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.     

In vitro and in vivo interaction between trophozoites of Entamoeba histolytica and gerbil lymphoid cells.

The in vitro and in vivo antiamoebic cytotoxic effects of peritoneal exudate cells and mesenteric lymph node lymphocytes of gerbils with cecal amoebiasis or those immunized with amoebic extract were investigated. A differential effect of the lymphoid cells against trophozoites of nonpathogenic and pathogenic strains of Entamoeba histolytica was observed. Nonpathogenic amoebae were more susceptible to killing by lymphoid cells than pathogenic amoebae in vitro and in vivo in infected or immunized animals. These data suggest that during the course of cecal amoebiasis in gerbils, a differential stimulation or depletion of cytotoxic cells in the lymphoreticular tissues occurs, resulting in an impaired cell-mediated immune response.     

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.     

CpG-oligodeoxynucleotide is a potent adjuvant with an Entamoeba histolytica Gal-inhibitable lectin vaccine against amoebic liver abscess in gerbils

The protozoan parasite Entamoeba histolytica causes invasive amoebiasis characterized by amoebic dysentery and liver abscesses (ALA). The E. histolytica galactose/N-acetyl-D-galactosamine-inhibitable lectin (Gal-lectin), an immunogenic surface molecule involved in colonization and invasion, is a promising vaccine candidate against amoebiasis. Gal-lectin is known to induce Th1 cytokines in macrophages and spleen cells in vitro, and a Th1 response is thought to be protective against ALA. In this study, we report the use of cytosine guanine oligodeoxynucleotide (CpG-ODN) as adjuvant to augment Th1 responses against Gal-lectin in the gerbil model of ALA. Gerbils were vaccinated intramuscularly with the native Gal-lectin plus CpG-ODN or a paired non-CpG control GpC-ODN, and control gerbils received CpG-ODN alone. One week after the last boost gerbils were challenged intrahepatically with 10(6) amoebae. Gerbils receiving CpG-ODN as adjuvant with Gal-lectin were completely protected against the development of ALA, whereas 50% of gerbils receiving GpC-ODN and Gal-lectin developed ALA and 85% of controls developed ALA. Stronger lymphoproliferation in response to the Gal-lectin and higher prechallenge titers of serum Gal-lectin-specific antibodies, capable of blocking amoebic adherence, were observed when CpG-ODN was used as adjuvant. Gerbils vaccinated with CpG-ODN and Gal-lectin also had significantly higher levels of gamma interferon, interleukin-12 (IL-12), and IL-2 mRNA than controls. These data indicate that CpG-ODN can enhance the Th1 responses, which improve the protective effects of Gal-lectin. This is the first report of the use of CpG as a potent Th1 adjuvant with Gal-lectin to increase protection against ALA formation.     

Distribution of Entamoeba histolytica Gal/GalNAc lectin-specific antibody response in an endemic area

To demonstrate the dynamics of specific antibody isotypes against Entamoeba histolytica Gal/GalNAc adhesin and its correlation, if any, with the development of immunity, we evaluated subjects suffering from a spectrum of amoebic infections ranging from amoebic liver abscess (ALA) to asymptomatic cyst passers. The quantitative analysis of antibody levels was done in the sera by enzyme-linked immunosorbent assay. Gal/GalNAc adhesin-specific immunoglobulin G (IgG) was higher in ALA (and their follow-ups) and intestinal amoebiasis cases as compared with asymptomatic cyst passers (P < 0.05). Among the isotypes of IgG, high levels of IgG1 (60% of the total IgG concentration), suggestive of T-helper 2-type response, was associated with ALA cases. Intestinal amoebiasis cases and cyst passers had high percentage of IgG1 and IgG4 antibodies as compared with per cent IgG2 and IgG3 (of the total IgG), whereas follow-up cases of ALA had predominantly IgG2 and IgG3 isotypes of antibodies. Gal/GalNAc lectin-specific IgM antibodies were maximum in cases of intestinal amoebiasis. ALA cases and their follow-ups had significantly lower levels of Gal/GalNAc-specific IgM levels as compared with cyst passers (P < 0.05). Gal/GalNAc adhesin-specific IgA antibodies were raised maximally in intestinal amoebic infection cases. ALA cases and their 3-month follow-ups had significantly higher concentrations of lectin-specific IgA (P < 0.05) as compared with the healthy subjects.     

