Development of inflammation and augmented chemotactic responsiveness of murine peritoneal macrophages following treatment with Entamoeba histolytica trophozoites

The accumulation of inflammatory cells in the peritoneal cavity of C57BL/6 mice was examined following intraperitoneal injection of Entamoeba histolytica trophozoites. Two different strains of E. histolytica were used: a virulent strain (IP:0682:1) and a non-virulent strain (DKB). Injection of 10(6) trophozoites of either strain resulted in significant increases in the numbers of total peritoneal cells, macrophages and polymorphonuclear cells as compared to either saline-injected control mice or mice injected with 10-fold lower doses of trophozoites. The in vitro chemotactic response of macrophages from amoebae-induced exudates was also examined. Macrophages from mice treated with strain IP:0682:1 or DKB strain trophozoites were more responsive to complement-derived chemotactic factors than macrophages from saline-injected mice. This increase was significant on day 2 and persisted at enhanced levels until day 20 when the experiment was terminated. In addition, it was found that trophozoites activated normal mouse serum resulting in the production of serum-derived chemotactic activity.     

In vitro effect of human recombinant tumor necrosis factor on Entamoeba histolytica trophozoites

The effect of tumor necrosis factor (TNF) on E. histolytica trophozoites was examined by using three virulent (IP: 0682:1, HM-1: IMSS, 200: NIH) and one nonvirulent (DKB) strain of E. histolytica. Various concentrations of recombinant TNF were added to E. histolytica trophozoites and the total parasite numbers and their viability were periodically assessed by microscopic observation and trypan blue staining after incubation at 37 degrees C in a nonhumidified chamber. In this study, concentrations of 10(1)-10(6) units of TNF were used. Over a concentration range of 10(4)-10(6) units, the number of trophozoites was significantly lowered in the amoebic cultures containing TNF as compared to untreated controls. It was also found that the effect of TNF was dependent on the densities of both virulent and non-virulent strains of E. histolytica trophozoites in axenic conditions. TNF has no significant affect on the growth of amoebae at the lower starting number of amoebae. The amoebae cultured at the higher density were growth-inhibited significantly in comparison with the control groups. When the growth of the virulent and nonvirulent strains of amoebae was compared in TNF treated culture, it was found that TNF has an inhibitory effect on both the virulent and nonvirulent strans of E. histolytica.     

In vitro killing of Entamoeba histolytica trophozoites by interferon-gamma-activated mouse macrophages

The effect of murine interferon gamma (IFN-gamma) on macrophage activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 mice and preincubated with IFN-gamma and/or lipopolysaccharide (LPS). In vitro amoebicidal activity of these macrophages was determined by trypan blue exclusion test against a virulent strain of E. histolytica (IP:0682:1). It was found that in vitro amoebicidal activity was evident in macrophage monolayers treated with both IFN-gamma and LPS. Macrophages treated with IFN-gamma alone did not develop cytotoxic activity unless they were exposed to LPS as a second triggering signal. The ability of IFN-gamma to prime macrophages to respond to trigger signals of LPS and develop cytotoxicity increased with time of incubation, the highest response being observed after 24 h. There was a dose-dependent relationship between the concentrations of both IFN-gamma and LPS used to activate macrophages and the number of dead trophozoites. These data suggest that macrophages are important in host defense against amoebiasis.     

Entamoeba histolytica proteins modulate the respiratory burst potential by murine macrophages.

Reactive oxygen intermediates are important components of macrophage microbicidal mechanisms and pathogenesis of parasitic disease. The purpose of the present study was to investigate the effect of virulent Entamoeba histolytica (strain HM1-IMSS) on respiratory burst potential of macrophages. Pretreatment of elicited peritoneal macrophages (EPM) with crude soluble amoebic proteins from 1 to 6 hr was found to prime EPM for enhanced O2 and H2O2 release in response to phorbol myristate acetate (PMA) in a dose-dependent manner, whereas pretreatment with the same concentrations of the non-pathogenic E. histolytica-like Laredo strain was without priming effect. Low molecular weight (MW) amoebic proteins (27,000-67,000) purified by Sephacryl-200 column chromatography and subfractionated by diethylaminoethyl cellulose chromatography were 10-fold more potent than crude amoebic proteins in priming EPM for an enhanced respiratory burst potential. Both crude and purified amoebic proteins inhibited the priming effect of lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and antagonized the stimulating effect of PMA. Amoebic proteins by themselves were incapable of stimulating EPM respiratory burst. These findings demonstrate that amoebic proteins are capable of modulating the respiratory burst response of macrophages, suggesting an important role for them in the immunoregulation and pathogenesis of amoebiasis.     

Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization

Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma).     

Modulation of tumor necrosis factor production by macrophages in Entamoeba histolytica infection.

The macrophage-derived mediator tumor necrosis factor alpha (TNF) is a cytokine with pleiotropic effects. TNF exhibits potent immunologic and inflammatory properties in parasitic diseases. The present study examined the production of TNF by macrophages isolated from gerbils infected with Entamoeba histolytica and by naive macrophages in response to amoebae in vitro. Amoebic liver abscess-derived macrophages produced low constitutive basal levels of TNF; in response to lipopolysaccharide (LPS) stimulation, TNF production was enhanced by 14-, 11-, and 6-fold at 10, 20, and 30 days postinfection, respectively. Amoebic liver abscess-derived macrophages pretreated with either recombinant gamma interferon (IFN-gamma) or the cyclooxygenase inhibitor indomethacin augmented TNF production in response to soluble amoebic proteins and LPS. Kupffer cells and peritoneal and spleen macrophages from infected animals did not release TNF constitutively in vitro. However, TNF production in response to LPS stimulation was significantly higher at 10 and 20 days postinfection. Macrophages from infected and naive animals pretreated with recombinant IFN-gamma or indomethacin produced increased amounts of TNF in response to LPS but not in response to soluble amoebic protein stimulation. Pretreatment of naive macrophages with amoebic proteins inhibited LPS-induced TNF production by 69 to 79%; the effect of the amoebic proteins was partially reversed by indomethacin pretreatment. In contrast, IFN-gamma- and LPS-activated naive macrophages produced enhanced levels of TNF in response to live amoebae and soluble amoebic proteins. Our results demonstrate that TNF production by macrophages is altered during E. histolytica infection and in response to amoebae and suggest a role for IFN-gamma and prostaglandin E2 in regulating TNF production during the infection.     

Susceptibility of Entamoeba histolytica to oxidants.

The effect of hydrogen peroxide and hypochlorite on culture forms of Entamoeba histolytica trophozoites was examined by using two strains of E. histolytica, virulent (IP:0682:1) and nonvirulent (DKB). The amoebae were incubated with various concentrations of hydrogen peroxide and hypochlorite, and their viability was determined at different times after incubation. When the viability of the virulent and nonvirulent strains was compared to different oxidant strengths, it became apparent that the virulent strain was less susceptible than the nonvirulent one to the cytotoxic effect of hydrogen peroxide and hypochlorite. Our studies further showed that the toxic effect was both time and dose dependent. To confirm that the killing of amoebae in this system was associated with the presence of hydrogen peroxide, amoebae were incubated with hydrogen peroxide and catalase. Catalase reduced the killing effect of hydrogen peroxide to the control level. These data confirmed previous observations of the susceptibility of amoebic trophozoites to hydrogen peroxide and also demonstrated susceptibility to hypochlorite.     

Virulence of enterohemorrhagic Escherichia coli O91:H21 clinical isolates in an orally infected mouse model.

