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Congenital toxoplasmosis involves Toxoplasma gondii type II strains in 95% of cases in France. We used spatial principal component analysis (sPCA) and 15 microsatellite markers to investigate the spatial genetic structure of type II strains involved in 240 cases of congenital toxoplasmosis in France from 2002 through 2009. Mailing addresses of patients were geo-referenced a posteriori in decimal degrees and categorized into urban or rural areas of residence. No spatial genetic structure was found for type II strains that infected mothers who were living in urban areas, but a global spatial genetic structure was found for those that infected mothers who were living in a rural environment. Our results suggest that sources of infection by T. gondii are different in rural and urban areas in France, and advocate for targeted messages in the prevention of toxoplasmosis according to the type of residence of susceptible people., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is the protozoan parasite responsible for toxoplasmosis, one of the most prevalent zoonoses worldwide. T. gondii infects humans through the ingestion of meat containing bradyzoites or through soil, food or water contaminated with oocysts. Soil contamination with oocysts is increasingly recognized as a major source of infection for humans, but has rarely been quantified directly. In this study, we investigated the spatial pattern of soil contamination with T. gondii over an area of 2.25 km(2) in a rural area of eastern France. The frequency and spatial distribution of T. gondii in soil was analyzed in relation with the factors that could influence the pattern of contamination: cats' frequency and spatial distribution and land use. According to a stratified random sampling Scheme 243 soil samples were collected. The detection of T. gondii oocysts was performed using a recent sensitive method based on concentration and quantitative PCR. Sensitivity was improved by analyzing four replicates at each sampling point. T. gondii was detected in 29.2% of samples. Soil contamination decreased with increasing distance from the core areas of cat home ranges (households and farms). However, it remained high at the periphery of the study site, beyond the boundaries of the largest cat home ranges, and was not related to land use. This pattern of contamination strongly supports the role of inhabited areas which concentrate cat populations as sources of risk for oocyst-induced infection for both humans and animals. Moreover, soil contamination was not restricted to areas of high cat density suggesting a large spatial scale of environmental contamination, which could result from T. gondii oocysts dissemination through rain washing or other mechanisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Toxoplasmosis is characterized by a complex epidemiology. The risk of infection for humans depends on their contact with infective oocysts in a contaminated environment and on the amount of tissue cysts located within consumed meat. Unfortunately, the prevalence of tissue cysts is largely unknown for game species. Although herbivorous game species are a source of infection for humans, the level of infection found in wildlife can also be used to estimate environmental contamination. The aim of this study was to estimate the prevalence of Toxoplasma gondii infection and analyze its temporal dynamics in one population of chamois (Rupicapra rupicapra), one of mouflon (Ovis gmelini musimon), and two of roe deer (Capreolus capreolus) in France, surveyed during a period of 6 to 28 years. Taking into account individual risk factors, we specifically analyzed the relationship between T. gondii prevalence and meteorological conditions that may influence oocyst survival. Serum samples from 101 chamois, 143 mouflons, and 1155 roe deer were tested for antibodies against T. gondii using the modified agglutination test (MAT), an enzyme-linked immunosorbent assay (ELISA) assay, or both. Using MAT with a threshold of 1:6, seroprevalence was 14.7% in mouflon, 16.8% in chamois, and 43.7% in roe deer. In mouflon and roe deer, seroprevalence was positively correlated with age and/or body mass, in accordance with the hypothesis that antibodies have long-term persistence. In roe deer, seropositivity differed between the two populations and changed linearly over time between 1983 and 2010, increasing by a factor 1.75 every 10 years. Moreover, in this species, the highest prevalences were found during dry and cold years or during warm and moist years, depending on the population. Our results suggest that the risk for people to acquire infection through game meat increases over time, but with high variability according to the population of origin and meteorological conditions of the year.

Toxoplasmosis is largely present in rural areas but its spatial distribution in this environment remains poorly known. In particular, it is unclear if areas of high density of cats, the only hosts excreting Toxoplasma gondii, constitute foci of high prevalence. To improve our understanding of the spatial distribution of T. gondii in rural areas, we performed a serological survey in rodents from two villages in France. We trapped 710 rodents including commensal rats and meadow or forest voles and mice. The presence of T. gondii was examined using PCR, mice inoculation and modified agglutination test for antibodies (MAT). We conducted multivariate and discriminant analyses to identify biological, ecological or spatial variables that could explain T. gondii serology in rodents. We then used a logistic regression to assess the relative influence of each explanatory variable. Overall seroprevalence was 4.1%. Commensal-rats were more infected (12.5%) than non-commensal species (3.7%). However, the major determinant of the risk of infection was the distance to the nearest farm (OR = 0.75 for 100 m), which explained the risk in all species or non-commensal species only. We contrast the role of species characteristics and that of the local environment, and discuss the risk of environmental contamination for humans.

