The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B₆C₃F₁ mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with ³H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni , which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba -mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba -specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.     

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.     

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.     

Acanthamoeba: biology and increasing importance in human health

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and is well recognized to produce serious human infections, including a blinding keratitis and a fatal encephalitis. This review presents our current understanding of the burden of Acanthamoeba infections on human health, their pathogenesis and pathophysiology, and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be targets for therapeutic interventions and/or the development of preventative measures.     

Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals

Knowledge that free-living amoebae are capable of causing human disease dates back some 50 years, prior to which time they were regarded as harmless soil organisms or, at most, commensals of mammals. First Naegleria fowleri, then Acanthamoeba spp. and Balamuthia mandrillaris, and finally Sappinia diploidea have been recognised as etiologic agents of encephalitis; Acanthamoeba spp. are also responsible for amoebic keratitis. Some of the infections are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitides), while others are non-opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitis occurring in immunocompetent humans). The amoebae have a cosmopolitan distribution in soil and water, providing multiple opportunities for contacts with humans and animals, as evidenced by antibody titers in surveyed human populations. Although, the numbers of infections caused by these amoebae are low in comparison to other protozoal parasitoses (trypanosomiasis, toxoplasmosis, malaria, etc.), the difficulty in diagnosing them, the challenge of finding optimal antimicrobial treatments and the morbidity and relatively high mortality associated with, in particular, the encephalitides have been a cause for concern for clinical and laboratory personnel and parasitologists. This review presents information about the individual amoebae: their morphologies and life-cycles, laboratory cultivation, ecology, epidemiology, nature of the infections and appropriate antimicrobial therapies, the immune response, and molecular diagnostic procedures that have been developed for identification of the amoebae in the environment and in clinical specimens.     

Epidemiology of free-living ameba infections

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred worldwide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

The interaction of Acanthamoeba castellanii cysts with macrophages and neutrophils

Acanthamoeba castellanii, a free-living amoeba, causes a sight-threatening form of keratitis. Even after extensive therapies, corneal damage can be severe, often requiring corneal transplantation to restore vision. However, A. castellanii cysts are not eliminated from the conjunctiva and stroma of humans and can excyst, resulting in infection of the corneal transplant. The aim of this study was to determine whether elements of the innate immune apparatus, neutrophils and macrophages, were capable of detecting and eliminating A. castellanii cysts and to examine the mechanism by which they kill the cysts. Results show that neither innate immune cell is attracted chemotactically to intact cysts, yet both were attracted to lysed cysts. Both macrophages and neutrophils were capable of killing significant numbers of cysts, yet neutrophils were 3-fold more efficient than macrophages. Activation of macrophages with lipopolysaccharide and interferon-gamma did not increase their cytolytic ability. Conditioned medium isolated from macrophages did not lyse the cysts; however, prevention of phagocytosis by cytochalasin D inhibited 100% of macrophage-mediated killing of the cysts. Conditioned medium from neutrophils did kill significant numbers of the cysts, and this killing was blocked by quercetin, a potent inhibitor of myeloperoxidase (MPO). These results indicate that neither macrophages nor neutrophils are chemoattracted to intact cysts, yet both are capable of killing the cysts. Macrophages killed the cysts by phagocytosis, whereas neutrophils killed cysts through the secretion of MPO.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Isolation and identification of Acanthamoeba species from thermal spring environments in southern Taiwan

Acanthamoeba species are free-living amoebae found in a range of environments. Within this genus, a number of species are recognized as human pathogens, potentially causing Acanthamoeba keratitis, granulomatous amoebic encephalitis, and chronic granulomatous lesions. In this study, 60 water samples were taken from four thermal spring recreation areas in southern Taiwan. We detected living Acanthamoeba spp. based on culture-confirmed detection combined with the molecular taxonomic identification method. Living Acanthamoeba spp. were detected in nine (15%) samples. The presence or absence of Acanthamoeba spp. in the water samples depended significantly on the pH value. The most frequently identified living Acanthamoeba genotype was T15 followed by T4, Acanthamoeba spp., and T2. Genotypes T2, T4, and T15 of Acanthamoeba, are responsible for Acanthamoeba keratitis as well as granulomatous amoebic encephalitis, and should therefore be considered a potential health risk associated with human activities in thermal spring environments.     

