Comparison of physiological, cytopathogenic and immunological properties between two environmental isolates of Acanthamoeba spp.

The aim of this study was to determine whether pathogenic and less-pathogenic isolates of environmental Acanthamoeba exhibit differences in adhesion to human erythrocytes. Based on physiological properties of temperature, tolerance, and rapid growth, Acanthamoeba were divided into pathogenic and less-pathogenic isolates. Acanthamoeba were tested for their ability to produce cytopathic effects (CPE) using two human cell lines, HEp-2 and KB cells. Both ameba isolates caused CPE to both cell lines with the same pattern without significant difference. Human erythrocytes from 20 healthy volunteers were used to study the erythrocyte reactivity of Acanthamoeba by co-incubation with trophozoites. The pathogenic Acanthamoeba exhibited significantly higher erythrocyte adhesion as compared to the less-pathogens (p<0.05). Erythrocyte activity occurred in the presence of plasma in all blood samples, suggesting the role of plasmatic components and contact-dependent mechanisms to produce host cell cytotoxicity. The present results showed correlation between the physiological properties and erythrocyte reactivity of Acanthamoeba.

Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells.

BACKGROUND: In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. OBJECTIVE: To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. METHOD: We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. RESULTS: Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. CONCLUSION: Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.

Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by down-regulating the class II transactivator CIITA.

Major histocompatibility complex (MHC) class II expression by microglia and astrocytes is critical for CD4+-mediated immune responses within the central nervous system. Here, we demonstrate that the obligate intracellular parasite, Toxoplasma gondii, down-regulates activation-induced MHC class II expression in human-derived glioblastoma cells as well as in primary astrocytes and microglia from cortices of rat fetuses. Down-regulation of MHC class II proteins was predominantly observed in parasite-positive, but not parasite-negative, host cells of T. gondii-infected cell cultures. MHC class II transcript levels induced by IFN-gamma alone or in combination with TNF-alpha were also clearly diminished after parasitic infection. Furthermore, T. gondii dose-dependently down-regulated the transcript levels of the class II transactivator CIITA. These results suggest that T. gondii partially evade CD4+-mediated intracerebral immune responses, a mechanism which may contribute to long-term persistence of the parasite within the CNS.

Detection and counting of Cryptosporidium parvum in HCT-8 cells by flowcytometry.

The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14%) than for IFA (i.e., 27-38% when performed by a single operator and 19-22% when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs.

Short-term exposure to membrane-active antibiotics inhibits Cryptosporidium parvum infection in cell culture.

A cell culture system and double fluorogenic staining were used to study the susceptibility of Cryptosporidium parvum to membrane-active antibiotics. Buforin II and magainin II exerted a cytotoxic effect on sporozoites but did not consistently affect oocyst viability. Lasalocid and nigericin demonstrated less activity against sporozoites but reduced the infectivity of oocysts.

In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones.

A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, Leishmania infanturn, Leishmania enriettii, and Leishmania major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.

Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis.

The dense granules of the intracellular protozoan Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole (PV) in which the parasite develops. The biogenesis of dense granules as well as the regulatory mechanisms controlling their specific exocytosis are still poorly understood. In this paper, we analyzed the secretory pathway of dense granule proteins (GRA proteins) in extracellular T. gondii through the effects of brefeldin A (BFA). Ultrastructural studies of BFA-treated parasites showed disassembly of the Golgi apparatus and accumulation of GRA proteins in a dilated vacuolar system connected to the nuclear envelope. BFA reversibly blocked the intracellular transport of the newly synthesized GRA proteins in a dose-dependent manner (blockade of 95% at 1 microg/ml of BFA). By contrast, discharge of GRA proteins from preformed dense granules was unaffected by BFA over a course of 60 min incubation. GRA protein secretion was dependent on incubation temperature as it only occurred above 26 degrees C and it could be stimulated by external factors. This stimulus might be provided by factor(s) present in the serum of the extracellular medium, as incubation of parasites in serum-free medium resulted in a dramatic decrease in protein secretion. Exocytosis can be restored in a dose-dependent fashion by serum addition (maximal stimulatory activity in the 30-200 kDa range) and was optimal at an extracellular pH of 6.5. Altogether, these results demonstrate that GRA proteins are exported through the Golgi apparatus via the classical secretory pathway and can be experimentally discharged from storage dense granules as regulated secretory proteins in response to specific stimulation, arguing in favor of a regulated component for dense granule exocytosis in T. gondii.

