Chemoattractant activity of Entamoeba histolytica for human polymorphonuclear neutrophils

The ability of axenic Entamoeba histolytica trophozoites and amoebic protein preparations to stimulate chemotaxis of human polymorphonuclear neutrophils (PMN) was evaluated. Virulent E. histolytica (strain HM1:IMSS) stimulated chemotaxis (delta distance = 0.55 +/- 0.02 mm, P less than 0.01 vs. control medium). Sonicated (100 micrograms protein/ml) or homogenized (500 micrograms protein/ml) virulent amoebae also significantly stimulated PMN chemotaxis, whereas preparations of the nonpathogenic "Entamoeba-like" Laredo strain did not stimulate chemotaxis. Preparations of subcellular fractions of E. histolytica demonstrated maximal stimulation of PMN chemotaxis existed in nonvesiculated membranes and the supernatant from plasma membranes.     

Movement of Naegleria fowleri Stimulated by Mammalian Cells In Vitro1

ABSTRACT. Naegleria fowleri amebae, but not those of N. australiensis, N. gruberi, or N. lovaniensis, demonstrated enhanced motility when placed in proximity to mammalian cells. Amebae of nonpathogenic species of Naegleria, however, were more motile in cell culture medium than the amebae of N. fowleri. The locomotory response of highly pathogenic mouse-passaged N. fowleri amebae to nerve cells was greater than axenically cultured amebae. The enhanced mobility elicited by whole nerve cells or disrupted nerve cells was not directed migration but chemokinetic. Naegleria fowleri responded to disrupted neuroblastoma cells more vigorously than to disrupted African green monkey kidney (Vero) cells.     

Entamoeba histolytica: collagenolytic activity and virulence

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Entamoeba histolytica: Collagenolytic Activity and Virulence1

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS, and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Differential stimulation of microglial pro-inflammatory cytokines by Acanthamoeba culbertsoni versus Acanthamoeba castellanii

Acanthamoeba spp. are opportunistic pathogens that cause granulomatous amebic encephalitis. We compared the highly pathogenic species A. culbertsoni to the relatively less pathogenic species A. castellanii for its capacity to elicit from neonatal rat microglia the gene expression of pro-inflammatory cytokines. Acanthamoeba culbertsoni elicited a robust cytokine gene response by neonatal rat microglia in vitro as compared to A. castellanii. The preponderant cytokine elicited at the mRNA and protein levels was interleukin-1beta. In addition, transmission electron microscopy revealed that microglial cells were capable of phagocytozing A. castellanii. In contrast, A. culbertsoni destroyed microglia. Collectively, these results suggest that a combined action of pro-inflammatory cytokines and destruction of host cells by amebae contribute to the pathology caused by the more pathogenic species.     

Ultrastructural Observations of Experimental Naegleria Meningoencephalitis in Mice: Intranuclear Inclusions in Amebae and Host Cells*

SYNOPSIS. Primary amebic meningoencephalitis was experimentally produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality, with average time of onset of symptoms or death occurring on the 7–8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.     

Ultrastructural observations of experimental Naegleria meningoencephalitis in mice: intranuclear inclusions in amebae and host cells.

Primary amebic meningoencephalitis was experimentallly produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality with average time of onset of symtoms of death occurring on the 7-8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Electron microscope studies of experimentalEntamoeba histolytica infection in the guinea pig

Early cellular and vascular changes in response to invasion of lamina propria byEntamoeba histolytica were studied sequentially, at the ultrastructural level, in germfree guinea pigs inoculated intracecally with amebae and enteric flora derived from patients with acute amebic colitis. Approximately one week post-inoculation the animals developed acute colitis with mucosal invasion by trophic amebae. Although epithelial cells at the sites of amebic invasion showed progressive cytoplasmic changes and desquamation resulting in microerosions, most mesenchymal elements in the lamina propria appeared normal without cytopathic changes even when in direct contact with invading amebae. Only the polymorpho-nuclear leukocytes (PMN) apposed or topographically close to amebae exhibited degenerative changes which were characterized by condensation of nucleoplasm and cytoplasm, extracellular release of cytoplasmic components including granules, and, finally, lysis of cell membranes. Capillaries and venules in the lamina propria showed a variety of changes such as swelling and gap formation at the intercellular endothelial junctions and more rarely at the fenestrae. Blood vessels physically close to amebae showed formation of endothelial cytoplasmic blebs which pinched off into the vascular or extravascular space. Platelet and fibrin thromboses were common in the more severely damaged capillaries and venules. Fragments or clumps of fibrin-like material were found also in the extracellular spaces. Amebic invasion of the lamina propria, then, is accompanied by continued epithelial shedding, PMN degeneration, and changes in both capillaries and venules consisting of endothelial damage and occlusive thrombosis. The vascular changes appeared to be closely related to PMN degeneration resulting from interaction of PMN with invading amebae.     

