicate that zinc alters the functionality of the ameba in vitro as reflected by a decrease in replication and adhesion and in vivo as manifested by inhibition of amebic pathogenicity. Received: 15 November 1998 / Accepted: 16 December 1998     

Immunization with the Entamoeba histolytica Surface Metalloprotease EhMSP-1 Protects Hamsters from Amebic Liver Abscess

Diarrhea and amebic liver abscesses due to invasive Entamoeba histolytica infections are an important cause of morbidity and mortality in the developing world. Entamoeba histolytica adherence and cell migration, two phenotypes linked to virulence, are both aberrant in trophozoites deficient in the metallosurface protease EhMSP-1, which is a homologue of the Leishmania vaccine candidate leishmanolysin (GP63). We examined the potential of EhMSP-1 for use as a vaccine antigen to protect against amebic liver abscesses. First, existing serum samples from South Africans naturally infected with E. histolytica were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of EhMSP-1-specific IgG. Nine of 12 (75%) people with anti-E. histolytica IgG also had EhMSP-1-specific IgG antibodies. We next used a hamster model of amebic liver abscess to determine the effect of immunization with a mixture of four recombinant EhMSP-1 protein fragments. EhMSP-1 immunization stimulated a robust IgG antibody response. Furthermore, EhMSP-1 immunization of hamsters reduced development of severe amebic liver abscesses following intrahepatic injection of E. histolytica by a combined rate of 68% in two independent animal experiments. Purified IgG from immunized compared to control animals bound to the surface of E. histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. We concluded that EhMSP-1 is a promising antigen that warrants further study to determine its full potential as a target for therapy and/or prevention of invasive amebiasis.     

Protection of hamsters from amebic liver abscess formation by a monoclonal antibody to a 150-kDa surface lectin of Entamoeba histolytica

We examined the effects of passive immunization with a monoclonal antibody (EH3015) that recognizes a 150-kDa surface lectin of Entamoeba histolytica on amebic liver-abscess formation in hamsters. The hamsters were inoculated i.p with 0.1, 1.0, or 10 mg of EH3015 at 24 h prior to an intrahepatic challenge with 10⁵ trophozoites of E. histolytica. In hamsters treated with 1.0 and 10 mg of EH3015 the incidence of liver abscesses was significantly reduced. These results demonstrate that monoclonal antibody EH3015 can prevent the development of amebic liver abscesses and that the 150-kDa lectin may be a protective antigen on the surface of E. histolytica. Received: 5 May 1998 / Accepted: 28 May 1998     

Amebic liver abscess in a european patient: Zymodeme classification ofentamoeba histolytica

Summary This is a case report of a 36-year-old patient who developed an amebic liver abscess after a stay in the Sudan. He was first misdiagnosed as having pneumonia of the right lower lobe. Following establishment of the correct diagnosis, the patient recovered fully after metronidazole treatment. The fecal culture in Robinson's medium yielded extensive growth ofEntamoeba histolytica. Electrophoretic characterization proved it to be a zymodeme XIX, which is one of the zymodemes associated with pathogenicity in the host. This first report of a zymodeme classification ofE. histolytica in Germany should initiate further epidemiological studies.Abkürzungsverzeichnis E. histolytica Entamoeba histolytica - GPI glucose phosphate isomerase - ME malic enzyme - PGM phosphoglucomutase - HK hexokinase - MIFC Merthiolate-iodine-formaldehyde concentration technique     

Cloning and characterization of Entamoeba histolytica antigens recognized by human secretory IgA antibodies

To identify the Entamoeba histolytica antigens capable of inducing secretory IgA (sIgA) responses in humans, a cDNA library from the strain HM1:IMSS was immuno-screened with saliva from patients with intestinal amebiasis or amebic liver abscess. Clones isolated with sIgA antibodies from patients with intestinal amebiasis corresponded to the known serine-rich protein isoform, a 29 kDa cysteine-rich protein and 1-α elongation factor. Clones corresponding to enolase, cyclophilin, ribosomal protein L23a, and an Hsp70 family protein were isolated with sIgA from a patient with amebic liver abscess. A glutamic acid-rich peptide (EhGARP) positive with sIgA from a patient with amebic liver abscess was also isolated; for EhGARP, no homologs were found in the protein databases. The antigens isolated are potentially useful in the development of an oral vaccine or new diagnostic tools for amebiasis. Received: 14 June 1999 / Accepted: 5 October 1999     

Nitric oxide production and nitric oxide synthase immunoreactivity in <Emphasis Type="Italic">Naegleria fowleri</Emphasis>

