Giant membrane rings/loops in the cytosol of P. falciparum-infected erythrocytes and their relation to the parasite.

Striking morphological transformations characterize the invasion of a red blood cell by the malaria parasite. Shortly after the infection, parasite-induced membranes appear in the cytosol of the affected host erythrocyte. One intensely investigated membrane type, commonly called Maurer's clefts, has a slit-like morphology and can be arranged in the form of extended three-dimensional membrane stacks or networks. Here we report the three-dimensional reconstruction of a second membrane type, giant or extended membrane rings/loops, that have only occasionally been described on single ultrathin sections, however that have never been systematically examined so far. Serial ultrathin sectioning of P. falciparum-infected red blood cells, subsequent three-dimensional reconstructions, and in addition examination of Giemsa-stained blood films revealed that intraerythrocytic membrane rings/loops are not isolated structures but are locally in contact with the parasite. They consist either of the parasitophorous vacuolar membrane alone or contain the parasitophorous vacuolar membrane including the plasma membrane of the parasite and small amounts of parasite cytoplasm. We demonstrate that membrane rings/loops represent surface extensions of the parasite that maybe involved in ring stage parasite formation and Maurer's cleft generation at least in a subset of infected red blood cells.

A promising diagnostic tool for toxoplasmic encephalitis: tachyzoite/bradyzoite stage-specific RT-PCR.

OBJECTIVES: To determine the diagnostic accuracy, technical benefit, and clinical application of the duplex reverse transcription-PCR (duplex RT-PCR) assay specific to bradyzoite (BAG1) and tachyzoite (SAG1) genes, for diagnosing toxoplasmic encephalitis (TE) in HIV-infected patients, using the US Centers for Disease Control and Prevention (CDC) recommended diagnostic criteria as the reference standard. METHODS: Advanced HIV-infected individuals with central nervous system opportunistic infections were enrolled in a prospective study, performed from July 2007 to January 2009; patients were classified as TE- or non-TE subjects in accordance with the CDC recommended criteria. Blood and cerebrospinal fluid samples were assayed by duplex RT-PCR to detect tachyzoite, bradyzoite, both, or none. RESULTS: A total of 61 advanced AIDS patients were included in the study, eight with TE and 53 as non-TE subjects. The duplex RT-PCR assay showed high diagnostic accuracy, with 100% specificity and positive predictive value, as well as 87.5% sensitivity. Its efficacy reached 98.3%. This diagnostic method was rapid, needed only moderately skilled technicians, and was four times cheaper than procedures used in the CDC diagnostic recommendations. It worked very well for blood samples, even after drug treatment had been started. CONCLUSIONS: The duplex RT-PCR assay is simple and rapid, and provides high efficacy with lower costs than the reference standard procedures. This is a promising alternative diagnostic tool for TE in HIV/AIDS individuals, especially in resource-limited settings.

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice.

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.

The complex morphology of Maurer's clefts: from discovery to three-dimensional reconstructions.

Over 100 years ago, Georg Maurer wrote one of the finest scientific accounts of what is now known as Maurer's dots, or clefts, describing the intracellular changes in red blood cells infected with Plasmodium falciparum. Maurer's clefts have since attracted much attention, and they form an intriguing aspect of parasite biology that may hold the key to the mechanisms by which the intracellular parasite alters red blood cell properties, leading to host pathogenesis and death. This review will focus on the description of the morphology of these clefts, from the first light-microscopic report up to recent three-dimensional reconstructions. Detailed knowledge of these structures should further our understanding of their functions.

Growth of benign and malignant schwannoma xenografts in severe combined immunodeficiency mice.

OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.

Malaria in a holoendemic area of Burkina Faso: a cross-sectional study.

A malaria survey of the entire population of a village in Western Burkina Faso (n=1,561) was conducted to assess malaria endemicity. The study population was examined for symptoms characteristic of malaria including fever, anaemia, splenomegaly and parasites present in thick blood films. In the overall study population, the prevalence of Plasmodium spp. infection by microscopic examination of thick blood films was 79.0% (1,233/1,561). In a subcohort with 201 individuals, PCR techniques found a prevalence rate for all Plasmodium spp. of 92.0% (185/201), while microscopy found one of 80.6% (162/201). A combination of both methods gives a rate of 95.5% (192/201). Though univariate logistic analyses of elevated body temperature, anaemia, splenomegaly and age showed them all to be predictors of or risk factors for an infection, only elevated body temperature and age were predictors in multivariate logistic analysis. However, the symptom of splenomegaly did show a highly significant association with infection by multiple species of Plasmodium.

