Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean Fran?ois Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.     

Development of an in vitro model of Toxoplasma gondii cyst formation

Abstract Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.     

Chagas' disease and AIDS

Chagas' disease caused by Trypanosoma cruzi is an opportunistic infection in the setting of HIV/AIDS. Some individuals with HIV and chronic T. cruzi infection may experience a reactivation, which is most commonly manifested by meningoencephalitis. A reactivation myocarditis is the second most common manifestation. These presentations may be difficult to distinguish from toxoplasmosis in individuals with HIV/AIDS. The overlap of HIV and Trypanosoma cruzi infection occurs not only in endemic areas but also in non-endemic areas of North America and Europe where the diagnosis may be even more difficult. The pathological features, diagnosis and the role of cytokines in the pathogenesis of the disease are discussed.     

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34⁺ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.     

The control of calcium current reactivation by catecholamines and acetylcholine in single guinea-pig ventricular myocytes

The time course of reactivation of the calcium current in isolated single cardiac cells is complex. The rising phase is sigmoid and there is an overshoot. Catecholamines increase the initial rate of reactivation but reduce or abolish the overshoot. This combination of effects results in a 'crossover', so that the net effect of adrenaline depends on the pulse interval used. Acetylcholine not only reduces the current amplitude, it also substantially slows recovery. At short intervals the effect of acetylcholine is therefore very large. Agents that increase intracellular cyclic AMP levels affect the amplitude of the current but do not have a large effect on the reactivation time course. It is suggested that the autonomic transmitters exert their effects by controlling the local calcium concentration near the inner surface of the channels. This is supported by the fact that there are natural variations in reactivation time course between different cells and that these are correlated with their calcium loading, as judged by other electrophysiological criteria, such as the speed of calcium current inactivation and the presence of the calcium-dependent slow inward current.     

Cytokine and antibody responses of reactivated murine toxoplasmosis upon administration of dexamathasone

Toxoplasma gondii has been shown to result in life-threatening encephalitis in immunocompromised patients after reactivation of dormant parasites. In order to obtain information on immune responses related to this phenomenon, BALB/c mice were infected with 25 cysts of the 76K strain of T. gondii, then, treated orally with dexamethasone (Toxo/Dexa-treated group) in order to reactivate the chronic toxoplasmosis. None of the T. gondii-infected mice died during the experimental periods, whereas the Toxo/Dexa-treated mice evidenced a significant attenuation of survival periods. Toxoplasma-specific IgG2a, IgA and IgM titers in sera were significantly depressed in the Toxo/Dexa-treated mice; however, the IgG1 sera titers were similar to those seen in the Toxoplasma-infected mice. The percentages of CD4+ and CD8 alpha + T cells in the Toxo/Dexa-treated mice were significantly reduced 2 weeks after dexamethasone treatment. IFN-gamma and IL-10 production levels in the Toxo/Dexa-treated mice were depressed significantly, whereas IL-4 production was increased temporarily. The expression levels of the Toxoplasma-specific P30 and B1 genes were found to have been increased in the Toxo/Dexa-treated mice in comparison with the Toxoplasmainfected mice. Collectively, the findings of this study demonstrate that reactivation of murine toxoplasmosis as the result of dexamethasone treatment induced a depression in Th1 immune responses, whereas Th2 immune responses were not significantly influenced.     

Use of IgG Avidity test in case definitions of toxoplasmosis in pregnancy

A survey network for congenital toxoplasmosis (TOXO-NET) was set up in December 1996 in Piedmont (Italy). Participants were asked to classify the infections in pregnant mothers and newborns by the criteria of the European Network on Congenital Toxoplasmosis published by Lebech in 1996. Because the IgG Avidity test is largely employed as a 2nd level test in toxoplasmosis diagnosis and it could be helpful to date infection, the co-ordinators of TOXO-NET suggested including it in the "case definition" of "probable" infection and "unlikely" infection. 117 cases of toxoplasmosis in pregnancy divided into the risk categories under Lebech's criteria were re-examined using the "new" case definitions. 77 out of 117 (65.8%) Toxoplasma gondii infections during pregnancy could be defined with only one serum sample using the IgG Avidity test. The IgG Avidity test proved a useful method to classify the Toxoplasma gondii infections in pregnancy, especially when we had only one serum sample.     

Toxoplasmosis and Epilepsy — Systematic Review and Meta Analysis

Background

Toxoplasmosis is an important, widespread, parasitic infection caused by Toxoplasma gondii. The chronic infection in immunocompetent patients, usually considered as asymptomatic, is now suspected to be a risk factor for various neurological disorders, including epilepsy. We aimed to conduct a systematic review and meta-analysis of the available literature to estimate the risk of epilepsy due to toxoplasmosis.

Methods

A systematic literature search was conducted of several databases and journals to identify studies published in English or French, without date restriction, which looked at toxoplasmosis (as exposure) and epilepsy (as disease) and met certain other inclusion criteria. The search was based on keywords and suitable combinations in English and French. Fixed and random effects models were used to determine odds ratios, and statistical significance was set at 5.0%.

