The role of the cytoskeleton in host cell invasion by Toxoplasma gondii

The protozoan parasite Toxoplasma gondii provides a model system for studying invasion by intracellular parasites belonging to the phylum Apicomplexa. Taking advantage of the versatility of T. gondii for genetic and cell biological studies, we have shown that parasite motility and cell invasion are powered by an actin-myosin based motor in the parasite. Unlike bacterial cell uptake, parasite invasion does not involve significant alterations in the host cell cytoskeleton. Instead, invasion is an active process of penetration into the host cell by the parasite. The force for cell penetration is provided by a unique form of substrate-dependent motility termed gliding. Gliding motility is characterized by the rearward capping of surface membrane proteins that propels the parasite forward in a helical spiral. Both actin and myosin are localized beneath the plasma membrane in the parasite where they presumably combine to produce the force necessary for motility. During cell invasion, the rearward capping of cell surface receptors envelopes the parasite in a unique vacuole derived from the host cell plasma membrane. This system offers insights into force generation and motility in a simple organism that is also an important human pathogen.     

The role of mononuclear phagocytes and dendritic cells in allergic inflammation

Mononuclear-phagocytic system is a diffuse network of cells which includes monoblasts and promonocytes of the bone marrow, blood monocytes, as well as free and fixed tissue macrophage cells. In different tissues and organs macrophages acquire different morphological and functional properties under the influence of the local tissue factors. Interaction of macrophages with other cells and molecules is performed via the large number of different receptors resulting in activation of the macrophage cell, accompanied by a series of morphological and metabolic changes which potentiate all its functions. Activated macrophage cells were found in certain diseases. Macrophages and dendritic cells are associated with all aspects of immunity. Owing to their capacity to undergo phagocytosis they are of the utmost importance for unspecific defense from microorganisms. As accessory cells they also participate in cellular and humoral immunity, being at the same time effector cells owing to their capacity of antigen presentation. Moreover, they also participate in immune response regulation owing to their influence on the function of other cells, including mast cells, basophilic leukocytes and T lymphocytes, in which they may influence differentiation toward Th1 or Th2 and cytokine milieu favorable for allergic reaction. Dendritic cells are the most important antigen-presenting cells and thus, they play a major role in activation of helper T lymphocytes, and mode of antigen presentation is significant for regulation of the nature and intensity of the immune response. Pulmonary macrophage cells have been most thoroughly studied, and the observed changeability of their functional and morphological characteristics is of the utmost importance for studying of the pathogenetic properties and regulation of the chronic inflammatory response in bronchial asthma.     

Induction of resistance to Toxoplasma gondii in human macrophages by soluble lymphocyte products

Autoradiography and light microscopy were used to study the effects of lymphocyte culture supernatants, prepared under a variety of conditions, on the course of intracellular Toxoplasma gondii infection in human monocyte-derived macrophages in vitro. Supernatants prepared by incubating lymphocytes of dye test- (DT) positive subjects with T. gondii lysate antigen (TLA), lymphocytes of DT/negative subjects with streptokinase-streptodornase (SK-SD), or both populations of lymphocytes with concanavalin A (Con A) were capable of activating macrophages to inhibit or kill intracellular T. gondii. Supernatants prepared with the homologous antigen (TLA) to the target organism appeared more active in conferring resistance to infection with T. gondii on macrophages than those prepared with a heterologous antigen (SK-SD) or mitogen (Con A). The number of lymphocytes was critical in preparing active supernatants. These results suggest that soluble lymphocyte mediators can activate human macrophages in vitro to inhibit or kill T. gondii.     

