Neutrophil extracellular traps and MPO in models of susceptibility and resistance against Entamoeba histolytica.

The main effector mechanisms of neutrophils are the release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO). In this work, we evaluated the role of NETs and the activity of MPO in the interactions of rodent neutrophils with amoebae and in amoebic liver abscess (ALA)-resistant and ALA-susceptible models. We showed with in vitro assays that mice produced greater amounts of NETs and MPO than did hamsters, and the elastase activity was high in both models. However, the inhibition of NETs and MPO promoted an increase in amoeba viability in the mice. The mouse ALAs showed a more profound presence of NETs and MPO than did the hamster ALAs. We concluded that both effector mechanisms were essential for the amoebic damage and could prevent the formation of ALAs in the resistant model.

Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors.

Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemagglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis.

Modulation of innate immune response by the vagus nerve in experimental hepatic amebiasis in rats.

The parasympathetic nervous system has a crucial role in immunomodulation of the vagus nerve, its structure provides a pathogen detection system, and a negative feedback to the immune system after the pathogenic agent has been eliminated. Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica, considered the third leading cause of death in the world. The rats are used as a natural resistance model to amoebic liver infection. The aim of this study is to analyze the interaction of Entamoeba histolytica with neutrophils, macrophages, and NK cells in livers of intact and vagotomized rats. Six groups were studied (n = 4): Intact (I), Intact + amoeba (IA), Sham (S), Sham + amoeba (SA), Vagotomized (V) and Vagotomized + amoeba (VA). Animals were sacrificed at 8 h post-inoculation of E. histolytica. Then, livers were obtained and fixed in 4% paraformaldehyde. Tissue liver slides were stained with H-E, PAS and Masson. The best development time for E. histolytica infection was at 8 h. Amoeba was identified with a monoclonal anti-220 kDa E. histolytica lectin. Neutrophils (N) were identified with rabbit anti-human neutrophil myeloperoxidase, macrophages (Mɸ) with anti-CD68 antibody and NK cells (NK) with anti-NK. Stomachs weight and liver glycogen were higher in V. Collagen increased in VA, whereas vascular and neutrophilic areas were decreased. There were fewer N, Mɸ, NK around the amoeba in the following order IA > SA > VA (p < 0.05 between IA and VA). In conclusion, these results suggest that the absence of parasympathetic innervation affects the participation of neutrophils, macrophages and NK cells in the innate immune response, apparently by parasympathetic inhibition on the cellular functions and probably for participation in sympathetic activity.

A study on HLA-DR/DQ typing in adult Malay patients with acute amoebic liver abscess.

INTRODUCTION: Amoebiasis is a parasitic disease caused by Entamoeba histolytica that may lead to death in developing countries. Few important risk factors have been identified in the development of amoebic liver abscess (ALA). There are limited reports that suggest an association between antigens of the major histocompatibility complex (MHC) particularly class II antigens and ALA development. This present work aimed at studying the possible association of HLA antigens with ALA and disease severity. Results of the study may serve as a guide for further immunological studies dealing with E. histolytica. METHODS: This preliminary study involved two groups of subjects: 20 ALA patients in the experimental group and 40 healthy individuals in the control group. Cases were selected from adult Malay patients confirmed with ALA based on clinical signs and symptoms, radiological findings, microbiological findings and who were admitted to the medical or surgical ward, Hospital USM, Kelantan. Venous blood was obtained from each patient and HLA typing was then conducted using polymerase chain reaction specific primer sequence. RESULTS: HLA DR12 was most frequently found in the healthy control and ALA groups at 40% and 55% respectively. HLA DQ7 and DQ8 were found to have the highest percentage in the ALA group at 65%. In the control group, HLA DQ8 (57.5%) had the highest percentage. CONCLUSION: HLA antigens play a role in acquisition of ALA and provide understanding of the disease outcome.

TNFα-mediated liver destruction by Kupffer cells and Ly6Chi monocytes during Entamoeba histolytica infection.

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.

Immune markers characteristic for asymptomatically infected and diseased Entamoeba histolytica individuals and their relation to sex.