Patients treated for amebic liver abscess develop cell-mediated immune responses effective in vitro against Entamoeba histolytica

We studied the afferent and efferent cell-mediated immune response in 15 patients treated for amebic liver abscess. Patients had a lower T4 to T8 ratio (1.25 +/- 0.65) compared with age- and sex-matched controls (1.89 +/- 0.44, p less than 0.01) due to a decrease in T4-"helper" cells and an increase in T8-"suppressor" cells (p less than 0.01). The in vitro proliferative response of patient T lymphocytes to the plant mitogen concanavalin A (Con A) was depressed; responses to phytohemagglutinin were not. The proliferative response of patient lymphocytes to an amebic soluble protein preparation (SPP) was greater than the mitogenic response seen in control lymphocytes (mean of 68,300 delta cpm and 22,300 delta cpm, respectively, p less than 0.001), correlated with the T4 to T8 ratio (p less than 0.05) and the duration of time from initiation of antiamebic therapy (p less than 0.01). Supernatants from patient lymphocytes exposed to the amebic SPP activated normal monocyte-derived macrophages to kill virulent axenic E. histolytica trophozoites (p less than 0.001); patient monocyte-derived macrophages activated by Con A-elicited lymphokine could also kill amebae. Finally, when incubated with the amebic SPP for 5 days, T lymphocytes from patients were able to kill virulent amebae (p less than 0.005); patient T lymphocytes not exposed to the amebic SPP or control T lymphocytes incubated for 5 days with the amebic SPP were not cytotoxic to E. histolytica trophozoites. In summary, after cure of amebic liver abscess, specific cell-mediated immune mechanisms develop that are effective in vitro against the parasite.     

Intranasal immunization of mice against Naegleria fowleri

The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 x 10(3) amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10(3) amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.     

Amebiasis in institutions for the mentally retarded in Kanagawa Prefecture, Japan

A parasitologic survey of 620 mentally retarded patients, institutionalized in five different facilities in Kanagawa Prefecture, revealed a high incidence (12.6%) of infection with Entamoeba histolytica. A concomitant serologic survey, by the indirect fluorescent antibody test, gave a much higher incidence (26.5%). Moreover, most zymodeme patterns of the amebae isolated from infected individuals were of a pathogenic type (Zymodeme II). Our findings demonstrate that the mentally retarded in Japan, as in the United States, still are plagued by a high rate of amebic infection.     

Entamoeba histolytica: is conversion of "nonpathogenic" amebae to the "pathogenic" form a real phenomenon?

Entamoeba histolytica isolates have been shown to fall into two groups based on isoenzyme analysis. These groupings ("pathogenic" and "nonpathogenic") correlate well with the clinical course of the infection. A controversy exists over whether isoenzyme patterns are stable or whether under certain circumstances an isolate can convert from one form to the other. Resolution of this uncertainty is of importance since the nonpathogenic pattern has never been observed in amebae isolated from cases of active disease. This implies that, if the patterns are stable, carriers of amebae with this nonpathogenic pattern may never develop invasive disease. Although we set out to study isoenzyme conversion, we have been unable to replicate the two published accounts of this phenomenon. We have examined all of the variables proposed to be involved in the triggering of conversion, both individually and in combination. In none of the experiments was an alteration in the isoenzyme pattern observed. We now believe that isoenzyme patterns are stable and that all available evidence, other than the reported conversions, points to pathogenic and nonpathogenic E. histolytica being distinct species.     

Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.     