Study of receptor-mediated neurotoxins released by HIV-1-infected mononuclear phagocytes found in human brain

Although there is growing evidence that neurotoxic molecules produced by HIV-1-infected mononuclear phagocytes damage neurons, the precise mechanisms of neuronal attack remain uncertain. One class of cytotoxin involves neuronal injury mediated via the NMDA receptor. We examined blood monocytes and brain mononuclear cells isolated at autopsy from HIV-1-infected individuals for the ability to release NMDA-like neuron-killing factors. We found that a neurotoxic amine, NTox, was produced by blood monocytes and by brain mononuclear phagocytes infected with retrovirus. In vivo injections of minute quantities of NTox produced selective damage to hippocampal pyramidal neurons. NTox can be extracted directly from brain tissues infected with HIV-1 and showed structural features similar to wasp and spider venoms. In contrast to NTox, HIV-1 infection did not increase the release of the NMDA excitotoxin quinolinic acid (QUIN) from mononuclear cells. Although we found modest elevations of QUIN in the CSF of HIV-1-infected individuals, the increases were likely attributable to entry through damaged blood-brain barrier. Taken together, our data pinpoint NTox, rather than QUIN, as a major NMDA receptor-directed toxin associated with neuro-AIDS.     

Regulation of 5-lipoxygenase activity in mononuclear phagocytes: characterization of an endogenous cytosolic inhibitor

The proinflammatory leukotrienes (LT) play important roles in host defense and disease states. However, no endogenous mechanisms to downregulate 5-lipoxygenase (5-LO), the enzyme catalyzing LT synthesis, have been described. We observed that the cytosolic fraction of rat alveolar macrophages (AMs) and peritoneal macrophages (PMs), and of peripheral blood monocytes (PBMs) contain substantial amounts of 5-LO protein, but little detectable 5-LO activity. We therefore examined these mononuclear phagocyte (MNP) cytosolic fractions for inhibitory activity against 5-LO. MNP cytosol dose-dependently reduced the 5-LO activity in neutrophil (PMN) cytosol and AM membrane. Furthermore, MNP cytosol dose-dependently prolonged the lag phase of soybean lipoxygenase (LO) without affecting the rate of product formation. This effect was overcome by subsequent addition of 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD), suggesting that the active factor scavenges hydroperoxides. Inactivation by boiling and roteinase K suggest that is a protein. We speculate that this cytosolic factor(s) may serve as an endogenous means for the down-regulation of 5-LO in macrophages.     

Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells

A therapeutic trial, involving 130 Schistosoma mansoni-infected children, with no previous history of antischistosomal treatment, was carried out to evaluate the efficacy of two different dose regimens of praziquantel. The study was carried out because low cure rates were described in this recently established (1990) S. mansoni focus in northern Senegal, following treatment with a standard dosage of 40 mg/kg. The subjects were randomly allocated into two groups: one group (1) received 40 mg/kg in one oral dose, the other group (2) was treated with two oral doses of 30 mg/kg at a 6-hr interval. Parasitologic examination and circulating anodic antigen (CAA) detection were performed before, 10 days, three, six, and 21 weeks after chemotherapy. No significant differences in cure rates were found between the two groups. Six weeks after treatment, 34% and 44% of the individuals were found to be stool negative in group 1 and group 2, respectively. However, only 10-15% became completely negative according to the serum CAA antigen assay. Mean egg counts were reduced by 99% in both groups. Antigen detection confirmed the parasitologic results. Fewer side effects were observed in the group treated with 2 x 30 mg/kg, which may be explained by split dosage administration. Our study shows that the low cure rates observed in this area could not be improved by using a higher dosage of praziquantel.     

Innate immunity to Toxoplasma gondii is influenced by gender and is associated with differences in interleukin-12 and gamma interferon production

Given that differences between the sexes in relative susceptibility to parasitic infections have been noted, this study further elucidates the mechanisms responsible by demonstrating that male SCID mice are more resistant than female mice to infection with Toxoplasma gondii and that this difference correlates with enhanced innate immune responses in these animals. Male SCID mice exhibited longer survival times, lower parasite burdens, and less severe pathological changes postinfection. An immunological basis for these differences is demonstrated in that these animals produced interleukin-12 more rapidly and exhibited higher levels of gamma interferon earlier postinfection.     