BACKGROUND: The protozoan parasite Entamoeba histolytica (E. histolytica) usually asymptomatically colonizes the human intestine. In the minority of the cases, the parasite evades from the gut and can induce severe symptoms like colitis or amebic liver abscess (ALA). Interestingly, ALA predominates in adult men despite a higher prevalence of the parasite in women. The present study aimed to identify characteristic serum markers in a unique cohort of clearly defined asymptomatically infected E. histolytica individuals in comparison to patients with an E. histolytica liver manifestation of both sex. METHODS: The following study groups were investigated: ALA patients (n = 38), healthy asymptomatic E. histolytica carriers (AC) (n = 44), and healthy E. dispar-infected controls (n = 24) out of an amebiasis endemic area. E. histolytica-specific immunoglobulin G (IgG) and the IgG subclasses against proteinaceous and non-proteinaceous amebic antigens were measured by ELISA. Serum cytokine and chemokine levels were investigated using a flow cytometry bead-based multiplex immunoassay. RESULTS: The IgG results revealed that not only ALA patients, but also AC, developed high E. histolytica-specific titers of IgG and all IgG subclasses as well as IgA. IgG and IgG2 titers against the glycolipid E. histolytica lipophosphoglycan were highest in ALA patients. As in ALA patients, high cytokine levels of interleukin (IL-) 4 were detected in AC compared to E. dispar infected individuals, while IL-6 was exclusively elevated in ALA patients. IL-10 was lower in AC compared to ALA patients. Equal serum levels of CCL2 were found in all study groups but ALA patients showed decreased levels of CCL3. Sex dependent analysis of the data indicated significantly higher IgG and IgG1 titers in female AC compared to male AC. CCL2, the chemokine involved in immunopathology in the mouse model for the disease, was higher in male AC compared to female AC. CONCLUSION: In this study we characterize for the first time an asymptomatic carrier stage in amebiasis that is associated with a significant immune reaction and provide immunological markers that might give first hints towards an understanding of immune mechanisms underlying the control or development of invasive amebiasis.

Evaluation of Entamoeba histolytica recombinant phosphoglucomutase protein for serodiagnosis of amoebic liver abscess.

BACKGROUND: Amoebic liver abscess (ALA) is the most frequent clinical presentation of extra-intestinal amoebiasis. The diagnosis of ALA is typically based on the developing clinical symptoms, characteristic changes on radiological imaging and serology. Numerous serological tests have been introduced for the diagnosis of ALA, either detecting circulating amoebic antigens or antibodies. However those tests show some pitfalls in their efficacy and/or the preparation of the tests are costly and tedious. The commercial IHA kit that used crude antigen was reported to be useful in diagnosis of ALA, however high antibody background in endemic areas may cause problems in its interpretation. Thus, discovery of well-defined antigen(s) is urgently needed to improve the weaknesses of current serodiagnostic tests. METHODS: Crude antigen of E. histolytica was analysed by 2-DE and Western blot to identify a protein of diagnostic potential for ALA. The corresponding gene of the antigenic protein was then cloned, expressed and the purified recombinant protein was subsequently evaluated for serodiagnosis of ALA in an indirect ELISA format. RESULTS: Analysis of crude antigen showed that phosphoglucomutase (PGM) has the diagnostic potential. Recombinant PGM (rPGM) showed 79.17% (19/24) sensitivity and 86.67% (195/225) specificity in diagnosis of ALA based on the COV of mean +1SD. There was no significant difference between rPGM-ELISA and IHA diagnostic kit in the diagnosis of ALA in terms of sensitivity and specificity at p-value < 0.05. CONCLUSION: In conclusion, rPGM-ELISA is found to be useful for serodiagnosis of ALA. Future studies will determine whether rPGM-ELISA also detects antibodies produced in amoebic dysentery and asymptomatic cases.

Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

In vivo programmed cell death of Entamoeba histolytica trophozoites in a hamster model of amoebic liver abscess.

Entamoeba histolytica trophozoites can induce host cell apoptosis, which correlates with the virulence of the parasite. This phenomenon has been seen during the resolution of an inflammatory response and the survival of the parasites. Other studies have shown that E. histolytica trophozoites undergo programmed cell death (PCD) in vitro, but how this process occurs within the mammalian host cell remains unclear. Here, we studied the PCD of E. histolytica trophozoites as part of an in vivo event related to the inflammatory reaction and the host-parasite interaction. Morphological study of amoebic liver abscesses showed only a few E. histolytica trophozoites with peroxidase-positive nuclei identified by terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick end labelling (TUNEL). To better understand PCD following the interaction between amoebae and inflammatory cells, we designed a novel in vivo model using a dialysis bag containing E. histolytica trophozoites, which was surgically placed inside the peritoneal cavity of a hamster and left to interact with the host's exudate components. Amoebae collected from bags were then examined by TUNEL assay, fluorescence-activated cell sorting (FACS) and transmission electron microscopy. Nuclear condensation and DNA fragmentation of E. histolytica trophozoites were observed after exposure to peritoneal exudates, which were mainly composed of neutrophils and macrophages. Our results suggest that production of nitric oxide by inflammatory cells could be involved in PCD of trophozoites. In this modified in vivo system, PCD appears to play a prominent role in the host-parasite interaction and parasite cell death.

Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess.

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jalpha18(-/-) mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using alpha-galactosylceramide (alpha-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d(-/-) mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-gamma but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28:0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-gamma production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to alpha-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.