Free-living ameba Naegleria fowleri produces an acute and fatal infectious disease called primary amebic meningoencephalitis (PAM), whose pathophysiological mechanism is largely unknown. The aim of this study was to investigate the role of nitric oxide (NO) in PAM. Although NO has a cytotoxic effect on various parasites, it is produced by others as part of the pathology, as is the case with Entamoeba histolytica. To test for the production of NO, we analyzed whether antibodies against mammalian NO synthase isoforms (neuronal, inducible, and endothelial) presented immunoreactivity to N. fowleri proteins. We found that the trophozoites produced NO in vitro. The Western blot results, which showed N. fowleri trophozoites, contained proteins that share epitopes with the three described mammalian NOS, but have relative molecular weights different than those described in the literature, suggesting that N. fowleri may contain undescribed NOS isoforms. Moreover, we found that trophozoites reacted to the NOS2 antibody, in amebic cultures as well as in the mouse brain infected with N. fowleri, suggesting that nitric oxide may participate in the pathogenesis of PAM. Further research aimed at determining whether N. fowleri contains active novel NOS isoforms could lead to the design of new therapies against this parasite.     

Human amebic liver abscess: expression of intercellular adhesion molecules 1 and 2 and of von Willebrand factor in endothelial cells

Liver invasion by amebas with production of amebic liver abscess (ALA) is the most common extraintestinal lesion produced by the protozoan parasite Entamoeba histolytica. This hepatic damage is characterized by the presence of extensive tissue necrosis. However, little is known about the parasite and host factors involved in the process of tissue damage. During the early establishment of amebas in the liver parenchyma as well as during the extension of the tissue necrosis, parasites interact with sinusoidal endothelial cells. As a consequence of ameba-endothelial cell interactions, the latter can be activated and express proinflammatory factors that could be related to tissue destruction. We studied by immunohistochemistry the localization of antigenic molecules of E. histolytica trophozoites and of molecules such as intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and von willebrand factor in activated endothelial cells of human ALA, which could be related to the pathophysiological mechanisms of tissue destruction in amebiasis. Received: 11 November 1996 / Accepted: 18 December 1996     

Host-pathogen interaction in amebiasis and progress in vaccine development

Entamoeba histolytica, the causative organism of invasive intestinal and extraintestinal amebiasis, infects approximately 50 million people each year, causing an estimated 40 to 100 thousand deaths annually. Because amebae only infect humans and some higher non-human primates, an anti-amebic vaccine could theoretically eradicate the organism. Uncontrolled epidemiologic studies indicate that acquired immunity to amebic infection probably occurs and that such a vaccine might be feasible. Application of molecular biologic techniques has led to rapid progress towards understanding howEntamoeba histolytica causes disease, and to the identification of several amebic proteins associated with virulence. These proteins are now being evaluated as potential vaccine components. Parenteral and oral vaccine preparations containing recombinant amebic proteins have been effective in preventing disease in a gerbil model of amebic liver abscess. Although systemic and mucosal cellular and humoral immunity both appear to play a role in protection againstEntamoeba histolytica, the relative importance of each in the human immune response remains unknown. No animal model of intestinal amebiasis currently exists, moreover, so it has been impossible to evaluate protection against colonization and colitis. Further investigation of the fundamental mechanisms by whichEntamoeba histolytica causes disease and of the human immune response to amebic infection is necessary to assess the true feasibility of an anti-amebic vaccine.     

Sensitivity and specificity of a new commercial enzyme-linked immunoassay kit for detecting Entamoeba histolyticaIgG antibodies in serum samples

The study presented here was conducted to evaluate the performance of the newly available RIDASCREEN Set (R-Biopharm AG, Darmstadt, Germany) for the detection of immunoglobulin G antibodies against Entamoeba histolytica. The sensitivity and specificity of this new enzyme-linked immunosorbent assay were evaluated using a panel of sera from 239 individuals. The assay was positive for 43 of 44 patients with invasive amebiasis, including all 18 patients with amebic liver abscess, while it was negative for 190 of 195 adult controls who were either healthy individuals or patients with other parasitic diseases. The kit was found to be highly specific (97.4%) and sensitive (97.7%) for detecting antibodies against E. histolytica in humans. Although antibody titers in patients with amebic liver abscess tend to be higher on average than in patients with invasive amebiasis, it is not possible to distinguish the two forms solely based on the results of this commercial test.     

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced byEntamoeba histolytica

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 ofEntamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (<1000 leukocytes/mm³), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains ofE. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulentE. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, andE. invadens) was generally proportional to their proteinase activity; however,E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential ofEntamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.     