Toxoplasma gondii: an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum.     

In control of its fate: gene regulation in Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii is an important opportunistic pathogen in animals and man. In parallel to its clinical significance, T. gondii is also receiving considerable attention as an attractive model organism for intracellular parasitism. Regulation of gene expression at various levels underlies the intricate interplay between the parasite and its host cell, as well as the interconversions between different life-stages. In this article we will discuss some of what is currently known about gene organization and gene regulation in T. gondii as well as some of the tools available to dissect the parasite at a molecular level.     

Effects of ropivacaine on eicosanoid release from human granulocytes and endothelial cells in vitro

OBJECTIVE: To examine the effects of ropivacaine, currently being investigated for treatment of ulcerative colitis, on the release of arachidonic acid metabolites. MATERIAL: Human granulocytes and endothelial cells. TREATMENT: Ropivacaine, lidocaine, hydrocortisone, 5-aminosalicylic acid or acetylsalicylic acid (10-1000 microM). METHODS: Leukotriene B4, 5-hydroxyeicosatetraenoic acid, 6-keto PGF1 alpha and 15-hydroxyeicosatetraenoic acid were measured using immuno assays. Wilcoxon signed rank test was used for statistical calculations. RESULTS: Ropivacaine dose-dependently inhibited zymosan-induced release of leukotriene B4 and 5-hydroxyeicosatetraenoic acid whereas the release after ionophore stimulation was not affected. Ropivacaine was more potent than 5-aminosalicylic acid but less potent compared to hydrocortisone. Ropivacaine had only a weak inhibitory effect on the release of 15-hydroxyeicosatetraenoic acid from zymosan- or ionophore-stimulated cells. In contrast to hydrocortisone and 5-aminosalicylic acid, ropivacaine only weakly affected the release of 6-keto PGF1 alpha after stimulation with thrombin. CONCLUSIONS: The inhibited release of 5-lipoxygenase products may account for some of the anti-inflammatory effects of ropivacaine seen in the treatment of ulcerative colitis.     

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.     

Release of gelatinase and superoxide from human mononuclear phagocytes in response to particulate Tamm Horsfall protein

This study describes the in vitro activation of human mononuclear phagocytes by particulate Tamm Horsfall protein (THP). Peripheral blood monocytes phagocytosed THP particles with the accompanying release of superoxide radicals, N-acetyl-beta-D-glucosaminidase, and neutral metalloproteinase. Immunoprecipitation and substrate gel analysis identified the neutral proteinase as a 95-kd gelatinase. A comparison with other particulate ligands highlighted the specificity of the response to THP and showed that the magnitude of the response was comparable with that obtained with lipopolysaccharide (100 micrograms/ml). Parallel studies using peritoneal macrophages resulted in a similar pattern of enzyme release and reactive oxygen species synthesis. THP has been implicated in the pathogenesis of tubulointerstitial nephritis associated with reflux nephropathy. The present study indicates that an inflammatory response initiated by a neutrophil-THP interaction may be extended into a chronic phase via the activation of mononuclear phagocytes. The subsequent release of reactive oxygen metabolites and proteinases may contribute to the tissue damage and fibrosis associated with chronic immune-mediated tubulointerstitial nephritis.     

Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T. gondii

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.     

Toxoplasma gondii: metabolism of intracellular tachyzoites is affected by host cell ATP production