Absence of CD38 delays arrival of neutrophils to the liver and innate immune response development during hepatic amoebiasis by Entamoeba histolytica.

To define the role of CD38 in the migration of neutrophils to the liver and consequently in the induction of an innate immune response during murine hepatic amoebiasis by Entamoeba histolytica, we examined amoebic liver abscess development (ALA), presence of amoebae and neutrophils, and expression levels of cytokines and other inflammation mediators mRNA, in infected wild-type and CD38 Knockout (CD38KO) C57BL/6J mice. Results showed that CD38KO mice undergo a delay in ALA development in comparison with the wild-type strain. The presence of amoebae lasted longer in CD38(-/-), and although neutrophils arrived to the liver in both strains, there was a clear difference in the time between the two strains; whereas in the wild-type strain, neutrophils arrived at early times (6-12 h), in the CD38KO strain, neutrophils arrived later (48-72 h). Cytokines profile during the innate immune response development (TNF-α, IL-1ß, IL-6) was, for WT mice concomitant with, and preceded, for CD38KO mice, the time in which neutrophils were present in the liver lesion. In conclusion, CD38 is important for neutrophils migration during hepatic amoebiasis, and in turn, these cells play an important role in the innate immune response.

Expression of cytokines and their regulation during amoebic liver abscess development.

Amoebic liver abscess (ALA) is the most important extraintestinal complication of Entamoeba histolytica infection. Amoebic liver abscess development causes severe destruction of the liver tissue concomitant with a strong inflammatory reaction. We analyse the in situ expression of TNF-α, IFN-γ, IL-1ß, 1L-8 and IL-10 at different stages of ALA development in a susceptible animal model. Results showed that after inoculation, neutrophils (PMN) and some macrophages infiltrated the liver and were positive for TNF-α and IFN-γ at the acute phase of amoeba infection. The presence of these cytokines was transient and decreased as tissue damage progressed. In contrast, IL-1ß and IL-8 were detected mainly in neutrophils and macrophages from the periods of acute infection to subacute and chronic infection and decreased when granulomas were formed. The IL-10 was expressed in PMN and mononuclear cells and only during a short period at the onset of acute infection. The qRT-PCR of mRNA revealed a relationship with the expression of the cytokines in cells found in the ALA. Furthermore, our data suggest that IL-10 does not regulate local production of these cytokines. Our results indicate that an exacerbated inflammatory milieu is established and contributes to liver tissue damage and probably supports the survival of the parasites.

Modulation of amoebic hepatic abscess by the parasympathetic system.

The neuro-immune network, in which the vagus nerve is involved, provides feedback between its afferent branches for signalling central nervous system from sites of injury through cytokines and its efferent branches, which release acetylcholine, an anti-inflammatory neurotransmitter. For gain insight into the parasympathetic mechanisms participating in the inflammatory response in the liver, we studied the effects of a vagotomy on the innate immune response in hamsters with amoebic liver abscess. At 7 days post-infection, compared to the control, liver parasympathectomy resulted in a larger abscess size, a greater production of collagen fibres, fewer trophozoites, increased serum levels of IL-10 and IFN-γ and increased numbers of IL-10 and IFN-γ-positive cells in situ, with no change in the number of macrophages and NK cells. Data indicate that the vagotomy disrupted the inflammatory response, causing an increase in the response against infection, then could favour the innervation of the liver by the sympathetic nervous system and would then take the control of the immune response by stimulating the conversion of macrophages to epithelioid cells; and through increased IL-10 production would induce the hepatic stellar cells to become myofibroblast collagen producer cells, thus forming a barrier of collagen and blocking trophozoite migration.

Host-microbe interactions and defense mechanisms in the development of amoebic liver abscesses.

SUMMARY: Amoebiasis by Entamoeba histolytica is a major public health problem in developing countries and leads to several thousand deaths per year. The parasite invades the intestine (provoking diarrhea and dysentery) and the liver, where it forms abscesses (amoebic liver abscesses [ALAs]). The liver is the organ responsible for filtering blood coming from the intestinal tract, a task that implies a particular structure and immune features. Amoebae use the portal route and break through the sinusoidal endothelial barrier to reach the hepatic parenchyma. When faced with systemic and cell-mediated defenses, trophozoites adapt to their new environment and modulate host responses, leading to parasite survival and the formation of inflammatory foci. Cytopathogenic effects and the onset of inflammation may be caused by diffusible products originating from parasites and/or immune cells either by their secretion or by their release after cell death. Liver infection thus results from the interplay between E. histolytica and hepatic cells. Despite its importance in terms of public health burden, the lack of integrated data on ALA genesis means that we have only an incomplete description of the initiation and development of hepatic amoebiasis. Here, we review the main steps of ALA development as well as the responses triggered in both the host and the parasite. Transcriptome studies highlighted parasite factors involved in adherence to human cells, cytopathogenic effects, and adaptative and stress responses. An understanding of their role in ALA development will help to unravel the host-pathogen interactions and their evolution throughout the infection.