Diagnosis of Amebic Liver Abscess and Amebic Colitis by Detection of Entamoeba histolytica DNA in Blood,Urine, and Saliva by a Real-Time PCR Assay

The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.Entamoeba histolytica is a protozoan parasite that causes amebic diarrhea, colitis, and amebic liver abscess (ALA), mostly in developing countries (5, 7, 22, 25). Eighty percent of infected individuals remain asymptomatic carriers, while the other 20% develop clinically overt disease (7, 9, 22, 25). About 50 million symptomatic cases of amebiasis occur worldwide each year, resulting in 40,000 to 100,000 deaths annually (25). Mortality from amebiasis is mainly due to extra-amebic colitis, of which ALA is the most common.It is difficult to differentiate ALA from pyogenic liver abscess or other space-occupying lesions of the liver. Imaging techniques such as ultrasound, computed tomography, and magnetic resonance have excellent sensitivities for the detection of liver abscess arising from any cause, but there are no findings specific for ALA (13). Further complicating the diagnosis is the fact that most patients with an ALA do not have coexistent intestinal infection with E. histolytica (11). Therefore, detection of E. histolytica antigen or DNA in stool samples is not very helpful for the diagnosis of ALA (1, 6, 8, 12).The current means for diagnosis of ALA is the detection of antiamebic antibody by serological tests combined with aspiration of the abscess. The presence of serum antibodies against E. histolytica and the absence of bacteria in the abscess fluid are consistent with an ALA. A drawback of serologic tests is that the serum antibody levels in people from areas of endemicity remain positive for years after infection with E. histolytica (3, 16, 23). Therefore, antiamebic antibodies in the serum may be due to amebiasis in the past, limiting their specificity for the diagnosis of ALA. A further limitation to the current approach to ALA diagnosis is that collection of liver abscess pus is an invasive procedure that requires technical expertise and can be done only in specialized hospitals.Several groups have reported the detection of E. histolytica DNA in liver abscess pus, stool, and other clinical samples by PCR (14, 15, 18, 19, 21, 24, 26). A real-time PCR assay has also been used for detection of E. histolytica DNA in stool and liver abscess pus specimens (2, 10, 20). Real-time PCR has never been used for detection of E. histolytica DNA in urine, saliva, and blood specimens of ALA patients. In this study, we evaluated a real-time PCR assay to detect E. histolytica DNA in blood, urine, and saliva samples of amebic liver abscess and colitis patients in Bangladesh.     

Experience with aspiration in cases of amebic liver abscess in an endemic area

The study presented here was performed to evaluate the need for aspiration in patients with amebic liver abscess (ALA). Patients older than 12 years with a diagnosis of ALA based on clinical features, ultrasound results, and positive amebic serology were included in the study (n=144). Serological testing was performed to detect the presence of immunoglobin G antibody against Entamoeba histolytica, and a value of more than 0.4 optical density units was considered positive. All patients were given intravenous metronidazole (500 mg every 8 h) and their clinical progress and need for abscess aspiration was documented. Fever, pain in the upper abdomen, and tender hepatomegaly was seen in 133 (92.3%), 128 (88.8%), and 144 (100%) patients, respectively. Multiple abscesses were seen in 40 (27.7%) patients. Six (4.1%) patients died. Seventy-one (49.3%) patients responded to metronidazole alone. A total of 73 (50.69%) patients required aspiration of the abscess. This study shows that almost 50% of the patients with amebic liver abscess failed to respond to metronidazole and required aspiration.     

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica

We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2–5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose-specific adhesin(s) are essential for amebic cytotoxicity. Received: 8 June 1999 / Accepted: 21 July 1999     

Interaction between trophozoites ofEntamoeba histolytica and the human intestinal cell line HT-29 in the presence or absence of leukocytes

Studies on the interaction between trophozoites ofEntamoeba histolytica of pathogenic or nonpathogenic origin and epithelial cells of the human intestine can contribute to the understanding of the pathogenesis of invasive amoebiasis. We have examined the interaction of virulentE. histolytica with the human colonic carcinoma cell line HT-29. Differentiated HT-29 cells are comparable to the mucosa cells to whichE. histolytica attaches physiologically. Adherence betweenE. histolytica trophozoites and HT-29 cells was effectively inhibited by glycoconjugates containing galactose, indicating the importance of the 170-kDa lectin ofE. histolytica in binding to intestinal cells. Adherence was not significantly inhibited by glycoconjugates containingN-acetyl-glucosamine, indicating that the 220-kDa lectin ofE. histolytica is not involved in binding to HT-29 cells. The destruction of HT-29 cells by pathogenicE. histolytica was dependent on adherence. The destruction was enhanced when polymorphonuclear granulocytes were added to theE. histolytica trophozoites.     

Expression of amoebapores is required for full expression of Entamoeba histolytica virulence in amebic liver abscess but is not necessary for the induction of inflammation or tissue damage in amebic colitis

Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(-) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.