Comparison of indirect fluorescent-antibody amoebic serology with counterimmunoelectrophoresis and indirect hemagglutination amoebic serologies.

Patients ranged from those with no prior diagnosis of or suspected exposure to Entamoeba histolytica to those with proven amoebic liver abscesses (extraintestinal disease). A comparison of serologies from patients with proven and suspected amoebiasis or possible past exposure revealed good correlation between the indirect fluorescent antibody (IFA) procedure and the other methods used, counterimmunoelectrophoresis and indirect hemagglutination. Titers from patients with proven extraintestinal amoebiasis were in the expected high range previously reported by other authors. Patients with clinical histories suggestive of exposure to E. histolytica but no proven disease had lower titers which indicated possible background exposure. The IFA procedure provides a rapid method of antibody detection; results obtained on an emergency basis provide essential information in making the diagnosis of amoebic abscess, pyogenic abscess, or tumor. The IFA procedure is rapid, reliable reproducible, and relatively inexpensive to perform, provided a good source of antigen is consistently available.     

Eicosanoid production by peritoneal and splenic macrophages in mice depleted of bone marrow by 89Sr

Previous studies showed that the prostaglandin-forming macrophages (M phi) induced in the spleens of CBA/J mice by intraperitoneal administration of Corynebacterium parvum (CP) could not be demonstrated following the depletion of bone marrow and blood monocytes with 89Sr. The present study compares prostaglandin E2 (PGE2), leukotriene C4 (LTC4), and LTB4 release by splenic and resident peritoneal M phi in 89Sr-treated mice and 88Sr controls following in vivo CP and in vitro incubation with zymosan, calcium ionophore A23187, or phorbol ester (PMA). Intraperitoneal administration of CP resulted in the appearance of PGE2- and LTB4-releasing M phi in the spleens of control but not 89Sr mice. The incorporation and quantitative distribution of 3H-arachidonic acid into membrane lipids, however, were comparable in test and control mice. Neither zymosan nor any of the other stimulatory agents was able to effect significant release of PGE2 in vitro. No release of LTC4 by splenic M phi was detectable under experimental or control conditions. In contrast, the capacity of resident peritoneal M phi to release PGE2, LTC4, and LTB4 was apparently unaffected by 89Sr-induced bone marrow and monocyte depletion with virtually no demonstrable elicitation. Resident peritoneal M phi removed after CP in such mice showed a dramatic decrease in PGE2 release when incubated in vitro with zymosan, A23187, or PMA. These results, taken with earlier findings, demonstrate characteristically different phenotypic expression of metabolism of certain eicosanoids by splenic M phi from the spleen and the peritoneal cavity and suggest in addition that the induction of PGE2-synthesizing M phi in the spleen by CP is dependent on either an immigrant cell originating in the bone marrow or a regulatory agent derived from a bone marrow cell.     

Immunochemical characterisation of a 29-Kda surface-associated molecule of Entamoeba histolytica and its recognition by serum from patients with amoebiasis.

A 29-Kda cytotoxic molecule of axenically-grown pathogenic Entamoeba histolytica (strain HM1) was purified from an amoebic extract by immuno-affinity chromatography with monoclonal antibodies. Immunoreactivity of the purified 29-Kda molecule altered significantly (p less than 0.01) after exposure to heat or trypsin, but remained unaltered after treatment with sodium metaperiodate. The 29-Kda molecule was recognised by serum from each of 13 patients with amoebic liver abscess. In an ELISA system, the molecule produced significantly higher (p less than 0.01) OD readings with these serum samples than with samples from asymptomatic cyst passers. No serum from healthy subjects or from patients with idiopathic ulcerative colitis or giardiasis had antibodies that reacted with the 29-Kda molecule. The immune response to the 29-Kda amoebic protein in man may indicate a specific role for this molecule in invasive amoebiasis.     

