Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica

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

Entamoeba histolytica extract and interferon-gamma activation of macrophage-mediated amoebicidal function.

The effect of recombinant murine interferon-gamma (IFN-gamma) and E. histolytica extract (E.h.E.) on macrophage (M phi) activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 and A/J mice and preincubated with IFN-gamma and/or E.h.E. It was found that amoebicidal activity could be induced in both C57BL/6 and A/J-derived macrophages by pretreatment with IFN-gamma and E.h.E. Pretreatment of the M phi with E. histolytica extract or IFN-gamma alone did not result in the activation of significant cytotoxic activity against E. histolytica trophozoites. In the presence of IFN-gamma, E.h.E. had a dose-dependent effect on the activation of M phi amoebicidal function.     

Antibody-dependent macrophage-mediated activity against Helicobacter pylori in the absence of complement

Helicobacter pylori is a Gram-negative bacterium, which chronically infects the stomach. Little is known about the immune mechanisms limiting the spread of infection and/or contributing to protection after experimental immunization. In this study, we investigated the hypothesis that specific antibodies and host cells cooperate in the immunity against H. pylori. Antibody-dependent cellular activity against H. pylori was assessed using specific immune serum, or purified IgG, in an in vitro assay, with peritoneal cells as effector cells. The natural antibacterial activity of peritoneal cells was significantly augmented by H. pylori-specific antibodies in a dose-dependent manner. A novel finding was that this killing effect did not require functional complement. Most of the bactericidal activity was associated with cells that were adherent, DX5(-), CD19(-), CD11c(-), Thy-1.2(-), CD11b(+) and CD16/32(+), indicating that the main effector population was represented by macrophages. Similar antibacterial killing was obtained with the macrophage cell line GG2EE. Cytochalasin D significantly impaired this antibacterial activity, suggesting that phagocytosis plays a major role in the antibody-mediated H. pylori killing.     

Antibody-dependent cell-mediated cytotoxicity against varicella-zoster virus-infected targets.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against cryopreserved varicella-zoster virus-infected human foreskin fibroblasts was detected in a 51Cr release assay. Target cells, samples of seropositive or seronegative sera, and mononuclear cells obtained by Ficoll-Hypaque centrifugation of human peripheral blood were added to microtiter plate wells and allowed to incubate at 37 degrees C for 4 h. Fibroblasts infected for 48 to 96 h were susceptible to ADCC. Effector cells from seropositive and seronegative normal children were equally active in the assay. Antibody titers were determined by testing serial dilutions of sera in the ADCC assay. Zoster immune globulin had a titer of 204,800. Sera from 40 naturally seropositive individuals were compared by assays for ADCC and fluorescent antibody to membrane antigen. All sera that were negative by fluorescent antibody to membrane antigen (less than 2) were also negative by ADCC (less than 20). All sera that were positive by fluorescent antibody to membrane antigen were also positive by ADCC, but titers of individual sera were frequently 5 to 20 times higher in the ADCC assay.     

Antibody-dependent cell-mediated cytotoxicity against Tamm-Horsfall protein in acute pyelonephritis

The cytotoxic activity of leukocytes from humans and rats with pyelonephritis were examined in an antibody-dependent cell-mediated cytotoxicity assay (ADCC) with CrCl3-treated erythrocytes coated with Tamm-Horsfall (TH) as target cells. The specificity of the ADCC was confirmed by absorption with TH urinary glycoprotein and inhibition of the ADCC activity seen with polyclonal rabbit anti-TH antisera by monoclonal mouse antibodies. The ADCC activity detected in children with acute pyelonephritis was low in the initial phase of the disease, but increased significantly 9 days after the start of antibacterial treatment. In rats with experimental pyelonephritis, ADCC activity decreased significantly with increased duration of infection. Depletion of cells adhering to carbonyl iron led to higher ADCC activity. During the course of the infection the difference in ADCC activity between effector cell preparations depleted using carbonyl iron and those not depleted decreased. The decreased ADCC activity demonstrated during acute pyelonephritis may point to mechanisms operating to diminish the risk of tissue damage.     

Antibody-dependent cell-mediated cytotoxicity against HLA-A, HLA-B and HLA-DR specificities

HLA specificity of alloantibodies involved in antibody-dependent cell-mediated cytotoxicity (ADCC) was investigated using PHA-induced lymphoblasts ( PLB ) from peripheral blood and Epstein-Barr virus-induced human lymphoblastoid cell lines (LCL). The ADCC activity with PLB sensitized by HLA-A or HLA-B antibodies was relatively low, but higher than that of control. DR antibodies completely failed to sensitize PLB for ADCC. LCL sensitized by corresponding DR antibodies gave strong ADCC reactions, independent from their D phenotypes. The ADCC reactions against LCL were abolished by absorption of the DR antibodies from the alloantisera. Thus the ADCC with LCL would provide a procedure to study the role of DR antibodies in chronic rejection of long-term renal allografts.     

Antibody-dependent cellular cytotoxicity in poikilotherms

⁵¹Cr-chromate labelled chicken red blood cells, treated with rabbit (anti-chicken red blood cell) serum, are lysed in vitro, in the absence of complement, by spleen cells from Xenopus laevis, Ambystoma mexicanum or Lacerta viridis. Optimal conditions for lysis by Xenopus spleen cells were determined. The phenomenon seems homologous with antibody-dependent cellular cytotoxicity (ADCC) mediated by mammalian or avian K cells. The phylogenetic significance of the finding is discussed.     