Maurer's clefts: a novel multi-functional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes.

Discovered in 1902 by Georg Maurer as a peculiar dotted staining pattern observable by light microscopy in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum, the function of Maurer's clefts have remained obscure for more than a century. The growing interest in protein sorting and trafficking processes in malarial parasites has recently aroused the Maurer's clefts from their deep slumber. Mounting evidence suggests that Maurer's clefts are a secretory organelle, which the parasite establishes within its host erythrocyte, but outside its own confines, to route parasite proteins across the host cell cytoplasm to the erythrocyte surface where they play a role in nutrient uptake and immune evasion processes. Moreover, Maurer's clefts seem to play a role in cell signaling, merozoite egress, phospholipid biosynthesis and, possibly, other biochemical pathways. Here, we review our current knowledge of the ultrastructure of Maurer's clefts, their proteinaceous composition and their function in protein trafficking.

Association of Plasmodium falciparum isolates encoding the p. Falciparum chloroquine resistance transporter gene K76T polymorphism with anemia and splenomegaly, but not with multiple infections.

The aim of the study was to assess whether infections with Plasmodium falciparum isolates encoding the P. falciparum chloroquine resistance transporter (pfcrt) gene K76T polymorphism, a molecular marker for chloroquine resistance, are associated with multiple infections, age, or clinical signs of malaria in a semi-immune population in a holoendemic area of Burkina Faso. The parameters of interest were investigated in 210 P. falciparum-positive inhabitants. Logistic regression analysis showed that pfcrt K76T-carrying isolates are significantly more likely to cause anemia and splenomegaly. Furthermore, we found that infections with P. falciparum isolates encoding pfcrt K76T are dependent on age rather than multiple infections. Our findings suggest that pfcrt K76T might serve as a valuable marker for assessing the long-term clinical effect of chronic infections with chloroquine-resistant P. falciparum isolates in populations, without the need of drug efficacy trials.

Comparison of five DNA extraction methods and optimization of a b1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain.

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide. Different techniques have been developed for T. gondii detection. At present, polymerase chain reaction (PCR) has been widely used. However, PCR for identifying T. gondii remains unsatisfactory in many laboratories because of lack of standardization and variations in efficiency. In the present study, we optimized a nested PCR protocol (n-PCR) in order to compare the amplification of T. gondii DNA, after being extracted from mouse brain by five different DNA extraction methods including phenol chloroform, QIAamp DNA minikit, Genomic DNA purification kit and Chelex with or without proteinase K. All DNA extraction methods were able to extract DNA from a single tissue cyst from mouse brain. However, among the five DNA extraction methods, the Chelex without proteinase K appeared to be the most rapid and easiest.

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections.

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.

Maurer's clefts--a novel secretory organelle?

During intra-erythrocytic development, the human malarial parasite Plasmodium falciparum extensively remodels its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane, but is, however, faced with a major problem: the lack of an endogenous protein trafficking machinery within the host erythrocyte. Thus, in order to export proteins the parasite has to install its own protein export system within the host erythrocyte. A growing body of evidence suggests that Maurer's clefts, parasite-derived membranous structures in the cytosol of the host cell, are a crucial component of this protein sorting and trafficking machinery. In this review we summarize our current understanding of the ultra-structure of Maurer's clefts and their role in protein transport process.

Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants.

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.

Evidence for trafficking of PfEMP1 to the surface of P. falciparum-infected erythrocytes via a complex membrane network.

The human malarial parasite Plasmodium falciparum exports virulence determinants, such as the P. falciparum erythrocyte membrane protein 1 (PfEMP1), beyond its own periplasmatic boundaries to the surface of its host erythrocyte. This is remarkable given that erythrocytes lack a secretory pathway. Here we present evidence for a continuous membrane network of parasite origin in the erythrocyte cytoplasm. Co-localizations with antibodies against PfEMP1, PfExp-1, Pf332 and PfSbpl at the light and electron microscopical level indicate that this membrane network is composed of structures that have been previously described as tubovesicular membrane network (TVM), Maurer's clefts and membrane whorls. This membrane network could also be visualized in vivo by vital staining of infected erythrocytes with the fluorescent dye LysoSensor Green DND-153. At sites where the membrane network abuts the erythrocyte plasma membrane we observed small vesicles of 15-25 nm in size, which seem to bud from and/or fuse with the membrane network and the erythrocyte plasma membrane, respectively. On the basis of our data we hypothesize that this membrane network of parasite origin represents a novel secretory organelle that is involved in the trafficking of PfEMP1 across the erythrocyte cytoplasm.