The origin of histological changes in amebiasis]

On microscopic examination of histological specimens from an autopsy case with severe amoebiasis, extensive ulcerative-necrotic lesions in the colon were found associated with an amoebic peritonitis and severe degenerative lesions of the ganglion-cells of the plexus myentericus induced by Entamoeba histolytica. There was also an infiltration of a mesenteric lymphnode by amoebae carried by the lymph through the vasa afferentiae lymphaticae. In the liver several granulomas (amoebic granulomas) and numerous small necrotic areas, surrounded by a dense leucocytic infiltration, were observed. The origin of the lesions in the colon could be attributed to the motility of the amoebae and the secretion of lytic substances (ferments) by the amoebae, whereas the lesions in the liver (amoebic granulomas and necrotic areas) must be considered as the residues of the destroyed amoebae which were brought by the circulation into the liver parenchyma. These lesions are similar to those seen in experimentally produced amoebiasis.     

Entamoeba histolytica antigen-specific induction of human immunodeficiency virus replication.

Replication of human immunodeficiency virus (HIV) may be initiated in infected lymphocytes by antigenic or mitogenic stimulation. A soluble protein derived from an invasive strain of Entamoeba histolytica (amoebic antigen [AA]) was used to study the lymphoblastic responses of T lymphocytes derived from 8 HIV-seronegative homosexual men (controls) and 15 HIV-seropositive homosexual men (patients). The soluble protein was also used in long-term cultures as a stimulus for HIV replication. No control or patient produced detectable lymphoblastic responses to AA in a 6-day tritiated-thymidine incorporation assay. Of 15 patients, 5 (33%) produced HIV p24 (ranging from 31 pg/ml to 151 ng/ml) in response to AA in 30-day cell cultures. HIV p24 was expressed in three of seven patients in response to AA but not to the T-lymphocyte mitogen phytohemagglutinin. Implications for managing HIV-infected patients are discussed.     

Protection of gerbils from amebic liver abscess by vaccination with a 25-mer peptide derived from the cysteine-rich region of Entamoeba histolytica galactose-specific adherence lectin

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality through intestinal infection and amebic liver abscess. Here we show that immunization of gerbils with a single keyhole limpet hemocyanin-coupled 25-mer peptide derived from the 170-kDa subunit of the E. histolytica galactose-binding adhesin is sufficient to confer substantial protection against experimentally induced amebic liver abscesses. Vaccination provided total protection in 5 of 15 immunized gerbils, and abscesses were significantly smaller (P < 0.01) in the remaining vaccinated animals. The degree of protection correlated with the titer of antibodies to the peptide, and results of passive transfer experiments performed with SCID mice were consistent with a role for antibodies in protection. In addition, parenteral or oral vaccination of gerbils with 13-amino-acid subfragments of the peptide N-terminally fused to the B subunit of cholera toxin also significantly inhibited liver abscess formation (P < 0.05). These data indicate that small peptides derived from the galactose-binding adhesin administered by the parenteral or oral route can provide protection against amebic liver abscess and should be considered as components of a subunit vaccine against invasive amoebiasis.     

Alterations in eicosanoid production by rat alveolar type II cells isolated after silica-induced lung injury.

Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-keto-PGF1 alpha or thromboxane B2 (TxB2). Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE2 and 6-keto-PGF1 alpha production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE2 between the normotrophic and the hypertrophic type II cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the alternative pathway of complement by Entamoeba histolytica.

A cytopathogenic effect was observed when Entamoeba histolytica was exposed to human sera from individuals with no clinical history or laboratory evidence of amoebiasis. Absorption studies showed that the effect was not due to natural antibodies. Studies performed using ethylenediamine tetracetic acid (EDTA), cobra venom factor (CoF) and heat-inactivation at 56 degrees C, indicated that the cytopathogenic effect was complement dependent. Furthermore, by using ethylene glycol tetracetic acid (EGTA) and Mg++, zymosan, heat-inactivation at 50 degrees C to destroy the activity of factor B of the alternative pathway, as well as electrophoretic studies with anti-human factor B, it was possible to determine that E. histolytica activated the properdin pathway. Finally, complement determinations indicated that incubation of E. histolytica with normal human serum consumed complement. The diminution in CH50 correlated with a consumption of C3 but not of C1, C4 and C2. It was concluded from these results that trophozoites of E. histolytica activate the alternative pathway of the human complement system.     

