Toxoplasma gondii-vertebrate cell interactions. II. The intracellular reproductive phase.

The reproduction of Toxoplasma gondii RH-strain in vertebrate cells was studied in a controlled-environment culture system. The lag period before reproduction and the doubling time of individual parasites were determined using a least-squares linear regression method of analysis which does not artificially constrain the data. In the majority of cases, the time intercept of the linear regression line was either zero, implying the lack of a lag phase before reproduction, or negative, implying the parasite had completed part of its reproductive cycle before entering the host cell. The mean doubling time of T. gondii is 10.9 h in bovine embryo skeletal muscle cells and 8.3 h in HeLa cells. This difference is not significant at the 5% level. The population doubling times of mouse-derived parasites is best described by a gamma distribution.     

Trypanosoma cruzi: interaction with vertebrate cells in vitro. IV. Environmental temperature effects

The effects of 4 environmental temperatures (29, 32, 35, and 38 C) on the interaction between Trypanosoma cruzi and bovine embryo skeletal muscle cells were quantified. Three aspects of the interaction (penetration of host cells by trypomastigotes, the lag period prior to the reproductive phase, and the reproductive phase) were markedly affected by temperature. There was a linear increase in the number of trypomastigotes penetrating cells in the 29–35 C range. Temperatures above 35 C can be considered supraoptimal as no further increase in the rate of penetration occurred. The lag period decreased linearly as temperature increased in the 29–35 C range; at 38 C, the lag period was markedly shortened. The doubling time of amastigotes increased linearly as temperature increased in the 32–38 C range; at 29 C, the doubling time was markedly lengthened. At all temperatures, parasites reproduced for 9 generations before cell rupture. The changes in lag period and doubling time complemented each other in the 32–38 C range. Thus, there was essentially no change in the overall length of the intracellular cycle which lasted 6.1 to 6.5 days. At 29 C, however, the cycle was lengthened to 8.9 days. Thermodynamic analysis revealed marked differences, characterized by a negative activation energy and negative enthalpy, between the reproductive phase of parasites within vertebrate cells and the vertebrate cells themselves. However, the thermodynamic parameters of parasites reproducing extracellularly in liquid medium and intracellularly were the same.     

Long-term culture of porcine esophageal epithelial cells without use of feeder layers

Epithelial cells from normal pig esophagus survived in culture for about 4 months undergoing about 12 passages and nearly 40 doublings before showing signs of slow proliferation and senescence. Epithelial cells did not show any attachment and proliferation in serum free media, compared to cells supplemented with 10% serum, where the doubling time was between 48 and 60h. Fibroblasts never became the prominent cell type in these cultures at any given time point. The epithelial cells reacted with antibodies to keratin AE1/AE3, keratin 14 and to involucrin, the differentiating marker.     

Trypanosoma cruzi: Interaction with Vertebrate Cells. DNA Synthesis and Growth of Intracellular Amastigotes and their Relationship to Host Cell DNA Synthesis and Growth

SYNOPSIS DNA synthesis of intracellular Trypanosoma cruzi amastigotes, following the infection of bovine embryo skeletal muscle (BESM) cells, was studied by autoradiography. After penetration, there was a prereplicative lag period (∼12 h) followed by a synchronous round of DNA synthesis which was found to be independent of parasite number/BESM cell and the host cell DNA synthesis cycle. Parasite reproduction occurred, for the first time, at ∼ 21 h postinfection. It was concluded that T. cruzi trypomastigotes are in the G₁/G, phase of their cell division cycle and that after penetration parasite reproduction occurs independent of events controlling host cell DNA synthesis and growth. The early synchronous growth of intracellular amastigotes should facilitate further studies on the biochemical events controlling trypomastigote-to-amastigote transformation and amastigote reproduction. A further application is envisaged for studies on the mode of action of drugs with trypanocidal activity.     

Toxoplasma gondii-Vertebrate Cell Interactions. I. The Influence of Bicarbonate Ion, CO2, pH and Host Cell Culture Age on the Invasion of Vertebrate Cells In Vitro

SYNOPSIS A controlled-environment culture system was used to show that both physical and biologic parameters can influence the penetration of vertebrate cells by Toxoplasma gondii. The optimum bicarbonate ion concentration for the penetration of bovine embryo skeletal muscle (BESM) cells is 36.25 mM. Higher or lower bicarbonate ion concentrations are increasingly inhibitory to penetration. As CO₂ increases in the range from 0.5–3.7 mM, penetration is progressively inhibited. No relationship was found between penetration and pH in the pH range of 6.949–7.765. The culture age of the BESM cells directly influenced the ability of the parasites to penetrate the cells. Older BESM cells were more refractory to penetration than younger cells.     