Evaluation of taxonomic validity of four species of Acanthamoeba: A. divionensis, A. paradivionensis, A. mauritaniensis, and A. rhysodes, inferred from molecular analyses

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.     

Entamoeba histolytica: chemoattractant activity for gerbil neutrophils in vivo and in vitro

The kinetics of the host's cellular response in the peritoneal cavity of gerbils toward axenic pathogenic and nonpathogenic Entamoeba histolytica strains were examined. Amebae contained in diffusion chambers or free in the peritoneum elicited a neutrophilic response accompanied by decreased levels of macrophages and lymphocytes. Pathogenic amebae (IP:0682:1 strain) elicited a neutrophilic response greater than the nonpathogenic DKB and "entamoeba-like" Laredo amebae. The neutrophil eliciting factor was found in high levels in disrupted freeze-thawed amebae (53% elicited neutrophils vs 8% for control), glutaraldehyde fixed amebae (45%) and amebic membranes (65%), and low levels in conditioned amebic medium (15%) and the supernatant fraction of amebae (16%). The factor was heat stable to high temperature (100 C for 30 min) and at various pH (6 to 9). The neutrophil eliciting factor in amebic membranes was lowered following pretreatment for 30 min with 1% immune and nonimmune gerbil or human sera (34-48% lowered neutrophil response vs control), acidic pH (less than 3, 69%), proteolytic digestion [trypsin (68%) and alpha-chymotrypsin (72%), 100 micrograms/ml], and 2% Triton X-100 (75%). Peritoneal neutrophils isolated following stimulation with amebic membranes or thioglycollate medium demonstrated higher chemotaxis in vitro toward live pathogenic amebae and amebic membranes (IP:0682:1 strain) compared to either the supernatant fraction or the nonpathogenic DKB or Laredo amebae. The results of this study indicate that membrane bound proteins of pathogenic amebae are chemotactic for gerbil neutrophils which may be important in the pathogenesis and pathology of amebiasis.     

Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii

Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.     

Weak cytotoxic activity of miltefosine against clinical isolates of Acanthamoeba spp

Hexadecylphosphocholine (miltefosine) is an anticancer drug active in vitro against various protozoan parasites, and recently used for the treatment of disseminated Acanthamoeba infection. In the present study, we present results of weak cytotoxic activity of this potential amoebicidal agent for 2 of 3 clinical isolates of Acanthamoeba spp. Although the inhibition effect for all tested concentrations was apparent, and showed 100% eradication of trophozoites of Acanthamoeba castellanii strain at a concentration of 62.5 μM after 24 hr, the strains Acanthamoeba sp. and Acanthamoeba lugdunensis exhibited low sensitivity to hexadecylphosphocholine, even in high concentrations. The determined minimal trophocidal concentrations were 250 μM for Acanthamoeba sp. and 500 μM for A. lugdunensis after 24 hr of exposure. Although hexadecylphosphocholine is a potential agent for treatment of Acanthamoeba keratitis and systemic infections, in clinical practice the possible insusceptibility of the amoebic strain should be considered for optimizing therapy.     

Prolactin-cytokine network in the defence against Acanthamoeba castellanii in murine microglia [corrected

In the central nervous system, cytokine-primed microglia play a pivotal role in host defence against parasite infections. In this study, the effect of recombinant (r) prolactin (rPRL) and rIFN-gamma on A. castellanii infection in murine microglia was examined. Priming of microglia with rPRL and rIFN-gamma synergistically triggered, in a dose-dependent manner, amebastatic activity and the release of endogenous IL-1alpha, IL-1beta, IL-6 and TNF-alpha. More than 53, 57, 49, 89 or 96 % of amebastatic activity was abrogated by anti-PRLR, IFN-gammaR, IL-1beta (but not IL-1alpha), IL-6 or TNF-alpha antibodies, suggesting that these two receptors and proinflammatory cytokines participate in the anti-microbial function. Inasmuch as DPI and AET, both inhibitors of NO synthase, blocked amebastatic activity only during the priming process, the NO-dependent pathway itself appears not to be directly involved in the anti-parasitic capacity. These data suggest that the PRL/PRLR and IFN-gamma/IFN-gamma/R complexes, by the induction of the IFN-gammaR on microglia, up-regulate the release of endogenous TNF-alpha, IL-6 and IL-1beta by these cells, which could trigger anti-microbial activity against A. castellanii infection in the brain [corrected].     

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.     

Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.