[Cell-host-parasite interactions: biodiversity, pathogenesis, environment]. / Interactions cellules-hôtes-parasites: biodiversité, pathogénie, environnement.

The apicomplexan Toxoplasma gondii, an obligate intracellular parasite, can infect humans and a wide range of vertebrates leading to toxoplasmosis. This generally benign affection can causes severe life-threatening disease, particularly in immunocompromised patients and in children with congenital toxoplasmosis. Our research team works on cell-host-parasite interactions by studying biodiversity, pathogenic mechanisms and environment. We search to identify prognostic factors of disease and markers of resistance. This project is an integral part of our Research Institute (IFR53) which receives support from the Toxoplasma Biological Resource Center for constituting a bank of well characterized toxoplasma isolates for genotyping, clinical and epidemiological data. The involvement of metalloproteinases implicated during monocytic cell invasion and identification of ABC transporter proteins in T. gondii, factors implicated in resistance, need to be explored.

Signaling during the invasion of host cells by Toxoplasma gondii.

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.

Quantitative determination of free calcium in subcellular compartments, as probed by Indo-1 and confocal microspectrofluorometry.

Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+]i). To perform in situ calibrations of [Ca2+]i in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+]i, intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue-shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+]i, intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+]i. The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+]i determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+]i in the cytoplasm than in the nucleus (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of calcium and calmodulin in Toxoplasma gondii tachyzoite invasion.

The tachyzoite of Toxoplasma gondii must successfully invade a host cell before it can replicate. Depletion of the Ca2+ in the external medium (EGTA) reduced tachyzoite invasion, suggesting that the initial tachyzoite-host cell interaction is Ca2+ dependent. The interaction of tachyzoites with host cells was also inhibited by Ca2+ channel blockers (verapamil) and calmodulin antagonists (trifluoperazine, calmidazolium). The calmodulin concentrated at the apical end of the tachyzoite could be involved in cytoskeleton movement and conoid extrusion. Invasion also depends on changes in tachyzoite cytosolic calcium. Depletion of Ca2+ with A23187+EGTA and release of Ca2+ from intratachyzoite pools (nuclear and perinuclear areas) inhibited invasion. In contrast, Ca-ionophore and thapsigargin which increase host cell cytosolic Ca2+, significantly decreased tachyzoite invasion. We therefore suggest that the effect of the drug is significantly different from the localized Ca2+ signal that is produced after parasite attachment to its host cell receptors and leads to its internalization into the host cell.

ELIEDA (enzyme-linked-immuno-electro-diffusion-assay): application of a combined immunoelectrodiffusion and immunoenzyme method to the study of immune response in parasitic infections.

The sensitivity of the technique of immunoelectrodiffusion (IED) has been enhanced by combining it with an enzyme method; this also enabled the class of immunoglobulins involved in the reactions to be determined by treating the precipitated immune complexes with enzyme-labelled class-specific antisera. The method has been employed in studies of the immunology of parasitic infections and these confirmed its sensitivity and specificity. ELIEDA provides a valuable tool for analysing responses to complex immunogens.

An enzyme-linked immuno-filtration assay used to compare infant and maternal antibody profiles in toxoplasmosis.

Enzyme-linked immuno-filtration assay is carried out on a micropore membrane. This doubly analytical technique permits simultaneous study of antibody specificity by immunoprecipitation and characterisation of antibody isotypes by immuno-filtration with enzyme-labelled antibodies. Recognition of the same T. gondii antigenic constituent by IgG, IgA, IgM or IgE antibodies produces couplets (IgG-IgM; IgG-IgA) or triplets (IgG-IgM-IgA; IgG-IgM-IgE) which identify the functional fractions of the toxoplasmosis antigen. In acquired toxoplasmosis, the persistence of IgM antibody long after infestation puts in question the implication of recent infestation normally linked to detection of this isotype. For sera of comparable titres, comparison of immunological profiles by the method described demonstrates disparities in the composition of the specific antibody content as expressed in international units. Use of the same method to detect IgM antibodies or distinguish between transmitted maternal IgG and IgG antibodies synthesised by the foetus or neonate makes a diagnosis of congenital toxoplasmosis possible in 85% of cases during the first few days of life. With the method described the diagnosis may be made on average 5 months earlier than with classical techniques. In the course of surveillance for latent congenital toxoplasmosis, the appearance of IgM or IgE antibodies raises the possibility of complications (hydrocephalus, chorioretinitis). After cessation of treatment, a rise in IgG antibodies indicating persistence of infection is detected earlier by the present than by classical methods.

Detection of staphylococcal enterotoxin B. A comparative study of ELISA and ELIFA systems.

The enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunofiltration assay (ELIFA) sandwich methods have been compared for their ability to detect staphylococcal enterotoxin B (SEB) in biological samples. Using the avidin-biotin amplification system in the ELISA, high sensitivity was observed and staphylococcal protein A interference was completely prevented. In contrast, ELIFA was less sensitive than ELISA, but very easy to perform, fast and simple to read routinely.

Enzyme-linked immuno-filtration assay applied to accelerated immunodetection of gel transferred proteins on immobilizing matrices.

The enzyme-linked immunofiltration assay (ELIFA) has been used for the rapid detection of electrophoresed proteins transferred from gels to immobilizing matrices. By controlled filtration, ELIFA permits the saturation of nylon or nitrocellulose membranes and the immunodetection of blotted antigens to be carried out in 15 min. The method is simple, can be automated and requires no handling of membranes. It complements the well standardized steps of gel electrophoresis and semi-dry horizontal electroblotting which can themselves be carried out in less than an hour. The sensitivity is at least 1-5 ng. The same process can be extended to the accelerated characterization of glycoproteins using appropriate ligands, or to the identification of antigens in a variety of biological fluids.

Automated reading and processing of quantitative IgG, IgM, IgA, and IgE isotypic agglutination results in microplates. Development and application in parasitology-mycology.

Microplate agglutination techniques represent a simple and commonly used approach for the quantitative or qualitative isotypic analysis of specific antibodies. However, they require optical reading by the investigator and are thus prone to an important degree of variability. In order to solve some of the problems associated with the variability of optical readings, we have used an automatic reader scanning each of the 96 wells of a standard microplate in 32 different locations. The inherent advantages of the automatic reader were further maximized by coupling it to a dedicated computer running customized software designed to process data coming on-line from the spectrophotometer. This approach has been applied to the diagnosis of human toxoplasmosis and candidosis. Suspensions of Toxoplasma gondii tachyzoites or of sensitised erythrocytes were used for the determination of IgG antibodies or the quantification of IgM, IgA, or IgE specific isotypes. This procedure allows the simple and reproducible collection of objective results. Moreover, it permits a reduction in cut-off values and direct interpretation of results with automatic conversion of scores into titer, units, index, or into any other scale appropriate for standardization purposes.

An anti-human mu chain monoclonal antibody: use for detection of IgM antibodies to Toxoplasma gondii by reverse immunosorbent assay.

A precipitating anti-human mu chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1 kappa) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, lambda and kappa chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the C mu 2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the C mu 2 domain, such a McAb could be used for IgM capture in the detection of specific IgM to Toxoplasma gondii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human mu chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.

Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii.

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii.

The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actin-myosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca(2+)-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosin-CaM complex and the steps of a signaling pathway for parasite motility.

Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of toxoplasma gondii.

Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:1411-1421, 1998)

Membrane potential changes after infection of monocytes by Toxoplasma gondii.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.

Value of the comparative enzyme-linked immunofiltration assay for early neonatal diagnosis of congenital Toxoplasma infection.

BACKGROUND: The transplacental transfer of specific maternal IgG antibodies makes the diagnosis of congenital Toxoplasma infection quite difficult in the neonate. The enzyme-linked immunofiltration assay (ELIFA), comparing at delivery the immunologic profile of the mother's antibody response and that of her child, allows discrimination between IgG antibodies of maternal origin and IgGs synthesized by the fetus. OBJECTIVE: To evaluate the diagnostic reliability of the comparative ELIFA for diagnosing congenital Toxoplasma infection as well as the reliability of testing for IgM- and IgA-specific antibodies in cord blood. METHODS: From November, 1991, to December, 1995, an ELIFA was prospectively performed at delivery on blood samples obtained from 227 women with primary Toxoplasma infection during pregnancy and from their infants. For each child the ELIFA result was evaluated in relation to the serologic follow-up: disappearance of specific anti-Toxoplasma gondii IgG antibodies in the absence of treatment before 12 months of age indicating an uninfected child, as opposed to persistence beyond 12 months of age indicative of a congenital infection. RESULTS: Of 227 children 139 were lost to follow-up. Among the 88 children available for follow up, the ELIFA was negative in 70 infants, 69 of whom were confirmed to be uninfected. Thirteen of these 69 cord blood ELIFA-negative samples were positive for anti-T. gondii IgM and/or IgA detected by means of a conventional immunosorbent agglutination assay. Of the remaining 18 children (representing 75% of all new cases of congenital toxoplasmosis diagnosed during the study period at our institution), the ELIFA was positive in 16, negative in 1 and inconclusive in 1. CONCLUSIONS: The ELIFA test is a valuable tool for diagnosing congenital T. gondii infection and in differentiating between true neonatal infection and cord blood contamination. In our experience the diagnostic sensitivity of the ELIFA test was 94.1% and the specificity was 98.6%. The cord blood was contaminated by specific maternal anti-T. gondii IgA and/or IgM in as many as 20% of the cases.