Principal findings

Six studies were identified, with an estimated total of 2888 subjects, of whom 1280 had epilepsy (477 positive for toxoplasmosis) and 1608 did not (503 positive for toxoplasmosis). The common odds ratio (calculated) by random effects model was 2.25 (95% CI 1.27–3.9), p = 0.005.

Conclusions

Despite the limited number of studies, and a lack of high-quality data, toxoplasmosis should continue to be regarded as an epilepsy risk factor. More and better studies are needed to determine the real impact of this parasite on the occurrence of epilepsy.     

Depletion of CD4+ T cells but not inhibition of the protective activity of IFN-gamma prevents cure of toxoplasmosis mediated by drug therapy in mice.

Toxoplasmosis is a frequent opportunistic infection in patients with AIDS. In these patients the major immune deficiencies are a severe depletion of CD4+ T lymphocytes and an impaired capacity to produce IFN-gamma. A mouse model was developed and used to study the effects that depletion of CD4+ T cells and/or inhibition of the protective activity of IFN-gamma have on the effectiveness of the drug therapy for toxoplasmosis. Infection of mice with a lethal inoculum of Toxoplasma gondii cysts followed by treatment with the hydroxynaphthoquinone 566C80 or with sulfadiazine resulted in 100% survival whereas untreated controls had 100% mortality within 15 days of infection. Administration of antiserum to IFN-gamma resulted in early death of untreated mice and in 30% mortality in those treated. Administration of mAb to CD4+ T cells followed by infection with T. gondii prevented the development of both antibody and cell-mediated immune responses against the parasite. These mice resisted the acute infection while undergoing specific treatment. Discontinuation of the treatment, however, resulted in reactivation of the infection and the majority of the animals died within 17 days of suspension of the treatment. Administration of antiserum to IFN-gamma or to CD4+ T cells 24 h but not 15 days after conclusion of the treatment also resulted in mortality. These results indicate that successful treatment of toxoplasmosis depends on the status of the immune system, particularly of CD4+ T cells. Although it is speculative to compare results obtained in mice to the situation in humans, our work suggests that restoration of a competent immune response is of crucial importance for a successful treatment of toxoplasmosis in immunocompromised individuals.     

Performance of Polymerase Chain Reaction Analysis of the Amniotic Fluid of Pregnant Women for Diagnosis of Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis

IntroductionCongenital infection caused by Toxoplasma gondii can cause serious damage that can be diagnosed in utero or at birth, although most infants are asymptomatic at birth. Prenatal diagnosis of congenital toxoplasmosis considerably improves the prognosis and outcome for infected infants. For this reason, an assay for the quick, sensitive, and safe diagnosis of fetal toxoplasmosis is desirable.GoalTo systematically review the performance of polymerase chain reaction (PCR) analysis of the amniotic fluid of pregnant women with recent serological toxoplasmosis diagnoses for the diagnosis of fetal toxoplasmosis.MethodA systematic literature review was conducted via a search of electronic databases; the literature included primary studies of the diagnostic accuracy of PCR analysis of amniotic fluid from pregnant women who seroconverted during pregnancy. The PCR test was compared to a gold standard for diagnosis.ResultsA total of 1.269 summaries were obtained from the electronic database and reviewed, and 20 studies, comprising 4.171 samples, met the established inclusion criteria and were included in the review. The following results were obtained: studies about PCR assays for fetal toxoplasmosis are generally susceptible to bias; reports of the tests’ use lack critical information; the protocols varied among studies; the heterogeneity among studies was concentrated in the tests’ sensitivity; there was evidence that the sensitivity of the tests increases with time, as represented by the trimester; and there was more heterogeneity among studies in which there was more time between maternal diagnosis and fetal testing. The sensitivity of the method, if performed up to five weeks after maternal diagnosis, was 87% and specificity was 99%.ConclusionThe global sensitivity heterogeneity of the PCR test in this review was 66.5% (I²). The tests show low evidence of heterogeneity with a sensitivity of 87% and specificity of 99% when performed up to five weeks after maternal diagnosis. The test has a known performance and could be recommended for use up to five weeks after maternal diagnosis, when there is suspicion of fetal toxoplasmosis.     

An approach to real-time flexible scheduling

Two types of flexibility are important in manufacturing scheduling in general and in real-time scheduling in particular. The first is flexibility with respect to the criteria that can be considered in the scheduling decisions. The second is flexibility with respect to the trade-off between decision quality and computational burden: that is, the ability to arrive at a solution that makes maximum use of theavailable computational capacity and computation time. This paper describes a procedure which meets the above requirements. The procedure is justified using a theoretical analysis based on probability. Experimental results of the procedure's performance are also presented. The results show that random selection (which is used in the procedure) can play a useful role in the real-time scheduling problem.     