Enhanced cytotoxicity of IFN-gamma-producing CD4+ cytotoxic T lymphocytes specific for T. gondii-infected human melanoma cells.

CD4+ lines specific for Toxoplasma gondii-infected human melanoma P36 cells were established from PBL of a patient with chronic toxoplasmosis. CD4+ CTL lines were obtained by weekly in vitro stimulation with T. gondii-infected P36 cells that shared HLA-DR4 molecules with the patient. The lytic activity of CD4+ CTL lines against T. gondii-infected P36 or T. gondii-infected autologous EBV-transformed B lymphoma (EBV-Ya) was inhibited by anti-HLA-DR mAb, whereas anti-HLA-A, B, C mAb failed to block the lytic activity. Thus, the cytotoxicity of CD4+ CTL lines against T. gondii-infected P36 was restricted by HLA-DR molecules. In response to Ag-specific stimulation, CD4+ CTL lines produced significant levels of IFN-gamma. Exogenously added IFN-gamma up-regulated the surface expression of MHC class II, but not of class I in T. gondii-infected P36 cells. In addition, the CTL activity against T. gondii-infected P36 cells was augmented when target cells were co-cultured with IFN-gamma. These data indicate that CD4+ CTL-mediated cytotoxicity against T. gondii-infected melanocytes is enhanced by the autocrine production of IFN-gamma. Further, CD4+ CTL may play a role in the manifestation of toxoplasmic retinochoroiditis by killing T. gondii-infected melanocytes.

Demonstration of in vitro cultured exoerythrocytic schizonts and hypnozoites of Plasmodium vivax (southern China isolate) by an immunoperoxidase antibody technique.

By indirect immunoperoxidase staining, different forms of exoerythrocytic (EE) stage of Plasmodium vivax (Southern China isolates) are revealed in d8 cultured material. The mature schizonts are elongated in shape measuring 42-48 microns in diameter, immature schizonts 14-28 microns and hypnozoites 4-7 microns. EE schizonts are stained dark-brown only after conjugated by monoclonal antibody (McAb) 4B2 specific against erythrocytic stages of P. vivax while hypnozoites are only stained after conjugated by McAb 2F2 against sporozoite. These results show that the antigenic components of these two forms of EE plasmodia are quite different. The ratio of EE schizont and hypnozoite found within hepatoma cells (HepG2-A16) is 1.5 to 1. Referred to the clinical manifestations of the isolate, among 5 volunteers not radically cured, two had long incubation period (283 d and 304 d, respectively) and three relapsed 235, 260 and 365 days after the primary attack. These data are unanimous with the comparatively large ratio of hypnozoites in the cultured material.

Effect of monoclonal antibodies on the entry and development of Plasmodium vivax sporozoite in cultured cells.

We have observed the effect of monoclonal antibodies against sporozoites (2F2,NV3,2E10) and blood stages (4B2,8E3) of P. vivax on entry and subsequent development of P. vivax sporozoites in HepG2--A16 cells in vitro. The results demonstrated that inhibitory effects of monoclonal antibodies on the attachment and entry were found to be related to the antibody concentration. At 25 micrograms/ml, the percentages of inhibition of sporozoite invasion were 100% (2F2), 76%(NVS3) and 10.5% (2E10). Even if the invasion was not totally inhibited, the presence of abnormal exoerythrocytic schizonts suggested that continued effect of antibodies after sporozoite penetration still existed. No significant effects of 4B2 (4.5%) and 8E3 (3.4%) were recorded as compared with normal mouse serum. These findings indicate that inhibition of sporozoite invasion assay may be useful for determining the protective effect of anti-sporozoite antibodies in vitro.

[Observation on hypnozoite of different isolates of Plasmodium vivax in cultured materials].