Immunological studies in ulcerative colitis. IV. Origin of autoantibodies.

The incidence and height of antibody titers to colon, assayed by indirect hemagglutination with a heat stable colon extract from germ free rats, is significantly higher in sera from patients with ulcerative colitis than in those from healthy controls or from patients with amebic liver abscess or dysentery. While sera from ulcerative colitis patients and controls are indistinguishable in regard to incidence and height of antibody titers to Forsman antigen, Staphylococcus aureus S 209, Clostridium difficile, and several common strains of E. coli, they have elevated titers and increased incidence of antibodies to a heat stable antigen of E. coli O14. Patients with amebic dysentery have normal titers of such antibodies. Absorption of patients' sera with E. coli O14 antigen inhibits the colon directed hemagglutination reaction in approximately 30% of the cases tested. Likewise, the anti-E. coli O14 reaction can sometimes be inhibited with the colon extract. Other E. coli strains and other bacteria are inactive or have only weak inhibitory activity. Hemagglutination inhibition experiments show that germ free rat colon and E. coli O14 contain common structures, depicted by antibodies in the patients' sera. This pattern of reactivity closely resembles that seen in rats made autoimmune to colon by injection of newborn rabbit colon. E. coli O14 is known to carry a heterogenetic antigen present in lower concentration (or activity) in most Enterobacteriaceae. Hemagglutination inhibition experiments with rabbit antisera to E. coli O14 suggest that the antigen common for E. coli O14 and colon is related to this heterogenetic antigen. The findings imply that this antigen, which is constantly present in low concentrations in the human colon, may give rise to anticolon antibody formation in ulcerative colitis through breakage of tolerance. Since this antigen is present in healthy individuals as well, additional factors are required to explain the induction of anti-colon autoimmunity in ulcerative colitis.

Identification of an epitope on the Entamoeba histolytica 170-kD lectin conferring antibody-mediated protection against invasive amebiasis.

The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose- and N-acetylgalactosamine-inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens.

Entamoeba histolytica: expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain.

We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG's anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.

Gonadectomy inhibits development of experimental amoebic liver abscess in hamsters through downregulation of the inflammatory immune response.

Incidence of amoebic liver abscess (ALA) in human males is considerably higher than in females, suggesting a role for sex hormones in this parasite infection. We describe here the effect of hamster gonadectomization on the development of ALA. After monitoring the decrease of oestradiol in females and testosterone in males to undetectable levels by ELISA and Radio Immuno Assay (RIA) in serum, hamsters were intraportally infected with Entamoeba histolytica trophozoites and killed 7 days later. ALA was absent in 50% of male and 15% of female gonadectomized (Gdx) hamsters, in comparison with 100% infection in non-Gdx controls. This protection against ALA in Gdx hamsters was concomitant to a comparatively scarce inflammatory infiltrate and necrosis surrounding clusters of trophozoites in the liver tissue, as well as to a lack of response of spleen cells to Con A, evaluated in proliferation assays. As tissue damage in ALA has been associated with a local inflammatory Th1 response, we determined the profile of response in hamsters by immunohistochemistry on liver sections. In contrast to strong Th1 responses in non-Gdx animals, Gdx females and males exhibited Th2 and Th3 profiles of cytokines, respectively, suggesting that protection against ALA following gonadectomization, could be related to downregulation of liver Th1 response during amoebic infection.

Mechanisms of natural resistance of Balb/c mice to experimental liver amoebiasis.

Entamoeba histolytica is the parasite responsible for human amoebiasis. The analysis of the natural resistance mechanisms of some rodents to amoebic liver abscess (ALA) may reveal alternative pathogenicity mechanisms to those previously discovered in the experimental model of ALA in hamsters. In this work the natural resistance of BALB/c mice to ALA was explored by performing: (i) in vivo chemotaxis analysis with a specifically designed chamber; (ii) in vitro amoebic survival in fresh and decomplemented serum; (iii) histological temporal course analysis of ALA development in mice with different treatments (hypocomplementemic, hyperimmune and treated with iNOS and NADPH oxidase inhibitors) and (iv) mouse liver amoebic infection by both in situ implantation of ALA from hamsters and inoculation of parasites into the peritoneal cavity. The results show that E. histolytica clearance from the mouse liver is related to a low chemotactic activity of complement, which results in poor inflammatory response and parasite inability to cause tissue damage. Also, the absence of amoebic tropism for the mouse liver is correlated with resistance to experimental liver amoebiasis.

Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model.

BACKGROUND: Despite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce. METHODS: To study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations. RESULTS: Mice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins. CONCLUSIONS: The present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.