Escherichia coli K-12 strains producing high levels of Shiga-like toxin type II (SLT-II) but not SLT-I were previously shown to be virulent in an orally infected, streptomycin-treated mouse model. In this investigation, we tested the virulence of several SLT-II-producing enterohemorrhagic E. coli (EHEC) isolates from patients with hemorrhagic colitis or hemolytic uremic syndrome. All of the strains tested were able to colonize the mouse intestine. However, only two strains were consistently virulent for mice: O91:H21 strain B2F1 (Strr), which was previously shown to carry two copies of slt-II-related toxins, and O91:H21 strain H414-36/89 (Strr), which was found in this study to contain three genes from the slt-II group. The oral 50% lethal doses of strains B2F1 (Strr) and H414-36/89 (Strr) when fed to streptomycin-treated mice were less than 10 bacteria. Histological sections from moribund mice fed the O91:H21 strains demonstrated extensive renal tubular necrosis; however, hematological results were not consistent with a diagnosis of hemolytic uremic syndrome. The central role of SLT in the virulence of the O91:H21 EHEC strains was supported by the finding that streptomycin-treated mice preinoculated with monoclonal antibody specific for SLT-II survived oral challenge with either B2F1 (Strr) or H414-36/89 (Strr). The basis for the variation in virulence among the SLT-II-producing EHEC strains tested was not determined. However, a correlation between the capacity of an EHEC strain to grow in small intestinal mucus and lethality in the streptomycin-treated mice was observed.     

Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100 degrees C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2(-/-) mice failed to respond to the extracts, whereas those from TLR2(+/+) cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.     

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.     

Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

AIMS--To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS--Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS--Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS--Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.     

In vitro and in vivo studies of macrophage functions in amebiasis.

Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets.     

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.     

An Entamoeba sp. strain isolated from rhesus monkey is virulent but genetically different from Entamoeba histolytica

An Entamoeba sp. strain, P19-061405, was isolated from a rhesus monkey in Nepal and characterized genetically. The strain was initially identified as Entamoeba histolytica using PCR amplification of peroxiredoxin genes. However, sequence analysis of the 18S rRNA gene showed a 0.8% difference when compared to the reference E. histolytica HM-1:IMSS human strain. Differences were also observed in the 5.8S rRNA gene and the internal transcribed spacer (ITS) regions 1 and 2, and analysis of the serine-rich protein gene from the monkey strain showed unique codon usages compared to E. histolytica isolated from humans. The amino acid sequences of two hexokinases and two glucose phosphate isomerases also differed from those of E. histolytica. Isoenzyme analyses of these enzymes in the monkey strain showed different electrophoretic mobility patterns compared with E. histolytica isolates. Analysis of peroxiredoxin genes indicated the presence of at least seven different types of protein, none of which were identical to proteins in E. histolytica. When the trophozoites from the monkey strain were inoculated into the livers of hamsters, formation of amebic abscesses was observed 7 days after the injection. These results demonstrate that the strain is genetically different from E. histolytica and is virulent. Revival of the name Entamoeba nuttalli is proposed for the organism.     

Entamoeba histolytica depresses chemiluminescence in stimulated human polymorphonuclear leukocytes

Effect of Entamoeba histolytica proteinase/toxin (Ehp/t) on the luminol-dependent chemiluminescence (CL) in stimulated human polymorphonuclear leukocytes (PMN) was studied. The role of superoxide (SO) and hydroxyl (OH) anions in the Ehp/t-associated enhancement/inhibition of CL was also studied using specific scavengers and a biological response modifier, muramyldipeptide (MDP). Ehp/t was isolated from axenic trophozoites of the HM-1:IMSS strain of virulent strain of E. histolytica. Proteinase activity was assayed on a synthetic substrate, Z-arg-arg-AFC and cytotoxicity was tested on HeLa cell monolayers. PMN isolated from blood were incubated with Ehp/t prior to stimulation by phorbol myristateacetate (PMA, 2 micrograms/ml), serum-treated zymosan (2.5 mg/ml) and glucan (2 mg/ml). CL was monitored in an LKB (Wallac) Luminometer. Ehp/t was found to depress up to 90% of CL induced by PMA, glucan and zymosan. Such a depression was Ehp/t concentration-dependent. A 150 micrograms/ml concentration of Ehp/t, obtained from a 0.015-1.5 mg/ml concentration range tested at different incubation times and temperatures, was used in most of our experiments. Incubation time and temperature optima were 15 min and 37 degrees C, respectively. Ehp/t partially inhibited the CL associated with SO and OH. MDP, in the presence of Ehp/t, enhanced CL response in human PMN to about 67% with reference to normal CL without inhibitor. PMN were confirmed to play a vital role in amebic tissue invasion mechanisms.     

Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo.