Heterotrophic nutrition of the marine pennate diatom Navicula pavillardi Hustedt.

Navicula pavillardi Hustedt, a marine, littoral, pennate diatom, can grow in the dark on glutamate or on the complex organic supplements tryptone or yeast extract. Growth on glutamate in the dark took place without an initial lag phase, whereas growth on tryptone began only after a 2-day lag phase that could be abolished by the simultaneous presence of glucose. Lactate inhibited growth in the dark on glutamate, but not photoautotrophic growth. Relatively low concentrations of glutamine inhibited photoautotrophic growth. The observed doubling time for heterotrophic growth on glutamate or tryptone was about 70 h, compared with a doubling time of 24 h under optimal photoautotrophic conditions. Glucose did not decrease the doubling time in the dark on tryptone. The assimilation efficiency for glutamate was 41%. The estimated necessary uptake rate for glutamate to account for the observed heterotrophic doubling time on glutamate was close to those measured with isotope techniques. The kinetic parameters for glutamate uptake, which followed Michelis-Menten kinetics, were Ks = 0.018 mM, and Vmax = 7.0 X 10(-10) mumol per cell per minute. Although several amino acids served as sole nitrogen sources for photoautotrophic growth and were demonstrated by the use of isotope techniques to enter the cells, they could not be used as substrates for growth in the dark. Glucose was not taken up to a significant extent except by cells grown in the presence of tryptone. Lactate was taken up only by dark-grown cells. Results of preliminary studies on the metabolic fate of several uniformly labeled amino acids are presented.     

Trypanosoma Cruzi: Pattern of Rna Synthesis Following Infection of Vertebrate Cells

SYNOPSIS. the pattern of RNA synthesis of intracellular Trypanosoma cruzi amastigotes, immediately following infection of Lesch-Nyhan human fibroblasts, was studied by autoradiography. Amastigote RNA synthesis, determined by [₃H]guanine incorporation, was not detected until 2 h after infection. At 8 h postinfection more than 90% of intracellular amastigotes were labeled. It was verified that extracellular trypomastigotes also synthesized RNA. Therefore it was concluded that, if RNA is required for trypomastigote-to-amastigote transformation, this nucleic acid is already present in the trypomastigotes before infection of the vertebrate cell. It is probable that the RNA synthesized by amastigotes during the prereplicative lag period (the period between initial infection and the onset of DNA synthesis) is required for intracellular growth and reproduction.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Male diploid embryonic cell line ofDrosophila virilis

Summay A new established cell line 79f7Dv3g, ofDrosophila virilis consisting initially of male and female cells and represented now, after 6 yr of cultivation, only by male cells is described. The population doubling time is 36 h at 25° C. The cell culture is also able to grow in serum-free media for an indefinite time without special selection and has a population doubling time of 2 d.     

Comprehension of attachment and multiplication properties by observing individual cell behaviors in anchorage-dependent culture

The behavioral properties of cell attachment and division were characterized by direct observation of individual cells in the culture of murine fibroblasts. At the cell attachment stage in the culture for early 10 h, the extent of cell spreading, which was defined as a ratio of the projected area of each cell against its saturated value, had a relatively broad distribution at 0.25 h, and it shifted to a higher level with elapsed time up to 10 h with narrowing in the distribution. The critical value of the extent of cell spreading was determined to be 0.54 as a threshold at which a cell is assumed to complete its adhesion to culture surface. The ratio of the number of cells with the extent of cell spreading over 0.54 against the total number of examined cells fairly followed the profile of cell adhesion which was obtained by measuring the number of adherent cells on culture surface.

At the cell growth stage in the culture for 20–64 h, doubling time of cell population increased gradually as the culture progressed toward confluence. Generation times (or cell-dividing spans) of individual cells, however, did not show a discriminating dependency on cell concentration and culture time. To clarify the influence of local congestion on the cell division, the generation time was formulated as a function of the number of contact cells around each target cell. Applying the cell placement growth model to estimating the extent of contact inhibition, the reciprocal value of doubling time could be correlated with the average of reciprocal generation times, implying that the doubling time on a cell-population basis is explained by considering the variation in dividing spans of individual cells affected by local contact environment.     

