Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Morphological characteristics of liver abscesses according its etiology / Características morfológicas de los abscesos hepáticos según su etiología

Although infrequent in the diagnostics areas, liver abscesses are of significant relevance, which is related to the high morbidity and mortality figures they can cause if they are not detected and treated in time. Although multiple operational classifications are described, the one most recognized for its usefulness is that related to its causative agent (pyogenic or bacterial infection, hydatid origin or secondary to infected hydatidosis, and amoebic or by invasive amebiasis). The objective of this article was to generate a study report regarding the morphological characteristics of liver abscesses, characterizing them according to their etiology, as well as describing their study and the latest recommended treatments.

Los abscesos hepáticos son entidades nosológicas de baja frecuencia, pero de significativa relevancia, la que se relacionada con las elevadas cifras de morbilidad y mortalidad que pueden causar si no son detectados y tratados a tiempo. Si bien se describen múltiples clasificaciones operacionales, aquella más reconocida por su utilidad es la relacionada a su agente causal (piógenos o bacterianos, hidatídicos o secundarios a hidatidosis infectada, y amebianos o por amebiasis invasora). El objetivo de este artículo fue generar un documento de estudio respecto de las características morfológicas de los abscesos hepáticos, caracterizándolos de acuerdo con su etiología, así como describir su estudio y tratamiento recomendado de acuerdo con el estado del arte.

EhRab21 associates with the Golgi apparatus in Entamoeba histolytica.

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.

Curcumin Provides Hepatoprotection against Amoebic Liver Abscess Induced by Entamoeba histolytica in Hamster: Involvement of Nrf2/HO-1 and NF-κB/IL-1ß Signaling Pathways.

Amoebic liver abscess (ALA) is the most common extraintestinal amoebiasis caused by Entamoeba histolytica (E. histolytica). However, despite current knowledge and scientific advances about this infection, there are no effective treatments to prevent it. Herein, the antiamoebic capacity of curcumin in a hamster model was evaluated. Curcumin (150 mg/kg, p.o., daily during 10 days before infection) considerably prevents liver damage induced at 12 and 48 h post-intrahepatic inoculation of trophozoites and decreases ALT, ALP, and γ-GTP activities, and macroscopic and microscopic observations were consistent with these results. On the other hand, after one week of intraportal inoculation, liver damage was prevented by curcumin (150 mg/kg, p.o., daily, 20 days before amoebic inoculation and during the week of infection); liver/body weight ratios and tissue and histological stains showed normal appearance; in addition, the increases in ALT, ALP, and γ-GTP activities were prevented; the depletion of glycogen content induced by the amoebic damage was partially but significantly prevented, while NF-κB activity was inhibited and the expression of IL-1ß was reduced; Nrf2 production showed a tendency to increase it, and HO-1 protein was overexpressed. These results suggest for the first time that curcumin can be a compound with antiamoebic effect in the liver, suggesting that its daily use could help greatly decrease the incidence of this type of 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.

Minimally invasive management of hepatic cysts: indications and complications.

OBJECTIVE: Liver cysts are divided into congenital and acquired. Congenital cystic lesions include polycystic liver disease, simple cysts, duct related and ciliated hepatic foregut cysts. Acquired cystic lesions are divided into infectious and non-infectious. The infectious cysts are the hydatid cyst, the amoebic abscess, and the pyogenic abscess, whereas the non-infectious cysts are neoplastic cysts and false cysts. While modern medicine provides a lot of minimally invasive therapeutic modalities, there has emerged a pressing need for understanding the various types of liver cysts, the possible minimal therapeutic options along with their indications and complications. We aim is to clarify the role of minimally invasive techniques in the management of hepatic cysts. MATERIALS AND METHODS: A literature review was performed using the MEDLINE database. The search terms were: liver cyst, minimally invasive, laparoscopic, percutaneous, drainage and fenestration. We reviewed 82 English language publications articles, published until October 2017. RESULTS: Minimally invasive management of liver LC is an emerging field including many therapeutic modalities ranging from the percutaneous aspiration of pyogenic abscesses to laparoscopic hepatectomy for hepatic cystadenomas. The most used techniques are percutaneous drainage, laparoscopic fenestration, and laparoscopic hepatectomy. CONCLUSIONS: The application of the various minimally invasive approaches, as well as their indication and complications, depend on the type of the cystic lesion, its size and its position in the liver. Percutaneous drainage is mostly used in simple cysts, hydatid cysts, pyogenic abscesses and bilomas. Laparoscopic fenestration is mostly used in simple cysts and polycystic liver disease. Finally, laparoscopic hepatectomy is mostly used in polycystic liver disease, hydatid cysts, and cystadenomas.

AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment.

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli.

BACKGROUND: Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica. METHODOLOGY/PRINCIPAL FINDINGS: Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1ß) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro. CONCLUSIONS/SIGNIFICANCE: The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.

Occult Amebic Liver Abscess as Cause of Extensive Inferior Vena Cava and Hepatic Vein Thrombosis.

The most common extraintestinal complication of Entamoeba histolytica is amebic liver abscess (ALA). Hepatic vein and inferior vena cava (IVC) thrombosis are rare but well-documented complications of ALA, typically attributed to mechanical compression and inflammation associated with a large abscess. We present a case of a previously healthy 43-year-old Canadian man presenting with constitutional symptoms and right upper quadrant abdominal pain. He was found to have thrombophlebitis of the IVC, accessory right hepatic vein, and bilateral iliac veins. Extensive investigations for thrombophilia were negative. Magnetic resonance imaging of the liver demonstrated a 3.2-cm focal area of parenchymal abnormality that was reported as presumptive hepatocellular carcinoma, and a 1.9-cm lesion in the caudate lobe with diffusion restriction and peripheral rim enhancement. Despite multiple biopsy attempts, a histopathological diagnosis was not achieved. Abdominal pain and fever 4 months later prompted repeat ultrasound demonstrating a 10.4- × 12.0-cm rim-enhancing fluid attenuation lesion felt to represent a liver abscess. Thick dark "chocolate brown" drainage from the lesion and positive serology for E. histolytica confirmed the diagnosis of ALA acquired from a previous trip to Cuba. The patient was started on treatment with metronidazole and paromomycin and repeat abdominal ultrasound demonstrated resolution of the abscess. This case is the first to demonstrate extensive IVC thrombosis secondary to a relatively small occult ALA and emphasizes the thrombogenic potential of ALA. Amebic infection should be considered as a rare cause of IVC thrombosis in the correct clinical context.

Amoebic liver abscess in northern Sri Lanka: first report of immunological and molecular confirmation of aetiology.

BACKGROUND: Since 1985, amoebic liver abscess (ALA) has been a public health problem in northern Sri Lanka. Clinicians arrive at a diagnosis based on clinical and ultrasonographic findings, which cannot differentiate pyogenic liver abscess (PLA) from ALA. As the treatment and outcome of the ALA and PLA differs, determining the etiological agent is crucial. METHODS: All clinically diagnosed ALA patients admitted to the Teaching Hospital (TH) in Jaffna during the study period were included and the clinical features, haematological parameters, and ultrasound scanning findings were obtained. Aspirated pus, blood, and faecal samples from patients were also collected. Pus and faeces were examined microscopically for amoebae. Pus was cultured in Robinson's medium for amoebae, and MacConkey and blood agar for bacterial growth. ELISA kits were used for immunodiagnosis of Entamoeba histolytica infection. DNA was extracted from selected pus samples and amplified using nested PCR and the purified product was sequenced. RESULTS: From July 2012 to July 2015, 346 of 367 clinically diagnosed ALA patients admitted to Jaffna Teaching Hospital were enrolled in this study. Almost all patients (98.6%) were males with a history of heavy alcohol consumption (100%). The main clinical features were fever (100%), right hypochodric pain (100%), tender hepatomegaly (90%) and intercostal tenderness (60%). Most patients had leukocytosis (86.7%), elevated ESR (85.8%) and elevated alkaline phosphatase (72.3%). Most of the abscesses were in the right lobe (85.3%) and solitary (76.3%) in nature. Among the 221 (63.87%) drained abscesses, 93.2% were chocolate brown in colour with the mean volume of 41.22 ± 1.16 ml. Only four pus samples (2%) were positive for amoeba by culture and the rest of the pus and faecal samples were negative microscopically and by culture. Furthermore, all pus samples were negative for bacterial growth. Antibody against E. histolytica (99.7%) and the E. histolytica antigen were detected in the pus samples (100%). Moreover, PCR and sequencing confirmed these results. CONCLUSION: To our knowledge, this is the first report from Sri Lanka that provides immunological and molecular confirmation that Entamoeba histolytica is a common cause of liver abscesses in the region.

Amoebic liver abscess: an unusual cause for a right iliac fossa mass: a case report.