Environmental characteristics in water and sediments of two contrasted coastal Mediterranean lagoons, Bages-Sigean and Canet-St Nazaire, were measured over a three season survey. The urban pollution (treatment plant discharges) is very important in Canet-St Nazaire lagoon reflecting untreated sewages, while in Bages-Sigean, the northern part appears more impacted due to larger anthropogenic inputs. Dissolved Cd concentrations are on the whole similar in both lagoons, whereas Cu concentrations are by far higher in lagoon Canet-St Nazaire. Cu concentrations appear to be highly dependent on dissolved organic carbon whereas salinity seems to control Cd variations. Concerning the sediments, the confined northern part of lagoon Bages-Sigean shows organic carbon and total nitrogen enrichment whereas lipid concentrations are much higher in the Canet-St Nazaire lagoon. Cu complexation seems to be strongly related to organic matter as evidenced by the two significant positive relationships, on one hand between Cu and organic carbon, and on the other hand, between Cu and lipids. On the contrary, Cd concentrations appear to be mainly controlled by carbonates. PCBs and PBDEs were detected only in sediments and show relatively low concentrations compared to similar lagoon environments. Regarding the sediment quality guidelines, Cd, Cu and PCBs in both lagoons did not exceed any Probable Effect Concentration (PEC).

Toxoplasmosis is a major zoonosis, and its prevention requires multiple approaches due to the complex life-cycle of its causative agent, Toxoplasma gondii. Environmental contamination by oocysts is a key factor in the transmission of T. gondii to both humans and meat-producing animals; however, its spatial and temporal variations are poorly understood. We analysed the distribution of T. gondii seropositivity in a sample of 210 cats, including the European wildcat (Felis silvestris silvestris), the domestic cat (Felis silvestris catus) and their hybrids that were collected in Central and Eastern France between 1996 and 2006. We searched for spatial variability among communes and temporal variations among years to relate this variability to landscape and meteorological conditions, which can affect the population dynamics of rodent hosts and the survival of oocysts. The overall seroprevalence was 65.2% (95% CI: 58.6-71.4). As expected, adults were more often infected than young individuals, while the occurrence of infection was not related to cat genotypes. Seroprevalence correlated significantly with farm density and the North-Atlantic Oscillation index, which describes temporal variations of meteorological conditions at the continental scale. The highest seroprevalence values were obtained in areas with high farm densities and during years with cool and moist winters. These results suggest that both farming areas and years with cool and wet winters are associated with increased T. gondii seroprevalence in cats. As cat infection determines the environmental contamination by oocysts, climate and landscape characteristics should be taken into account to improve the risk analysis and prevention of T. gondii.

Assessing implicit learning in the continuous pursuit-tracking task usually concerns a repeated segment of target displacements masked by two random segments, as referred to as Pew's paradigm. Evidence for segment learning in this paradigm is scanty and contrasts with robust sequence learning in discrete tracking tasks. The present study investigates this issue with two experiments in which participants (N = 56) performed a continuous tracking task. Contrary to Pew's paradigm, participants were presented with a training sequence that was continuously cycled during 14 blocks of practice, but Block 12 in which a transfer sequence was introduced. Results demonstrate sequence learning in several conditions except in the condition that was obviously the most similar to previous studies failing to induce segment learning. Specifically, it is shown here that a target moving too slowly combined with variable time at which target reversal occurs prevents sequence learning. In addition, data from a post-experimental recognition test indicate that sequence learning was associated with explicit perceptual knowledge about the repetitive structure. We propose that learning repetition in a continuous tracking task is conditional on its capacity to (1) allow participants to detect the repeated regularities and (2) restrict feedback-based tracking strategies.

Foodborne infections are of public health importance and deeply impact the global economy. Consumption of bivalve mollusks generates risk for humans because these filtering aquatic invertebrates often concentrate microbial pathogens from their environment. Among them, Giardia, Cryptosporidium, and Toxoplasma are major parasites of humans and animals that may retain their infectivity in raw or undercooked mollusks. This review aims to detail current and future tools and methods for ascertaining the load and potential infectivity of these parasites in marine bivalve mollusks, including sampling strategies, parasite extraction procedures, and their characterization by using microscopy and/or molecular techniques. Method standardization should lead to better risk assessment of mollusks as a source of these major environmental parasitic pathogens and to the development of safety regulations, similar to those existing for bacterial and viral pathogens encountered in the same mollusk species.

Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC-MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied., (© C. Doliwa et al., published by EDP Sciences, 2013.)

Toxoplasma gondii is a protozoan parasite infecting humans and animals. Wild boars Sus scrofa are a potential source of human infection and an appropriate biological model for analyzing T. gondii dynamics in the environment. Here, we aimed to identify environmental factors explaining the seroprevalence of toxoplasmosis in French wild boar populations. Considering 938 individuals sampled from 377 'communes', overall seroprevalence was 23% (95% confidence interval: [22-24]). Using a Poisson regression, we found that the number of seropositive wild boars detected per 'commune' was positively associated with the presence of European wildcats (Felis silvestris) and moderate winter temperatures.