The hypnozoites of different isolates of P. vivax from Shenzhen and Shixin, Guangdong Province, Changsha, Hunan Province and Yincheng, Hubei Province of China in cultured materials were observed employing immunoperoxidase staining method. The percentages of hypnozoites among exoerythrocytic stages were 40.1, 43.5, 50.6 and 57.1%, respectively, indicating that the percentage of hypnozoites and the latitude are in positive correlation. The equation is y = -6.68 + 2.05x. When the difference in latitude of Plasmodium vivax isolate source was more than 5 degrees, a significant difference in the percentage of hypnozoites in various geographic isolates was found. However, the average diameters of hypnozoites and schizonts among various geographic isolates showed no significant difference. The results showed that in the regions north to the Yangtze River, the proportion of hypnozoite in the liver stage of P. vivax was larger than those in the southern China, being consistent with the clinical manifestations.

[In vitro cultivation of the exoerythrocytic stage of Plasmodium vivax (southern China isolate)].

An in vitro culture system for the exoerythrocytic (EE) stage of Plasmodium vivax was first developed in our laboratory in China. Anopheles stephensi mosquitoes were infected by membrane feeding with heparinized blood from a volunteer. After 14-18 days, the mosquito salivary glands were aseptically dissected in culture medium and ground in a tissue grinder to form sporozoite suspension. Sporozoites were counted and added to the cultures of monolayer hepatoma cells at the number of 4.9 x 10(4)-1.3 x 10(5) per cover glass. Sometimes, 8-10 pairs of infected gland were added directly to the cultured cells. On day 7, EE schizonts of P. vivax were found in cultures. In addition, normal erythrocytes (type o) were added to the cultures on day 10 at a concentration of 10(8) per dish. Fourteen days later, erythrocytes in culture supernatant were collected and thin blood films were made. Numerous intra-erythrocytic P. vivax parasites were identified on the films after Giemsa staining. Most intra-erythrocytic forms were rings and early large trophozoites. Schuffer's dots were present in many of the infected cells which were pale and obviously enlarged. These results indicated that the in vitro hepatic cycle of P. vivax was established.

[Comparative susceptibility of different cell lines for culture of Toxoplasma gondii in vitro].

In order to establish a useful cell culture system for T. gondii, we compared the degree of proliferation of T. gondii tachyzoites among 8 different cell lines; 2 kinds of normal animal cells (MDCK-canine kidney cells; Vero-monkey kidney cells) and 6 kinds of human tumor cells (A 549, PC 14-lung cancer cells; SNU 1, SNU 16, MKN 45-stomach cancer cells; HL-60-promyelocytic leukemia cells), through morphological observation and 3H-uracil uptake assay. The degree of susceptibility to infection with T. gondii tachyzoites was highest in A 549 and PC 14 cells, medium in Vero, HL-60, MDCK and SNU 1, and lowest in SNU 16 and MKN 45 cells. The kinetics of T. gondii multiplication during the post-infection 60 hours were highly dependent upon the dose of tachyzoites administered and the duration of cultivation. These results show that A 549 and PC 14 are the most suitable cell lines among the 8 tested for the growth and multiplication of T. gondii in vitro.

Plasmodium falciparum: diversity of isolates from Malawi in their cytoadherence to melanoma cells and monocytes in vitro.

We observed considerable diversity in the cytoadherence of Plasmodium falciparum isolates from Malawi to melanoma cells, U937 cells and human peripheral monocytes. Each isolate exhibited a unique cytoadherence profile for the three human cell types. These isolates generally adhered well to U937 cells and fresh monocytes, moderately to melanoma cells and poorly to TE 671, MIA-Pa-Ca, WI 38, PLC/PRF/5 and HeLa cells. An antimalarial immunoglobulin pool inhibited binding to melanoma cells by 50% or more and to U937 cells by 25% or less. There was no correlation between in vitro cytoadherence to the three cells and clinical disease. These results suggest that malarial adherence ligands exposed on the surface of infected erythrocytes vary from one isolate to another.

Antigen presentation by Toxoplasma-infected cells: antigen entry through cell membrane fusion.