Entamoeba histolytica infections are associated with a state of transient suppression of cell-mediated immunity. Macrophages, the most important cells in host defence and control of invasive amoebiasis, in infected animals have been found to be deficient in effector functions and accessory cell potential. However, little is known of the cellular mechanisms responsible for the down-regulation. This study investigated whether macrophage dysfunction in amoebiasis is associated with altered macrophage arachidonic acid (AA) metabolism. Resident peritoneal macrophages (PMO) from naive gerbils produced enhanced levels of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in response to live E. histolytica trophozoites, to diffusible excretory/secretory products released from live amoebae in millicells and to freeze-thawed soluble amoebic proteins that were inhibitable by indomethacin (INDO) and nordihydroguaiaretic acid (NDGA), respectively. In contrast to PMO from naive gerbils, PMO from animals with amoebic liver abscesses at 10, 20 and 30 days post-infection (p.i.) released high basal levels of PGE2 and LTC4. In response to zymosan stimulation, PMO from infected animals produced two- and fourfold less PGE2 and LTC4, respectively, as compared to uninfected controls. AMO showed high constitutive basal release of PGE2 and LTC4. In response to amoebic and zymosan stimulation, AMO at 10 days p.i. produced significantly higher levels of PGE2 than AMO at 20 days p.i., while AMO at 30 days p.i. were refractory to stimulation to produce higher than basal levels of PGE2. Early (10 days) and late (20-30 days) AMO were refractory to amoebic and zymosan stimulation for enhanced LTC4 release. Pretreatment of AMO with AA substrate restored optimal PGE2 and LTC4 biosynthesis, but the cells were generally unresponsive to zymosan stimulation to produce augmented levels of LTC4. These results strongly suggest that intrinsic or secreted products or both from E. histolytica can induce profound alteration of eicosanoid formation in cyclooxygenase and lipoxygenase pathways in macrophages from naive and infected animals and that AA metabolism by AMO is sequentially modified during the course of the disease.     

Cellular and humoral aspects of host resistance in murine salmonellosis.

Mice were challenged with a highly virulent strain of Salmonella typhimurium by intraperitoneal injections. At relatively low infecting doses, immunizations with either viable attenuated or heat killed Salm. typhimurium were found to be equally protective against otherwise fatal infections. Pre-opsonization of virulent salmonellae significantly increased the survival rate of mice infected with small numbers of the pathogen. By a cell culture method, peritoneal macrophages of mice were shown to be innately capable of destroying the ingested virulent Salm. typhimurium. Macrophages from previously infected mice did not appear to have any significant increase in their bactericidal activity against salmonellae, but they possessed cytophilic antibodies specific against the H and the O antigens of Salm. typhimurium. It is believed that humoral elements play an important role in acquired immunity in murine salmonellosis by opsonization of the pathogen.     

Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen.

The goal of our study was to obtain a highly conserved Entamoeba histolytica recombinant antigen for study as a subunit amebiasis vaccine. We screened a Uni-Zap cDNA library of E. histolytica (strain HM1:IMSS) with human immune sera and isolated a dominant 804-bp cDNA clone. A 33-kDa fusion protein expressed from the cDNA clone was determined by monoclonal antibody binding, DNA hybridization, and nucleotide sequence to be the complete E. histolytica 29-kDa antigen. Serum antibodies to the recombinant protein were detected by enzyme-linked immunosorbent assay in 80% of subjects from Egypt and South Africa with amebic liver abscess. Similar results were found with the native 29-kDa protein. Native and recombinant 29-kDa antigens induced proliferation of lymphocytes harvested from patients with amebic liver abscess (P < 0.01 compared with controls). Intraperitoneal immunization of gerbils with the recombinant fusion protein (10 micrograms) with Titermax adjuvant elicited an antigen-specific serum immunoglobulin G antibody response and was partially protective (54%) against intrahepatic challenge with 5 x 10(5) virulent axenic trophozoites (strain HM1:IMSS). In summary, the recombinant form of the E. histolytica 29-kDa antigen demonstrated serologic specificity for amebic liver abscess, exhibited conserved T-cell epitopes, and was effective as a subunit vaccine in an experimental animal model of amebic liver abscess.     

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.