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.

Monitoring of Toxoplasma infection in animals destined for human consumption is a great challenge for human toxoplasmosis prevention. This study aimed to compare results obtained from a naturally infected population of sheep using different tests and targeting an original matrix: meat samples and muscle fluids collected at the slaughterhouse. A commercial ELISA test was performed on diaphragm fluids from 419 ovine carcasses collected at the slaughterhouse. A MAT (modified agglutination test) was performed on heart fluids obtained from the same animals. In addition, all hearts were bioassayed in mice. Serological test agreement, the relative sensitivity of ELISA MAT and mouse bioassay as well as a correlation between titres and parasite isolation probability were statistically evaluated. The overall agreement (kappa coefficient=0.64) of ELISA on diaphragm fluids and MAT on heart fluids is substantial and subsequently both tests can be used for epidemiological studies. Relative sensitivity was higher for MAT performed on cardiac fluids (90%) than ELISA on diaphragm fluid (61%). For both serological tests, relative sensitivity is lower in lambs younger than 12 months. Relative sensitivity of mouse inoculation was 42%. A significant correlation was obtained between increasing MAT titres and probability to isolate live parasite from the heart. When the fluid titre was higher than 1:16, parasites were isolated in 65% of cases. When it was lower, isolation failed in 95% of the cases. According to our results, cardiac fluids appear to be a relevant matrix for toxoplasmosis survey in meat., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The protozoan parasites Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii are pathogens that are resistant to a number of environmental factors and pose significant risks to public health worldwide. Their environmental transmission is closely governed by the physicochemical properties of their cysts (Giardia) and oocysts (Cryptosporidium and Toxoplasma), allowing their transport, retention, and survival for months in water, soil, vegetables, and mollusks, which are the main reservoirs for human infection. Importantly, the cyst/oocyst wall plays a key role in that regard by exhibiting a complex polymeric coverage that determines the charge and hydrophobic characteristics of parasites' surfaces. Interaction forces between parasites and other environmental particles may be, in a first approximation, evaluated following the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloidal stability. However, due to the molecular topography and nano- to microstructure of the cyst/oocyst surface, non-DVLO hydrophobic forces together with additional steric attractive and/or repulsive forces may play a pivotal role in controlling the parasite behavior when the organism is subjected to various external conditions. Here, we review several parameters that enhance or hinder the adhesion of parasites to other particles and surfaces and address the role of fast-emerging techniques for mapping the cyst/oocyst surface, e.g., by measuring its topology and the generated interaction forces at the nano- to microscale. We discuss why characterizing these interactions could be a crucial step for managing the environmental matrices at risk of microbial pollution.

Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.

Toxoplasmosis is a world-wide infection caused by Toxoplasma gondii. Oocysts disseminated in the environment by infected cats provide a major source of infection for humans and intermediate hosts. The level of soil contamination and the dynamics of this contamination are mostly unknown due to the lack of sensitivity of detection method. Our aim was to improve the detection of T. gondii oocysts in soil samples by comparing three extraction protocols (A, B and C) on unsporulated and sporulated oocysts of different strains and ages, and by testing the effect of sporulation and soil characteristics on oocyst recovery using the most efficient method. The oocyst recovery obtained using protocol C, in which the flotation solution was placed under the sample solution after the dispersion step, was at least ten-fold higher than protocols A and B, in which the sample was just filtered before flotation. The efficiency of protocol C, tested on five artificial soil matrices and four natural soils inoculated with oocysts, was lowest in soils with high proportions of sand. We recommend the protocol C for field investigations, and we advise that results should be interpreted with caution, considering the effect of soil characteristics, especially sand content, on oocyst recovery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Consumption of sheep meat presents a risk of human contamination by Toxoplasma gondii. A nationwide study was conducted in France to evaluate the prevalence of Toxoplasma in fresh ovine meat. A sampling procedure was established to guarantee the representativity of consumption. As is the case for meat consumed, half of the samples were from France and half were imported from other countries. Animals were selected according to their age, as lamb (<12months) represents 90% of the meat consumed. Available data for French samples allowed the selection of 16 districts distributed in seven areas according to their density of production. Diaphragms and hearts from 433 sheep were collected. Diaphragms were collected from 398 imported carcasses. Fluids from hearts and diaphragms were tested serologically. All hearts were bioassayed in mice and parasite isolates were genotyped using PCR-restriction fragment length polymorphism and microsatellite markers. Prevalence estimates were calculated, taking into account uneven distribution of production and age. For French meat, the effect of area, age and their interactions was evaluated. The overall estimate of Toxoplasma seroprevalence was 17.7% (11.6-31.5%) for lambs and 89% (73.5-100%) for adults (P<0.0001). No significant difference was observed between imported and French meat. In France, seroprevalence in lambs showed an increasing North-western to Southern gradient. The proportion of French carcasses carrying live parasites according to bioassay results was estimated at 5.4% (3-7.5%) (45 genotype II; one genotype III). This study offers an accurate drawing of the toxoplasmosis pattern amongst sheep consumed in France and a model for a zoonosis hazard control survey., (2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The ATP-binding cassette (ABC) superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides, but also in cellular processes such as DNA repair, translation or regulation of gene expression. For many years, the role of ABC proteins was mainly investigated for their implication in drug resistance. However, recent studies focused rather on their physiological functions for the parasite. In this review, we present an overview of ABC proteins in major protozoan parasites including Leishmania, Trypanosoma, Plasmodium, Toxoplasma, Cryptosporidium and Entamoeba species. We will also discuss the role of characterized ABC transporters in the biology of the parasite and in drug resistance.