Activation of macrophages by Entamoeba histolytica extracts in mice

The effect of Entamoeba histolytica extracts on the production of inflammatory macrophages and the release of hydrogen peroxide (H2O2) and superoxide (O2-) from these cells was examined in C57BL/6 mice. Two different strains of E. histolytica, either virulent (IP:0682:1) or nonvirulent (DKB), were used in this study. The number of macrophages recovered from the peritoneal cavity of mice treated 5 days previously with 150 micrograms of either strain of amoebic extracts was significantly higher than in the saline-treated groups. Macrophages from mice treated with 150 micrograms of the IP:0682:1 strain of amoebic extracts exhibited a powerful burst of oxidative metabolis when triggered with phorbol myristate acetate (PMA). Extract from the non-virulent strain was much less effective in activating macrophages for the production of oxidative metabolites. Thus, a crude extract preparation from E. histolytica, particularly the virulent strain, induces a strong macrophage inflammatory response in which the cells also produce high levels of H2O2 and O2-.     

Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody.

Features of tissue damage in invasive amoebiasis, in particular polymorphonuclear neutrophil (PMN) degranulation and vasculitis, bear resemblance to that seen in Wegener's granulomatosis, the latter being associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We therefore tested sera from patients with confirmed amoebic liver abscess (ALA) for the presence of ANCA by means of an indirect fluorescent antibody test using pure neutrophils as substrate. ANCA was detected in 97.4% of amoebic sera; the pattern of staining was cytoplasmic, homogeneous, without central accentuation (C-ANCA). A proteinase 3 (PR3) ELISA demonstrated PR3 specificity in 75% of C-ANCA-positive ALA sera. Possible explanations are (i) a cross-reacting antibody to a component of Entamoeba histolytica, or (ii) an antibody to PMN components released, and possibly modified, by the action of E. histolytica on PMN. It is possible that this antibody contributes to the pathogenesis of invasive amoebiasis.     

Serological diagnosis of amoebiasis by immunofluorescence

A positive serological diagnosis of amoebiasis could be made by immunofluorescence in 66 of 78 established cases, taking a serum titre of 16 or higher as diagnostic: at this level there were no false positives among 94 control sera. The test is simple and may be carried out on amoebic smears stored for several months in 2-octanol. The serological activity is largely confined to the IgG immunoglobulin fraction and is specific for Entamoeba histolytica; cross reactions were not detected with other protozoa. Gel diffusion serological analysis permitted a positive diagnosis of amoebiasis in 60 of the 78 cases, and, combining this with the immunofluorescence test, raised the diagnostic score to 71 cases.     

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.     

The generation and cellular distribution of leukotriene C4 in human eosinophils stimulated by unopsonized zymosan and glucan particles

Human eosinophils (EOSs) stimulated under optimal conditions with 5 X 10(8) unopsonized zymosan particles at 37 degrees C for 30 minutes produced an average total immunoreactive leukotriene (LT) C4 of 1.6 ng per 10(6) EOSs, and 30% to 60% of the generated product remained cell associated. The dose-response characteristics of zymosan-induced LTC4 generation were different from those of phagocytosis, suggesting that the two events were independent. Pretreatment of EOSs with 10(-8) mol/L of formyl-methionyl-leucyl-phenylalanine for 30 minutes led to a twofold to fivefold augmentation of LTC4 generation by cells subsequently activated by unopsonized zymosan. Optimal EOS activation with 1 mumol/L of the calcium ionophore A23187 at 37 degrees C for 15 minutes produced more than 100 times greater quantities of LTC4 than with zymosan. The amount of immunoreactive LTC4 that remained cell associated after calcium ionophore A23187 stimulation reached a maximum after 5 minutes and then declined. Of the relatively small amount generated in the first minute, 71% was cell associated, but this figure declined to 9% after 15 minutes, by which time there had been a redistribution of the LTC4 to the supernatant. Inflammatory leukocytes may respond to zymosan because the cells recognize either one or both of its major polysaccharide components, glucan and mannan. Glucan, but not mannan, stimulated EOSs to generate LTC4 in a dose- and time-dependent manner. Under optimal conditions, there was no significant difference in the total quantities of LTC4 elaborated by EOSs stimulated by glucan and by unopsonized zymosan. This suggests that zymosan may induce leukotriene generation in the human EOS through a glucan recognition mechanism.     

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.