The antigen presentation of cells infected by Toxoplasma gondii (T.g.) for MHC class-I-restricted cytotoxic T cells (CTL) was blocked by treating the T.g.-infected cells with the fungal antibiotic brefeldin A. Electron microscopic analysis of T.g.-infected murine and human-antigen-presenting cells (APC) prepared by the combination of quick-freezing with the deep-etching or freeze-substitution methods revealed that the outer membrane of T.g. fused with some parts of the vacuolar membranes of APC to build up channel-like structures. Work station and cytofluorometry studies detected 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in cells infected by BCECF-labeled T.g., which strongly suggested the direct entry into cells of BCECF derived from T.g. These findings provide evidence that T.g. antigens enter the cytoplasm of APC through cell membrane fusion of T.g. and T.g.-infected APC, and that T.g.-infected APC present T.g. antigen for MHC class-I-restricted CTL through the endogenous pathway of antigen presentation.

Modulation of Pneumocystis carinii adherence to cultured lung cells by a mannose-dependent mechanism.

Pneumocystis carinii is an opportunistic parasite that attaches to the alveolar epithelium during the initiation of pneumonia. It is unknown whether P. carinii recognizes specific receptors on the surface of lung cells. Our study indicates that concanavalin A (Con A), a lectin that recognizes mannose-containing glycoproteins, binds to P. carinii organisms in a saturable manner with a binding affinity of Kd = 11 x 10(-6) mol/L and with 18.5 x 10(6) Con A binding sites per P. carinii organism. Con A binds predominantly to glycoprotein 120, a mannose-rich glycoprotein on the surface of P. carinii. Treatment of cultured target lung cells (A549 cells) with Con A resulted in dramatic reduction of P. carinii attachment, from 34.9% +/- 4.1% to 8.1% +/- 1.3% (p less than 0.001), suggesting that mannose-containing cell surface molecules may be important in P. carinii adherence to target lung cells. In contrast, treatment of P. carinii with Con A resulted in slightly increased adherence of P. carinii when compared with controls. The effects of Con A on P. carinii adherence were reversed when Con A treatments were conducted in the presence of excess mannose, the sugar ligand for Con A. Further, pretreatment of A549 cell monolayers with excess mannose (5000 micrograms/ml) resulted in significant reduction of P. carinii adherence to A549 cells, from 39.4% +/- 2.5% to 28.4% +/- 1.3% (p = 0.003). These studies, for the first time, implicate mannose-containing cell surface molecules as important mediators of attachment between P. carinii and target lung cells.

Cell cycle-dependent entry of Toxoplasma gondii into synchronized HL-60 cells.

The degree of attraction of Toxoplasma gondii to vertebrate cells varies with cell type and cell phase. Human promyelocytic leukemia cells, HL-60, were synchronized by double thymidine block method and co-cultured with Toxoplasma for 1 hr at each cell stage to investigate the cell cycle specific susceptibility of parasites to host cells. For 30 hr the average number of Toxoplasma that invaded was a little changed except at 3 hr from G1/S phase boundary which concurred with the peak point of DNA synthesis. At 3 hr which is a relatively short interval compared to whole S phase, modification of cells by parasitic invasion was most remarkable. The number of Toxoplasma that penetrated was increased to more than six times. The shape of the cells became sludgy and almost indiscernible by strong accessibility of parasites only for an hour of mid-S phase. The same fluctuation was also observed at the second peak of S phase but weakly. This suggests that there be surface molecules concerning with the attachment of Toxoplasma to the host cells, which is expressed at special point of S phase. Further studies on the specific protein or similar molecules related could be carried out using synchronized HL-60 cells.

Elimination of mycoplasmas from cell cultures by a novel soft agar technique.

Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12-21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1-3 days. In separate studies, it was shown that certain Mycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Toxoplasma gondii maintenance in tissue culture: a new efficient method for culturing RH tachyzoites.

We describe here a new tissue culture method for prolonged laboratory maintenance of tachyzoites of the highly virulent RH strain of Toxoplasma gondii. Using a rapidly proliferating murine tumor cell line (YAC-1), the method described is easy to perform and is as or more efficient (both in terms of yield and cost) than other traditional methods for maintenance of the parasite. Furthermore, upon prolonged maintenance (greater than 160 days) in YAC-1 tissue culture, the pathogenicity of the parasite, as well as its capacity to elicit an immune response, are comparable to that of organisms maintained in mice. We conclude therefore, that the method described herein is a suitable alternative to the traditional method of maintenance of virulent RH strain T. gondii tachyzoites.