Backgrounds and Aims: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment., Methodology/principal Findings: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats., Conclusions: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

Background: In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver., Methods: Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and beta-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected beta-galactosidase clusters., Results: In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, beta-galactosidase expression was maximum at day 7, but only a few (less than 1%) beta-galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75-fold higher in positive cell clusters than in the surrounding parenchyma., Conclusions: The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period.

The evaluation of Toxoplasma gondii isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6 and GRA7 are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6 and GRA7 from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6 and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasma infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7 locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6 and the GRA7 loci. Serum samples from humans infected with Toxoplasma strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.

We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.

Purpose: Metabolic inherited liver diseases are attractive targets for gene therapy. Recombinant lentiviruses are very powerful viral vectors able to infect nonmitotic cells. We wanted to develop a new surgical approach to improve gene transfer in adult liver using low viral doses., Materials and Methods: Adult rats were injected with 2.108 infectious particles of lentiviral vectors encoding the green fluorescent protein marker gene under control of a liver-specific promoter transthyretin. In the control group (n = 5), gene delivery was performed by inflow intraportal injection. In the surgical group (n = 5), liver was completely excluded from systemic circulation before viral injection in infrahepatic vena cava with high pressure., Results: At day 9, transduction efficiency was 14.35% in the surgical group 3 and 0.39% in the control group (P = .016). At month 2, the number of transduced hepatocytes decreased in the most part of rats, except in half of rats in the surgical group. Antibodies against green fluorescent protein were detected in all rats at month 2, except one in the surgical group., Conclusions: We developed a new surgical approach allowing an efficient transduction of hepatocytes in adult rats using lentivirus at low viral doses. We have now to control the immune response to permit long-term expression of transgene.

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)2 structural organization of the "full" ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.

In urban areas, there may be a high local risk of zoonosis due to high densities of stray cat populations. In this study, soil contamination by oocysts of Toxoplasma gondii was searched for, and its spatial distribution was analysed in relation to defecation behaviour of cats living in a high-density population present in one area of Lyon (France). Sixteen defecation sites were first identified. Cats were then repeatedly fed with marked food and the marked faeces were searched for in the defecation sites. Of 260 markers, 72 were recovered from 24 different cats. Defecation sites were frequented by up to 15 individuals. Soil samples were also examined in order to detect the presence of T. gondii using real-time PCR. The entire study area was then sampled according to cat density and vegetation cover type. Only three of 55 samples were positive and all came from defecation sites. In a second series of observations, 16 defecation sites were sampled. Eight of 62 samples tested positive, originating in five defecation sites. Laboratory experiments using experimental seeding of soil showed that the inoculated dose that can be detected in 50% of assays equals 100-1000oocysts/g, depending on the strain. This study shows that high concentrations of oocysts can be detected in soil samples using molecular methods and suggests that spatial distribution of contamination areas is highly heterogeneous. Positive samples were only found in some of the defecation sites, signifying that at-risk points for human and animal infection may be very localised.

Sulfadiazine, pyrimethamine, and atovaquone are widely used for the treatment of severe toxoplasmosis. Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I. We determined the in vitro activities of these drugs against 17 strains of Toxoplasma gondii belonging to various genotypes and examined the correlations among 50% inhibitory concentrations (IC50s), growth kinetics, strain genotypes, and mutations on drug target genes. Growth kinetics were determined in THP-1 cell cultures using real-time PCR. IC50s were determined in MRC-5 cell cultures using a T. gondii-specific enzyme-linked immunosorbent assay performed on cultures. Mutations in dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), and cytochrome b genes were determined by sequencing. Pyrimethamine IC50s ranged between 0.07 and 0.39 mg/liter, with no correlation with the strain genotype but a significant correlation with growth kinetics. Several mutations found on the DHFR gene were not linked to lower susceptibility. Atovaquone IC50s were in a narrow range of concentrations (mean, 0.06 +/- 0.02 mg/liter); no mutation was found on the cytochrome b gene. IC50s for sulfadiazine ranged between 3 and 18.9 mg/liter for 13 strains and were >50 mg/liter for three strains. High IC50s were not correlated to strain genotypes or growth kinetics. A new mutation of the DHPS gene was demonstrated in one of these strains. In conclusion, we found variability in the susceptibilities of T. gondii strains to pyrimethamine and atovaquone, with no evidence of drug resistance. A higher variability was found for sulfadiazine, with a possible resistance of three strains. No relationship was found between drug susceptibility and strain genotype.