Synthesis of ribosomal and transfer ribonucleic acids in yeast during a nutritional shift-up.

The growth rate of Saccharomyces cerevisiae was increased by adding a mixture of amino acids to cultures containing proline as the sole nitrogen source. The transition from balanced growth in the basal medium (doubling time 4 h) to balanced growth in the enriched medium (doubling time 2 h) took about 2-5 h. The rate of RNA accumulation increased soon after the enrichment to almost its final value. This increase began after a short lag of 10 to 15 min, therefore synthesis of new RNA polymerase molecules may be required before stable RNA production can increase. The different stable RNA species were not stimulated at different times after the enrichment, but all increased continuosly throughout the transition. The rRNA species accumulated in a co-ordinate fashion at a rate faster than the rate of tRNA accumulation.     

Basic studies on gene therapy of human malignant melanoma by use of the human interferon beta gene entrapped in cationic multilamellar liposomes. 1. Morphology and growth rate of six melanoma cell lines used in transfection experiments with the human interferon beta gene

Six cell lines of human malignant melanoma: A375, A375.2, G361, HMV-1, MM8.1 and WM115 were seeded at densities of 1 × 10⁴ cells/ml, 2 × 10⁴ cells/ml or 3 × 10⁴ cells/ml of RPMI medium supplemented with 10% fetal calf serum and antibiotics in a humidified atmosfere of 5% CO₂ at 37°C. A375 cells were also grown in Dulbecco's minimum Eagle's medium (DMEM medium). The morphology was studied by phase contrast light microscopy. At 4 days after seeding the colonies of A375 cells and HMV-1 cells were oval-shaped, the cells were polyhedrical and were making contact with each other regularly. The remaining cells were scattered, more elongated, and made contact randomly. G361 cells and MM8.1 cells tended to form superposed layers before 100% confluency was achieved. There were great differences in the growth rate and doubling time of melanoma cells. The doubling time in day 1 was short (around 6-12 h) in the case of A375, G361 and HMV-1 cells, longer (around 18h) in the case of MM8.1 cells and very long (ranging between 26 and 89 h) for A375.2 and WM115 cells. There were also differences in the doubling time of cells as a function of the cell density at seeding. On the other hand, except for MM8.1 cells, there were differences between the doubling time in day 2 compared to day 1.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation.

The central nervous system (CNS) of the intermediate host plays a central role in the lifelong persistence of Toxoplasma gondii as well as in the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised patients. In order to analyze the parasite-host interaction within the CNS, the host cell invasion, the intracellular replication, and the stage conversion from tachyzoites to bradyzoites was investigated in mixed cultures of dissociated CNS cells from cortices of Wistar rat embryos. Two days post infection (p.i.) with T. gondii tachyzoites, intracellular parasites were detected within neurons, astrocytes, and microglial cells as assessed by double immunofluorescence and confocal microscopy. Quantitative analyses revealed that approximately 10% of neurons and astrocytes were infected with T. gondii, while 30% of the microglial cells harbored intracellular parasites. However, the replication of T. gondii within microglial cells was considerably diminished, since 93% of the parasitophorous vacuoles (PV) contained only one to two parasites which often appeared degenerated. This toxoplasmacidal activity was not abrogated after treatment with NO synthase inhibitors or neutralization of IFN-gamma production. In contrast, 30% of the PV in neurons and astrocytes harbored clearly proliferating parasites with at least four to eight parasites per vacuole. Four days p.i. with tachyzoites of T. gondii, bradyzoites were detected within neurons, astrocytes, and microglial cells of untreated cell cultures. However, the majority of bradyzoite-containing vacuoles were located in neurons. Spontaneous differentiation to the bradyzoite stage was not inhibited after addition of NO synthase inhibitors or neutralization of IFN-gamma. In conclusion, our results indicate that intracerebral replication of T. gondii as well as spontaneous conversion from the tachyzoite to the bradyzoite stage is sustained predominantly by neurons and astrocytes, whereas microglial cells may effectively inhibit parasitic growth within the CNS.     

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.     

Functions of the various IgG Fc receptors in mediating killing of Toxoplasma gondii.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.     

Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion. METHODS: We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment. RESULTS: The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms. CONCLUSIONS: The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.