宠物弓形虫的感染及其防控

弓形虫病是一种世界性分布的人兽共患寄生虫病,对人类,尤其是妇女、儿童危害很大,估计全世界有1/3人受到该病的威胁。孕妇感染弓形虫后导致早产、流产、胎儿发育畸形;弓形虫是免疫功能低下患者的主要死亡原因之一。犬、猫是弓形虫的中间宿主和终末宿主,是人类感染弓形虫的主要来源。随着我国经济的迅速发展和人民生活水平的不断提高,城市中饲养犬、猫作为宠物的人越来越多,人、宠物间的亲密接触增加了弓形虫病传播给人的机会。加强对宠物犬、猫弓形虫病的研究及防控势在必行。本文就弓形虫的危害、宠物犬猫弓形虫感染及其防控措施作以综述。     

Is stage conversion the initiating event for reactivation of Toxoplasma gondii in brain tissue of AIDS patients?

Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood-brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.     

Toxoplasma gondii, "new" genotypes and virulence

Toxoplasma gondii has been described as a parasite with a low genetic diversity and a clonal population structure. The three main clonal lineages designated as type I, II or III largely predominate in Europe and North America. But strains not related to these main lineages circulate, notably, in other continents. They possess a shuffled combination of alleles that typify the three clonal types and unique polymorphisms detected by multilocus analysis. The population structure of Toxoplasma in these continents is also characterized by a higher genetic diversity associated with a lower linkage desequilibrium suggesting a role for genetic exchange. Due to their genomic diversity, it is difficult to draw global conclusions about their virulence. However, most of them are virulent in mice at isolation. Several reports also suggest a higher pathogenicity in humans and an association with ocular toxoplasmosis or severe cases of acquired toxoplasmosis in immunocompetent patients.     

Selective scenarios for the emergence of natural language

The recent blossoming of evolutionary linguistics has resulted in a variety of theories that attempt to provide a selective scenario for the evolution of early language. However, their overabundance makes many researchers sceptical of such theorising. Here, we suggest that a more rigorous approach is needed towards their construction although, despite justified scepticism, there is no agreement as to the criteria that should be used to determine the validity of the various competing theories. We attempt to fill this gap by providing criteria upon which the various historical narratives can be judged. Although individually none of these criteria are highly constraining, taken together they could provide a useful evolutionary framework for thinking about the evolution of human language.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

Toxoplasmosis: beyond animals to humans

The parasitic zoonosis toxoplasmosis, which was poorly understood before the advent of the HIV epidemic, has become a major clinical problem worldwide. Humans acquire toxoplasmosis from cats, from consuming raw or undercooked meat and from vertical transmission to the foetus through the placenta during pregnancy. Studies of the unique environmental factors in various communities indicate the important roles that eating habits and culture have on the transmission of this infection. The socioepidemiological aspects of toxoplasmosis are thought to be important contributing factors for the spread of this disease. Preventative measures should consider the cultures and beliefs of people in various communities more than solving poverty and giving orthodox health education.     

Control of the risk of human toxoplasmosis transmitted by meat

One-third of the human world population is infected with the protozoan parasite Toxoplasma gondii. Recent calculations of the disease burden of toxoplasmosis rank this foodborne disease at the same level as salmonellosis or campylobacteriosis. The high disease burden in combination with disappointing results of the currently available treatment options have led to a plea for more effective prevention. In this review we describe Toxoplasma as a hazard associated with the consumption of undercooked meat or meat products and provide an analysis of the various options to control the risk of human toxoplasmosis via this source. Monitoring and surveillance programs may be implemented for pre-harvest control of Toxoplasma infection of farm animals, with the reduction of environmental oocyst load as the most important milestone. Alternatively, Toxoplasma safe meat can be obtained through simple post-harvest decontamination procedures, whereby freezing the meat may currently be the best option, although new technologies using irradiation or high-pressure treatment may offer promising alternatives. Influence of culture, religion and food handling customs may predispose a certain type of meat as an important source of infection, indicating that prevention needs to be tailored according to social habits in different regions in the world. The rationale for more stringent control measures to prevent toxoplasmosis both from disease and economic points of view is emphasized.     

Protection against experimental toxoplasmosis by adoptive immunotherapy

The role of humoral and cell-mediated immunity against toxoplasmosis in experimentally infected guinea pigs was examined by using a syngeneic passive transfer system. Serum or spleen and lymph node cells from guinea pigs immune to infection with the RH strain of Toxoplasma gondii conferred partial protection against symptomatic disease in recipient guinea pigs. This result was based on the reduced dissemination or growth of T. gondii parasites from the primary inoculation site to various selected organ sites of the recipients of immune serum or cells. Similar levels of partial protection against disseminated toxoplasmosis occurred in animals infused with cell suspensions enriched for immune T cells, whereas treatment of immune cells with a monoclonal anti-guinea pig T cell antibody plus complement abolished their ability to transfer resistance. These findings provide substantial direct evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to toxoplasmosis.