Little is known about the prevalence or importance of Toxoplasma gondii infections in raptors. Sera from Eurasian Buzzards (Buteo buteo, n=14), Tawny Owls (Strix aluco, n=12), Barn Owls (Tyto alba, n=18), Eurasian Sparrowhawk (Accipiter nisus, n=1), and Common Kestrels (Falco tinnunculus, n=8) were examined for agglutinating antibodies using the modified agglutination test at 1:25 dilution. Antibodies were not detected in Common Kestrels and the Eurasian Sparrowhawk but were detected in 11 Eurasian Buzzards (79%), six Tawny Owls (50%), and two Barn Owls (11%). Toxoplasma gondii, genotype II, was isolated from the brain of an adult Tawny Owl.

Serum samples from 218 small mammals trapped in forest and grassland in the Ardennes region (North-eastern France) were tested for antibodies to Toxoplasma gondii. Using the modified agglutination test, positive results were found in 4/92 Apodemus sp., 3/64 Clethrionomys glareolus, 0/26 Microtus agrestis, 0/4 Micromys minutus, 3/5 Sorex sp., 2/9 Arvicola terrestris, and 7/18 Talpa europaea. Toxoplasma gondii was not isolated from the heart of seropositive individuals after bioassay in mice. Seroprevalence was significantly higher in large fossorial mammals living in grassland than in small forest mammals, probably related to ecological factors.

Background/aims: To optimise vaccination strategies for immunotherapy in the liver, we have generated a line of transgenic mice expressing beta-Galactosidase downstream of the alpha-fetoprotein promoter (AFP/betaGal)., Methods: betaGal expression was documented by qRT-PCR, enzyme activity and immunohistochemistry. betaGal-specific CD8+ T-cell activation in mice immunised with various vectors was measured by interferon-gamma ELISpot., Results: Like AFP, betaGal expression was detected in fetal hepatocytes and disappeared around birth. In adult mice, a CD8+ T-cell response to betaGal was observed after immunisation with betaGal adenovirus or plasmid DNA but not with betaGal protein or after retroviral infection. When betaGal was re-expressed in adult hepatocytes, immunisation with betaGal adenovirus triggered T-cell mediated elimination of betaGal-expressing hepatocytes. However, the response was weaker than in AFP/betaGal animals in which betaGal was only present around birth., Conclusions: In AFP/betaGal mice, betaGal is a fetal liver self-antigen. Interestingly, the basal tolerance to betaGal displayed by these animals is increased during liver re-expression of the self-antigen in adulthood. Adenoviral immunisation allows complete elimination of betaGal-expressing hepatocytes in spite of this increased peripheral tolerance. These results highlight the importance of tolerance against self-antigens and validate the AFP/betaGal mice as a good background to test immunotherapy strategies in hepatocarcinogenesis models.

The apicomplexan Toxoplasma gondii, an obligate intracellular parasite, can infect humans and a wide range of vertebrates. Following oral infection, the parasite invades tissues by crossing non-permissive biological barriers such as the placenta or the blood-brain barrier. But the molecular mechanisms underlying migration of T. gondii remain poorly characterized. The crossing of various basal membranes and infiltration into the extracellular matrix by T. gondii could involve matrix metalloproteinases (MMPs). We demonstrated a decrease in proMMP-2 and proMMP-9 secretion by THP-1 cells at 24 and 48h post invasion with regulation at the mRNA level throughout infection. This down regulation was associated with a decrease in TIMP-2 secretion and an inhibition of its expression. Moreover, results showed an activation of MT1-MMP; its expression was regulated after 6, 24, and 48h.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

Crigler-Najjar type 1 disease (CN-1) is a genetic disorder characterized by high levels of unconjugated bilirubin due to the absence of hepatic UDPglucuronosyltransferase (UGT1) activity. Here we show that in vivo neonatal hepatocyte transduction with a lentiviral vector expressing the defective enzyme resulted in long-term correction in Gunn rats, a model of CN-1. Lentiviral vectors harboring the human UGT1 cDNA (approved symbol UGT1A1) under the control of a liver-specific transthyretin promoter were produced. Two-day-old Gunn rats were injected with 50 microl of vector. Bilirubinemia was monitored at 6 weeks and monthly thereafter. At 6 weeks, bilirubinemia was completely normalized in treated animals, whereas it remained around 100 microM in control rats. The level of correction remained stable for up to 42 weeks. Large amounts of bilirubin conjugates were present in the bile of corrected animals. PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was 40% and transduced cells were not detected in extrahepatic tissues except bone marrow in some animals. This work represents the first demonstration of a complete and permanent correction of hyperbilirubinemia in Gunn rats using lentiviral vectors.