Leptin protects host cells from Entamoeba histolytica cytotoxicity by a STAT3-dependent mechanism

The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P < 1E-05), supporting the hypothesis that leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection.     

Characterization of Entamoeba histolytica alpha-actinin

We have cloned, expressed and characterized a alpha-actinin-like protein of Entamoeba histolytica. Analysis of the primary structure reveals that the essential domains of the alpha-actinin protein family are conserved: an N-terminus actin-binding domain, a C-terminus calcium-binding domain and a central helical rod domain. However, the rod domain of this Entamoeba protein is considerably shorter than the rod domain in alpha-actinins of higher organisms. The cloned Entamoeba 63 kDa protein is recognized by conventional alpha-actinin antibodies as well as binds and cross-links filamentous actin and calcium ions in the same manner as alpha-actinins. Despite the shorter rod domain this protein has conserved the most important functions of alpha-actinins. Therefore, it is suggested that this 63 kDa protein is an atypical and ancestral alpha-actinin.     

Purine-metabolising enzymes in Entamoeba histolytica

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.     

Chromatin organization in Entamoeba histolytica

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.     

Antibody-dependent cytotoxicity against Trypanosoma rhodesiense mediated through an alternative complement pathway.

A quantitative in vitro method was used to examine the role of classical and alternative pathways of complement activation in cytotoxicity against African trypanosomes by immune serum. This assay is based on the estimation of the extent of antibody-mediated cytotoxicity by measurement of inhibition of incorporation of [(3)H]leucine as an indicator of trypanosome metabolic integrity. To determine which pathway(s) is activated during cytotoxic events, complements sufficient in all components (C4S) and deficient in C4 (C4D) were used. Immune inhibition of [(3)H]leucine uptake by trypanosomes was observed in the presence of both complement sources. Treatment of C4S or C4D serum with cobra venom factor or disodium ethylenediaminetetraacetic acid abolished antibody-mediated cytotoxicity as well as immune hemolysis, thus suggesting the requirement for late-acting complement components. Ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid had no effect on inhibition of leucine incorporation, whereas immune hemolysis was inhibited, suggesting that cytotoxicity did not require C1 activation, a Ca(2+)-dependent event. The dependence of the cytotoxic process on Mg(2+) and not Ca(2+) ions and the fact that C4D guinea pig serum is fully active in trypanosome cytotoxicity indicate that an alternative pathway of complement activation is sufficient for activity.     

Immunization of guinea pigs against Entamoeba histolytica using glucan as an adjuvant

Beta 1-3 polyglucose or glucan, an extract of cell wall of Saccharomyces cerevisiae, has been successfully employed in this laboratory as an effective immunopotentiator in experimental studies on amoebiasis. An antigen extract from Entamoeba histolytica was combined with beta, 1-3 glucan for immunizing guinea pigs. In order to study the effectiveness of such vaccine preparations, several batches of guinea pigs were immunized with amoeba antigen alone, and in combination with various immunoadjuvants. Antigen inoculations were carried out via intraperitoneal route. Protective immune responses were obtained against amoeba antigen by using glucan as an adjuvant partner. The study showed that glucan can be safely used as an effective immune enhancer.     

A dot enzyme-linked immunosorbent assay for detection of antibodies against Entamoeba histolytica

A visual micro-dot enzyme-linked immunosorbent assay (ELISA) based on detection of antibodies against soluble antigens of axenically grown cultures of Entamoeba histolytica is described. The antigen was spotted on a nitrocellulose sheet, the unsaturated sites blocked with bovine serum albumin (BSA) and incubated with 3-fold dilutions of patient sera followed by incubation with protein A conjugated to peroxidase. Enzymic activity was evidenced using the substrate 4-chloro-1-naphthol. A positive reaction produced a blue spot. The sensitivity of the assay was better and comparable to the indirect haemagglutination assay (IHA) and plate ELISA. The entire assay could be completed within 3 h. Antigen-loaded and pre-blocked nitrocellulose strips could be stored up to 3 months at room temperature and 37 degrees C.     

Antibodies against Entamoeba histolytica in human milk and serum in Kenya.

A modified enzyme-linked immunosorbent assay (ELISA) technique has been developed for the detection of secretory immunoglobulin A (SIgA) antibodies against Entamoeba histolytica. By substituting phosphate-buffered saline-Triton for phosphate-buffered saline-Tween as the antibody incubating buffer, unspecific absorption of SIgA was avoided. As revealed by chessboard titrations, the optimal amount of antigen for coating was higher in SIgA than in IgG ELISA. A purified antigen from the membrane pellet fraction of E. histolytica gave equally good reactivity with SIgA and IgG antibodies and was used throughout. A total of 283 milk and 232 serum samples from three areas in Kenya were tested. The samples were collected in maternity hospitals on one of days 1 to 3 after parturition. All milk samples were tested for total SIgA. In one of the study areas (Machakos), the mean level of SIgA was significantly lower than in the other two areas (Mombasa and Nairobi). Eighty-seven of the milk samples (31%) were reactive in the test. The rate of positives was higher in Mombasa, where the SIgA levels were highest. Since both the frequency and the level of serum antibodies were similar in the three study areas, it is likely that the higher milk reactivity in Mombasa was mainly due to the higher SIgA concentrations observed. Antibodies were detected in 32 (14%) of the sera, mostly in low or moderate titers. Surprisingly few mothers had detectable antibodies in both milk and serum. In fact, the majority of positives were reactive in either milk or serum, with a predominance of milk positives. The background for this is probably complex, containing components such as differences in immunoglobulin concentrations in the samples, diversities in local and systemic antigenic stimulation responses, and level of immunological memory.