PURPOSE: To evaluate different-caliber biopsy cutting needles in terms of the benefits and potential risk of bleeding in a swine model. MATERIALS AND METHODS: A total of 190 sequential liver biopsy specimens were obtained in 11 Yorkshire pigs (weight, 50-70 lb [22.5-31.5 kg]) by using 14-, 18-, and 20-gauge cutting needles. For each biopsy procedure, blood loss was determined by weighing sponges used to absorb bleeding, and sample-tissue DNA content was measured with spectrofluorometry. Analysis of variance was used to compare results. RESULTS: The larger the caliber of needle, the greater the absolute blood loss (for 14-gauge, 1.69 g; for 18-gauge, 0.74 g; for 20-gauge, 0.32 g) and DNA content per sample (for 14 gauge, 40.38 microg; for 18-gauge, 12.18 microg; for 20-gauge, 5.86 microg). The ratio of blood loss to amount of DNA recovered did not differ among the different-caliber needles. To obtain the same amount of diagnostic tissue, more passes were needed with the smaller-caliber needles. CONCLUSION: Use of larger-caliber needles is more efficient despite the greater amount of blood loss, because more tissue can be recovered and because fewer passes are necessary, which reduces the chances of complications.     

Alterations of Toxoplasma gondii induced by 2',3'-dideoxyinosine in vitro

The time-course of action of the antiviral agent 2',3'-dideoxyinosine (ddI) against Toxoplasma gondii tachyzoites in vitro and its effects at the ultrastructural level were investigated. The very short latency of effect and high efficacy of ddI were evidenced by the fact that the drugs' effects on parasite growth occurred 2 hr after addition to the culture medium, and that an IL90 value of 0.5 microg/ml was reached after 72 hr. Although without apparent effect on uninfected cells, ddI clearly acted on the intracellular parasites, which tended to disappear. Remaining tachyzoites were almost exclusively extracellularly located and often exhibited a clustering of mitochondria-like bodies and subsequent deep alterations of their plasma membranes. These results confirm previous findings and emphasize the potential usefulness of ddI in the management of cerebral toxoplasmosis, a major health problem in acquired immune deficiency syndrome patients.     

Sensitized lymphocytes and CD40 ligation augment interleukin-12 production by human dendritic cells in response to Toxoplasma gondii

Dendritic cells (DC) are potent antigen-presenting cells that can stimulate T cell responses by secreting cytokines. During Toxoplasma gondii infection, host immunity is mediated by interferon-gamma, which is induced by interleukin-12 (IL-12). Whether T. gondii infection would stimulate human DC to produce IL-12 was determined. DC were generated from human peripheral blood mononuclear cells cultured with recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human IL-4. DC secreted high levels of IL-12 in response to lipopolysaccharide but not to either live T. gondii tachyzoites or soluble antigen. However, IL-12 production in response to T. gondii was observed when DC were cocultured in contact with lymphocytes isolated from seropositive donors. Ligation of CD40:CD154 was partially essential for IL-12 secretion. These data demonstrate that signals obtained from contact with sensitized lymphocytes are critical for human DC to secrete IL-12 in response to T. gondii.     

Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.     

Caprine toxoplasmosis: abortion, clinical signs, and distribution of Toxoplasma in tissues of goats fed Toxoplasma gondii oocysts

Thirteen goats (9 does and 4 bucks) were each inoculated orally with 10,000 infective Toxoplasma gondii oocysts. Three does and one buck were used as noninoculated controls. In 2 to 4 days after inoculation (DAI), inoculated goats became dull, pyrectic (40 to 41 C), and anorectic. Three goats died (10, 10, and 14 DAI) and two goats were killed (7 and 32 DAI) because they were moribund; also, 3 does aborted, 2 had weak kids, and 2 had dead fetuses. Toxoplasma was isolated from the placenta of three goats, and the fetal tissues of four goats. The control goats remained asymptomatic. The distribution of T gondii in blood and other tissues was studied by inoculation of mice with caprine tissues. Parasitemia was detected in 7 of 7 goats--beginning 4 DAI in 1 goat, 5 DAI in 5 goats, and 8 DAI in 1 goat. The parasitemia lasted 3 to 10 days. Toxoplasma was isolated from the milk of 2 goats at 12 and 14 DAI. Toxoplasma was isolated from 15 or more tissues of 5 goats killed 7 to 35 DAI and from 10 tissues of 2 goats killed 69 and 95 DAI.