Subcellular localization and quantitative analysis of Toxoplasma gondii target antigens of specific immunoglobulins G, M, A, and E.

The target antigens of specific immunoglobulins G, M, A, and E from patients with acquired acute toxoplasmosis were determined using immunocytochemistry. The relative repartition of these antigens in four cellular compartments of Toxoplasma (membrane complex, apical area, rhoptries, and dense granules) was quantitatively evaluated. Rhoptry antigens mainly react positively with IgA. Membrane, submembrane area (membrane complex), and rhoptry antigens are immunodominant for IgA and IgM. Apical area antigens are recognized by IgM two times more than IgG and IgA. IgE recognized only rhoptry antigens. The localization of pathogenetically antigenic components and their identification by the immune system appeared to be of importance for selection of immunodominant or recombinant antigens. Such localization would improve laboratory diagnosis of serious congenital toxoplasmosis or in immunocompromised patients with toxoplasmic complications after cyst reactivation.

Subcellular calcium localization in Toxoplasma gondii by electron microscopy and by X-ray and electron energy loss spectroscopies.

The localization of calcium in Toxoplasma gondii tachyzoites was studied at the ultrastructural level, with a cytochemical pyroantimonate precipitation method (PA) and controlled by EGTA chelating and EDX and EELS microanalyses. Appropriate conditions for material preparation, fixation and embedding, were defined. The proportion of precipitates that were either free or inside vacuoles and their distribution inside Toxoplasma appeared to be PA dose-dependent. Precipitation mainly occurred in the anterior pole of the Epon-embedded tachyzoites. EDX and EELS analyses showed that out of 30 PA precipitates inside tachyzoites, 78% contained Ca. In Melamine sections, 96% of the tachyzoites had intracellular precipitates and the membrane complex was stained; 25% of the tachyzoites inside host cells contained PA-Ca precipitates, but most of them were retained in the reticular network of the parasitophorous vacuole. Melamine embedding appeared to improve the preservation of calcium pyroantimonate precipitates.

Immunological study of hydatidosis. I. Evaluation of immunoelectrodiffusion tests and enzyme-linked immunoelectrodiffusion assay (ELIEDA) in human hydatidosis.

The comparative sensitivity, specificity and rapidity of immunoelectrodiffusion (IED) on cellulose acetate membranes and of immunoelectrophoresis (IEP) on agarose gel, were evaluated in the diagnosis of hydatidosis. Pooled crude fluid from several cattle hydatis cysts was used as antigen in tests on 1,750 non-hydatis and 400 hydatis sera obtained from patients with hepatic, pulmonary, splenic, peritoneal and cerebral hydatidosis before and after surgery. Coupled to a specific human reference serum for arc 5, IED shows a specificity comparable to that of IEP but its sensitivity is slightly higher and the amount of antigen needed is very small. The appearance of a typical "gloved finger" pattern in sera from patients with ruptured cysts emphasizes the interest of quick results (3 hours) obtained by this method. In order to increase the sensitivity of IED and to define the class of immunoglobulins involved in the antigen-antibody reaction, we have coupled this method to an enzymatic technique. The immune complexes precipitated by IED were treated with peroxidase-labelled antibodies specific to each class of human immunoglobulins. The specificity of this enzyme-linked immunoelectrodiffusion assay (ELIEDA) permits one to follow the immunologic evolution of hydatidosis and to identify IgM in ruptured hepatic cysts and IgA in pulmonary cysts.

Immunological studies in amebiasis: isotypic characterization of specific antibodies by enzyme-linked immunofiltration assay.

Fifty sera from 17 cases of invasive amebiasis (15 hepatic localizations, 1 ameboma, 1 diffused colic localization) were studied by enzyme-linked immunofiltration assay (ELIFA). IgM and IgE anti-Entamoeba histolytica were detected in 10 and 15, respectively, of the 17 patients studied. IgM and IgE antibodies were found simultaneously in 10 cases; these 2 isotypes were only recognized twice in the same serum by the same antigenic components. During post-therapy monitoring, in less than 90 days IgE or IgM-Ab regressed completely in half the cases. If they appeared or reappeared after the third month, prognosis was bad. In addition to its value for diagnosis and prognosis, the ELIFA allowed us to detect the functional antigenic components revealed more particularly by some IgG, IgM, IgE, or IgA antibodies.

Dirofilariasis of the breast in france.

Sections of a nongravid adult female Dirofilaria, probably D. repens, were found within a subcutaneous nodule excised from the breast of a 45-year-old Frenchwoman. This is the 29th case of D. repens in man in France and the first in which the parasite was located in the breast. The patient apparently acquired the infection while vacationing in the southeastern Mediterranean region of the country.