Crigler-Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-glucuronosyl transferase (UGT1), resulting in high levels of unconjugated bilirubin. CN1 is an attractive candidate disease for gene therapy. Here we show that in vivo neonatal hepatocyte transduction using recombinant oncoretroviral vectors results in long-term and complete phenotype correction in Gunn rats, a model for CN1. Two-day-old newborn Gunn rats were injected via the temporal vein with 200 microL UGT1 or control beta-galactosidase retroviral vectors. In UGT1-injected animals, bilirubinemia was normal at 6 weeks (3 micromol/L) and remained in the normal range (i.e., <10 micromol/L) for more than 34 weeks. In contrast, in beta-galactosidase-injected animals as well as in noninjected controls, bilirubinemia remained at a high level (i.e., >100 micromol/L) during the whole experimental follow-up. Large amounts of bilirubin monoglucuronides and diglucuronides were present in the bile of treated animals. Finally, polymerase chain reaction and reverse transcription polymerase chain reaction analysis as well as Western blot confirmed the presence and expression of UGT1 almost exclusively in the liver. The estimated proportion of transduced hepatocytes was in the range of 5% to 10%. In conclusion, complete and permanent correction of hyperbilirubinemia in newborn Gunn rats using retroviral vectors can be obtained, paving the way for future gene therapy for CN1.

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.

Gene therapy of inherited hepatic disease relies on sustained expression of the therapeutic transgene. In many instances, such expression will require immune tolerization to the non-self therapeutic transgene product. We previously demonstrated that a cytotoxic immune response eliminated hepatocytes after in vivo transduction using recombinant retroviral vectors. In the present study we investigated whether prior gene transfer to the retina, which is suspected to induce immune tolerance, could alleviate the immune response occurring after retrovirus mediated gene transfer to the liver. Retinal cells were transduced using adeno-associated viral vectors harbouring a beta-galactosidase transgene. Sixty days later, regenerating hepatocytes were transduced after partial hepatectomy using a recombinant retrovirus carrying the transgene. Three weeks later, anti beta-galactosidase antibodies were present in all animals. Elimination of the transduced hepatocytes eventually occurred in all animals by 2 months after liver gene transfer, although sustained beta-galactosidase expression was still present in the retina in 66% of the animals. We conclude that although the retina behaves as an immunoprivileged site, gene expression in the subretinal space is not sufficient to induce immune tolerance to a transgene product expressed in the liver., (Copyright 2004 Springer-Verlag)

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.

Background: Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes. This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross-presentation of the transgene product present as a contaminant in the viral stock. In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long-term transduction of hepatocytes using the nls-lacZ marker gene., Methods: Animals were divided in four groups. In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ. In group 2, using the same strategy, animals received retroviral supernatant that was passed through a beta-galactosidase affinity column to reduce beta-galactosidase contamination. In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection., Results: In group 1, 23.1% of animals had no immune response 2 months after gene delivery vs. 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4. Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant. This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response., Conclusions: Our present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus-mediated gene delivery to the liver., (Copyright 2003 John Wiley & Sons, Ltd.)

Intravenous delivery of nls-lacZ retroviral vectors to the regenerating liver triggers a cytotoxic immune response directed against transduced hepatocytes. We sought to determine whether prior immunization with retroviral vectors impacted on adeno-associated virus (AAV)-mediated muscular expression of the same transgene. The first group of rats first received nls-lacZ retroviral vectors intravenously after a partial hepatectomy. Thirty days later they received AAV vectors intramuscularly in both legs. In the second group, animals received the same vectors in the opposite sequence (i.e., AAV first and retroviruses 20 days later). In the first group, immune response occurred after retrovirus delivery with appearance of anti-beta-galactosidase antibodies and elimination of transduced hepatocytes. However, the immune response did not prevent sustained (9-month) beta-galactosidase expression in AAV-injected muscles. In the second group, AAV injections did not induce immune response and resulted in beta-galactosidase expression in myofibers. In this group, subsequent delivery of retroviral vectors triggered appearance of immune response and elimination of transduced hepatocytes. However, the immune response did not modify beta-galactosidase expression in AAV-transduced myofibers for up to 9 months. These results demonstrate a differential susceptibility between retrovirally transduced liver and AAV-transduced muscles to immune response against the transgene product.