Neutrophil-derived oxidants as mediators of chemical activation in bone marrow

Neutrophil-derived oxidants have been implicated in both damage to biomolecules and the metabolic activation of xenobiotics. Since the bone marrow is a relatively neutrophil-rich tissue which is subject to xenobiotic toxicity, we have characterized the oxidant generating capability of neutrophilic cells isolated from femurs of male C57BL/6J mice. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to neutrophil preparations (70 +/- 5% ring neutrophils and metamyelocytes) elicited superoxide anion generation, as indicated by superoxide dismutase (SOD)-inhibitable acetylated cytochrome c reduction, and oxidant-dependent chemiluminescence (CL) from luminol or lucigenin. The interaction of benzo[a]pyrene-7,8-dihydrodiol (BP-diol), a proximate carcinogenic metabolite of benzo[a]pyrene (BP), with TPA-stimulated bone marrow neutrophils resulted in azide-inhibitable CL (90%) indicative of its myeloperoxidase-dependent oxidation to an excited-state intermediate. Covalent binding of [3H]BP-diol to exogenous DNA was similarly increased 3-fold in the presence of TPA-stimulated bone marrow neutrophils. Recently, our laboratory has shown that in addition to CL, TPA-stimulated human polymorphonuclear leukocytes can activate BP-diol to an intermediate which covalently binds to DNA and elicits mutagenicity in Salmonella typhimurium TA100. These observations combined with our current results suggest a possible role for neutrophil-derived oxidants in the mechanisms of chemically-induced bone marrow toxicity.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Depletion of neutrophils in IL-10(-/-) mice delays clearance of gastric Helicobacter infection and decreases the Th1 immune response to Helicobacter

Gastric infection with Helicobacter induces a lymphocyte-rich mucosal inflammation that contains a minor population of neutrophilic granulocytes. The function of neutrophils in the local immune response to gastric Helicobacter infection remains unknown. To investigate this issue, we conducted experiments in neutrophil-depleted control wild-type (wt) and IL-10(-/-) mice infected with Helicobacter felis by gastric lavage. Infection of wt mice elicited a mild, focal gastritis and a Helicobacter-specific Th1 immune response. In wt mice Helicobacter colonization of the stomach was persistent and progressively increased during the 29 days of observation. Infection of IL-10(-/-) mice with H. felis elicited a severe chronic gastritis and a greatly enhanced Helicobacter-specific Th1 immune response, as compared with wt mice. After initial colonization, the IL-0(-/-) mice completely cleared Helicobacter from the stomach by day 8. The gastric inflammation in wt and IL-10(-/-) mice contained modest numbers of neutrophils. The intensity of gastric inflammation and the extent of Helicobacter colonization were similar in control and in neutrophil-depleted wt mice. In contrast, neutrophil depletion of Helicobacter-infected IL-10(-/-) mice decreased the severity of gastritis, modulated the Helicobacter-specific Th1 immune response, and delayed the clearance of bacteria from the stomach. These studies identify a role for neutrophils in the local and systemic immune response to gastric Helicobacter in IL-10(-/-) mice.     

Naegleria fowleri infection acquired by mice through swimming in amebae-contaminated water

Groups of mice were placed in water containing from 10(2) to 10(6) Naegleria fowleri amebae per ml and allowed to swim for 2.5 to 20 min. Mouse mortality ranged from 0 to 70% and was dependent upon the concentration of amebae per ml and the length of swimming exposure. That swimming mice can develop fatal naeglerial infection further confirms the mouse model for studying experimental primary amebic meningoencephalitis.     

Activation of complement by pathogenic and nonpathogenic Entamoeba histolytica

Previous studies had demonstrated that strains of Entamoeba histolytica isolated from patients with colitis or amebic liver abscess were resistant to complement-mediated killing, whereas strains from asymptomatic patients were readily lysed by non-immune serum. Both serum-sensitive and serum-resistant strains of E. histolytica depleted complement rapidly as assessed by CH50, C3, and C7, and C5-9 hemolytic activities. Activation of the alternative pathway was important in lysis of nonpathogenic strains, as demonstrated by lysis by NHS (60.9 +/- 15.6%) and NHS + 5 mM EGTA (59.3 +/- 4.5%) as well as by C4-deficient guinea pig serum (72.8 +/- 7.1%) and C2-deficient human serum (64.4 +/- 11.1%), but not by NHS + 5 mM EDTA. Classical pathway activation also occurs as both pathogenic and nonpathogenic strains deplete greater than 98% of C4 activity, although it is not necessary for lysis. Pathogenic strains are not lysed by either the classical or the alternative pathway. These results suggest that pathogenic strains of E. histolytica activate complement but are able to evade an important host defense, complement-mediated lysis.