BACKGROUND: Amoebic liver abscess is the most common extra intestinal manifestation of amoebiasis in tropical countries. It usually presents with right hypochondrial pain, fever and anorexia. Amoebic liver abscess has gained clinical significance due to the wide variety of clinical presentations which can cause diagnostic dilemmas and high mortality in untreated cases. CASE PRESENTATION: We report a case of a 63-year-old male with a history of anorexia for 3 weeks, fever for 4 days and examination findings of tender hepatomegaly with a liver span of 15 cm in the mid clavicular line and a firm irregular mass in the right iliac fossa. Ultrasound scan of the abdomen showed two large liver abscesses with one of them leaking into the peritoneal cavity causing a localized pus collection, which had been walled off in the right iliac fossa. He was treated with metronidazole and liver abscesses were drained percutaneously under ultrasound scan guidance. The diagnosis of Entamoeba histolytica infection was confirmed with the serology and subsequently by PCR from the aspirated material. He made an uneventful recovery with resolution of the symptoms and right iliac fossa mass. CONCLUSION: Recognition of variable presentation of amoebic liver abscess is vital, considering the curable nature of this disease and potentially fatal outcome of untreated abscess. An intra-abdominal mass in a patient with amoebic liver abscess should raise the suspicion of a localized collection of pus and impending generalized peritonitis. Early diagnosis and prompt intervention can prevent the dreaded complication of peritonitis and toxemia, and hence reduce the consequent morbidity and mortality.

Multiple Medium Amoebic Liver Abscesses Successfully Treated with Medication and Comprehensive Percutaneous Catheter Drainage.

Solitary small (<5 cm) amoebic liver abscesses in the right lobe are generally treated using medication alone, while large abscesses are typically treated via a combination of medication and drainage. However, the therapeutic indications for multiple medium (5-10 cm) amoebic liver abscesses remain unclear. We herein report the findings of a 53-year-old woman who was receiving lenalidomide for multiple myeloma and subsequently developed multiple amoebic abscesses. Metronidazole alone was unsuccessful, although metronidazole and repeated percutaneous catheter drainage of the right lobe, left lobe, and thorax proved to ultimately be successful. Therefore, the successful use of medication alone may be associated with the total combined abscess volume.

Gastrointestinal Fistulization in Amebic Liver Abscess.

BACKGROUND: Liver abscess is a common deep seated abscess in children; amebic liver abscess is associated with more local complications. CASE CHARACTERISTICS: We report two preschool children presenting with short history of pain, fever and right upper quadrant pain. The abscess communicated with gastro-intestinal tract (ascending colon in case 1 and duodenum in case 2), and diagnosis of amebic liver abscess was confirmed by DNA PCR. OUTCOME: Both children were successfully managed with intravenous antibiotics and catheter drainage. MESSAGE: Gastrointestinal fistulization may be rarely seen in amebic liver abscess. Conservative management with antibiotics, catheter drainage and supportive care may suffice.

First report of Entamoeba histolytica infection from Timor-Leste--acute amoebic colitis and concurrent late development of amoebic liver abscess in returned travellers to Australia.

This communication reports invasive amoebic colitis and late onset amoebic liver abscess in three members of a group of 12 Australian travellers to Timor-Leste (TL). This is the first report of Entamoeba histolytica infection from TL. Clinicians in Australia need to consider amoebiasis in the differential diagnosis in travellers returning with colitis, abdominal pain and fever. Presentation with amoebic liver abscess months after exposure is rare but should be suspected in symptomatic individuals with a relevant history of travel.

A review of the proposed role of neutrophils in rodent amebic liver abscess models.

Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2(-), H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response.

Endoscopic ultrasound-guided drainage of an amoebic liver abscess extending into the hepatic subcapsular space.

Here, we report a case of an amoebic liver abscess (ALA) successfully treated with endoscopic ultrasound (EUS)-guided liver abscess drainage (EUS-LAD). A 37-year-old male with a liver abscess was referred to our hospital due to disease progression despite receiving antibiotic therapy. Computed tomography showed an intrahepatic abscess extending into the hepatic subcapsular space. The abscess could not be punctured through the hepatic parenchyma percutaneously due to the presence of hepatic subcapsular lesions. Hence, EUS-LAD was performed via the transhepatic approach through the stomach. A 5-Fr pigtail-type nasocystic tube was inserted into the abscess through the hepatic parenchyma, with no procedure-related complications. The contents of the abscess had the appearance of anchovy paste which made us suspect an amoebic abscess; therefore, we started antibiotic therapy with metronidazole. Afterwards, serum anti-amoebic antibodies were found to be positive and the diagnosis of ALA was confirmed. Two weeks later, the size of the abscess decreased, and the patient's clinical symptoms disappeared. Hence, the tube was removed. There were no signs of recurrence during the follow-up period. The use of EUS-LAD for pyogenic or tuberculous abscesses has been reported previously. EUS-LAD for an ALA, similar to that for other liver abscesses, is an effective alternative to percutaneous transhepatic abscess drainage or surgical treatment.

Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.