Background and Objective: The practical value of immunological diagnosis of bird-breeder's disease (BBD) is controversial, because of difficulties in distinguishing active disease patients from simple contact subjects. The aim of this study was to determine the diagnostic and prognostic value of (a) presumed disease-associated antibodies precipitating pigeon antigens (immunoglobulin A (IgAp) and P2 component), (b) characterization of specific isotypes (IgG, IgM, and IgA), and (c) antibody kinetics after antigen eradication., Methods: 405 subjects (775 sera) in contact with birds were studied [by means of co-immunoelectrodiffusion (Co-IED) and enzyme-linked immunofiltration (ELIFA)] with soluble extracts of pigeon droppings and squab crop milk. These patients were divided into two groups based on the final clinical evaluation of the patients' physicians, which was taken as the gold standard (positive in 90 and negative in 315 cases)., Results: On the basis of this gold standard, the detection of presumed disease-associated precipitating antibodies by Co-IED had a specificity of 95.5%, a sensitivity of 98.7%, an accuracy of 98%, and positive and negative predictive values of 95.5% and 98.7%, respectively. Most of the patients with a final positive diagnosis of BBD had specific IgG, IgM, and IgA antibodies by ELIFA. After antigen eradication, anti IgAp and/or P2 antibodies disappeared more rapidly than other precipitating systems., Conclusion: Identification by Co-IED of precipitating immune complexes IgAp and/or P2 significantly reinforces the intrinsic credibility of immunological diagnosis of BBD. Compared to these presumed disease-associated precipitating antibodies, detection and time course of specific IgM, IgA antibodies, provided no additional diagnostic value or prognostic arguments to judge disease activity after antigen eradication.

Highly efficient retroviral-mediated gene transfer into hepatocytes in vivo has been previously reported in rats, but some reports described transient expression of the transgene that may be related to induction of an immune response against the transgene product. To devise a surgical approach to circumvent this drawback, two-thirds partial hepatectomy was performed in Wistar male rats to induce the hepatocyte division required to achieve retrovirus integration. Delivery of amphotrophic retroviral vectors (RVV) encoding Escherichia coli beta-galactosidase was performed 24 h after partial hepatectomy. In a first group (n = 11), gene delivery was performed by peripheral injection of 2 ml retrovirus-containing medium. For the second group (n = 11), asanguineous perfusion of the regenerating liver after complete vascular exclusion was carried out with 20 ml viral solution. Liver biopsies were performed sequentially in each group. In the first group, beta-galactosidase was expressed at day 7 in 7 +/- 6.3% of hepatocytes and the labeled hepatocytes had disappeared in less than 4 weeks. Polymerase chain reaction experiments demonstrated the elimination of the transduced cells and the appearance of antibodies against beta-galactosidase. Of the 11 rats in the second group, 8 were still able to express beta-galactosidase more than 6 weeks after asanguineous perfusion with no detectable antibody response. Asanguineous perfusion of the regenerating liver with RVV after complete vascular exclusion enabled long-term expression in rats and avoided the immune response present after peripheral delivery in most animals. These results suggest that the immune reaction is secondary to viral infection of antigen presenting cells. Asanguineous perfusion could thus be a way to perform gene therapy for inherited liver diseases without immunosuppressive therapy.

Gene transfer of reporter genes may trigger immune responses against the heterologous protein resulting in shortening of gene expression and inflammation. We generated transgenic rats expressing the lacZ gene under the control of the human immunodeficiency virus type 1 (HIV-1) long-terminal repeat (LTR) (HIV-lacZ) to obtain rats with undetectable transgene expression using histologic methods, thus avoiding interference with beta-galactosidase (beta-gal) expression from gene transfer, and displaying immune tolerance toward beta-gal. LacZ transgenic mice with tolerance toward beta-gal have already been used for gene transfer but rats constitute unique animal models with several advantages compared to mice. Two transgenic lines displayed low levels of beta-gal mRNA in most organs tested, as detected only by reverse transcription-polymerase chain reaction (RT-PCR). The protein was undetectable by immunohistology and was only detected in the thymus and spleen using a sensitive enzyme-linked immunosorbent assay (ELISA). HIV-lacZ transgenic rats displayed immune tolerance to beta-gal because immunization with beta-gal resulted in markedly lower cellular and antibody responses compared to wild-type controls, whereas immunization with a nonrelated antigen, keyhole limpet hemocyanin (KLH), resulted in comparable immune responses. The usefulness of this model in gene transfer was tested using a retroviral vector, which does not elicit destructive immune responses against transduced cells. Retroviral-mediated nlslacZ gene transfer in the liver resulted in nuclear beta-gal expression for longer than 12 months in HIV-lacZ transgenic rats, whereas wild-type controls showed nuclear beta-gal expression for less than 1 month. After gene transfer of nlslacZ to the liver, antibodies, cytotoxic T lymphocytes (CTLs), and proliferation against beta-gal were detected in wild-type controls but not in HIV-lacZ transgenic rats. In conclusion, HIV-lacZ transgenic rats displaying low beta-gal expression and immune tolerance toward beta-gal are a useful tool to analyze the spatial and temporal expression of the beta-gal protein in gene transfer experiments using lacZ as a reporter gene.

The in vitro regulation of tumour necrosis factor (TNF)-alpha receptors during Toxoplasma gondii infection of human MRC5 fibroblasts and human myelomonocytic THP-1 cells was investigated. Cells were infected with the virulent RH of T. gondii. TNFR membrane receptors were analysed by flow cytometry with biotinylated TNF-alpha. Shedding of the soluble form of TNFR1 and TNFR2 in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and expression of mRNA production of TNFR1 and TNFR2 was analysed by quantitative real-time polymerase chain reaction, 1 h after infection. In the MRC5 cell line, T. gondii infection did not induce any up- or down-regulation of membrane TNFRs, soluble TNFRs or mRNA of TNFRs. However, THP-1 cell infection with living parasites induced a significant soluble TNFR1 release by THP-1 cells after 1 h. We detected an approximately 50% up-regulation (P < 0.01) of soluble TNFR1 in infected THP-1 cells compared to controls. No change in soluble TNFR2 levels was observed in the same conditions. Moreover, infection decreased the level of TNF membrane receptors, but had no effect on TNFR1 and TNFR2 mRNA levels. TNFR modulation by T. gondii infection, in vitro, depends on the cell type. Furthermore, our data suggest that living parasites control the shedding of the soluble form of TNFR1. This mechanism may influence the role of TNF-alpha in toxoplasmosis.

Vectors derived from oncoretroviruses can transduce a small proportion of hepatocytes when injected in the regenerating liver. Transgene expression may be sustained for months without immune response. In striking contrast, we observed a rapid extinction when the intravenous injection of a high input of nuclear beta-galactosidase (beta-gal) expression vector, one day after partial hepatectomy, led to a significant proportion of transduced cells in the liver. Extinction was associated with liver inflammation on tissue sections and appearance of antibodies against the transgene product, while vector genomes became undetectable in liver tissue by PCR. These observations suggested the elimination of transduced cells by an immune response. Transgenic rats tolerant for cytoplasmic beta-gal, or normal rats depleted in CD8 T lymphocytes, steadily expressed the beta-gal vector. In the spleen of normal rats, we detected cytotoxic cells directed against cells expressing beta-gal after the injection of the beta-gal vector. In jaundiced Gunn rats deficient in bilirubin glucuronosyl transferase (BGT1) and treated with a human BGT1 cDNA expression vector, we observed the same kinetics of extinction as well as the appearance of anti-BGT1 antibodies. This study demonstrates that retrovirus-mediated gene transfer may induce cytotoxic T lymphocytes specifically directed against transgene-expressing cells.

Toxoplasma gondii is an intracellular, obligate protozoan parasite that actively invades host cells. Phospholipases A2 (PLA2) are widely distributed enzymes that exist in many isoforms [secretory (sPLA2), cytosolic (cPLA2)] in monocytic and other human cells (pancreatic, kidney, etc) and also in parasites. We examined the effects of inhibitors of sPLA2 type II and cPLA2 on the invasion of human monocytic cells (THP1) by T. gondii (RH strain). We also measured sPLA2 type II and cPLA2 enzyme activities and their modulation by interferon gamma (IFN gamma) in extracts of host cells and parasite. Inhibition of both parasite and THP1 sPLA2 type II, and of parasite cPLA2 reduced the number of infected cells. Enzyme assays and immunoblot analyses demonstrated T. gondii sPLA2 type II and cPLA2 activities and indicated that T. gondii increased the activity of THP1 sPLA2 type II. Incubation with IFN gamma (1,000 units/ml) for 20 h reduced the activities of sPLA2 and cPLA2 in infected and non-infected cells. Thus, IFN gamma blocks the activities of both THP1 and parasite sPLA2 and cPLA2 in membrane fractions, resulting in protection against active invasion by T. gondii.

Genetic labeling using recombinant retroviruses is a powerful strategy for the study of cell lineage in the liver. However, this type of vector is only able to infect dividing cells. The synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic in the adult rat liver. In this study, we used retroviral vectors carrying the nuclear targeted beta-galactosidase gene to selectively label and follow the fate of hepatocytes dividing on administration of CPA. Labeled cells as well as those in mitosis were preferentially located around the portal tract, whereas apoptotic bodies were predominant in the pericentral area. Labeled hepatocytes did not disappear after apoptosis, suggesting a preferential elimination of nontransduced cells. The presence of labeled binucleated hepatocytes showed the persistence of a binucleation process. Finally, we performed long-term analysis of labeled cells in transgenic animals tolerant for beta-galactosidase and treated with 2-acetylaminofluorene (2-AAF) to promote the growth of CPA-initiated hepatocytes. The presence of beta-galactosidase-positive hepatocyte clones showed that hepatocytes divided during treatment with 2-AAF. Only 3% of beta-galactosidase clones were positive for the placental form of glutathione S-transferase (GSTp), indicating the absence of a preferential appearance of preneoplastic foci in the population of beta-galactosidase-labeled hepatocytes. In conclusion, our results show that the mitogenic and tumor-initiating activities of CPA are directed toward different hepatocyte populations.