Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress.

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M L-cysteine HCl/0.2 M NaHCO3 solution at 37 °C in 100% CO2 atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro.

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria.

Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells.

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS: We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS: In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS: Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.

Dynamics of transcription of immunomodulatory genes in endothelial cells infected with different coccidian parasites.

Sporozoites of Eimeria bovis and tachyzoites of Neospora caninum and Toxoplasma gondii are able to invade and to replicate in endothelial cells. Here we report on responses of bovine umbilical vein endothelial cells (BUVEC) in vitro to these coccidial infections by determining mRNA levels of the CXC chemokines GRO-alpha, IL-8 and IP-10, the CC chemokines MCP-1 and RANTES and of GM-CSF, COX-2 and iNOS relative to the level of housekeeping gene (GAPDH) transcription. T. gondii and N. caninum tachyzoites caused profound transcriptional upregulation of all genes in question. In general, upregulation started 2-4 h p.i. and maximum transcript levels were observed 4 h p.i. GRO-alpha and IL-8 gene transcription had decreased to almost control levels by 12 h p.i.; in the case of the other chemokines enhanced transcript levels persisted longer or showed a biphasic time-course. A similar time-course to CC chemokines was observed for GM-CSF mRNA, whilst COX-2 gene transcript peaks were detected at 2-4 h p.i. and 48-72 h p.i. iNOS mRNA levels increased from 4 to 48 h p.i. In contrast, E. bovis sporozoites failed to induce the transcription of CXC chemokine genes and of COX-2, and only caused moderate transcription upregulation of the other genes considered. In conclusion, infections of BUVEC with these coccidian parasites result in host cell activation associated with enhanced transcription of genes encoding for proinflammatory and immunomodulatory molecules, which are important for innate immune reactions and the transition to adaptive immunity. Differences between E. bovis versus T. gondii and N. caninum may illustrate a particular evasion strategy of E. bovis sporozoites, which is related to their need to persist in the host cell for a long period of time and to the avoidance of inflammatory process-induction.

Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes.

Infection with Plasmodium falciparum during pregnancy leads to chondroitin 4-sulfate-mediated adhesion of the infected red blood cells (IRBCs) in the placenta, causing severe health complications to fetus and the mother. The IRBCs are also frequently found in low density in the umbilical cord of infected placentas. In this study, the CSPGs of umbilical vein and arteries were purified, characterized, and their localization and IRBC-binding abilities were studied. While a versican type CSPG was found both in the vein and arteries, a serglycin type CSPG was present exclusively in the vein. The CSPGs were present at significant level on the endothelial surface of the umbilical vein but not on that of arteries. Although the purified versican and serglycin type CSPGs could bind IRBCs, their binding abilities were significantly less compared to the low sulfated CSPGs of the placenta because of the predominance of 6-sulfated disaccharide moieties in the CS chains. Therefore, IRBCs were unable to bind efficiently onto the umbilical cord endothelial surface. Unexpectedly, however, the IRBCs adhered densely in the blood vessels of fetal villi in the placental tissue sections and sparingly in the blood spaces of the umbilical cord vein, presumably because the CSPG that can efficiently bind IRBCs is present at high levels in the fetal blood vessels and at very low levels in the umbilical cord blood vessels. Since the C4S-adherent IRBCs that enter the fetal blood vessels cannot adhere to the cord endothelial surface and parasites cannot efficiently grow due to fetal hemoglobin toxicity and protection by maternal antibodies, transplacental infection may be quickly cleared without clinical episodes.

Schistosoma mansoni egg attachment to cultured human umbilical vein endothelial cells: an in vitro model of an early step of parasite egg excretion.

To model the attachment of Schistosoma mansoni eggs to the endothelium of the mesenteric vasculature, the interaction between S. mansoni eggs and cultured human umbilical vein endothelial cells (HUVEC) in vitro was investigated. S. mansoni eggs rapidly attached to monolayers of both HUVEC and bovine aortic endothelial cells but more slowly to monolayers of cultured fibroblasts and smooth muscle cells. While both native and glutaraldehyde-fixed eggs attached equally well to HUVEC, eggs attached only to live, metabolically active HUVEC. Attachment was enhanced by both serum and plasma factors. In addition, platelet release products increased egg attachment by 75%. Preincubation of S. mansoni eggs with soluble egg antigens promoted attachment; in contrast, preincubation of HUVEC with the antigens inhibited attachment. These results suggest that interaction of S. mansoni eggs with HUVEC is an active process that can be modulated by molecules secreted by the egg and by platelets during egg extravasation.

Plasmodium falciparum: cytoadherence of malaria-infected erythrocytes to human brain capillary and umbilical vein endothelial cells--a comparative study of adhesive ligands.

The cytoadherence of Plasmodium falciparum-infected erythrocytes (FCR-3 line) to human brain capillary endothelial cells (HBEC), C32 amelanotic melanoma cells, and human umbilical vein endothelial cells (HUVEC) was studied. The adhesion of infected red cells was HBEC > amelanotic melanoma > HUVEC. The presence or absence of the adhesive ligands ICAM-1 (CD54 or intercellular adhesion molecule 1), ICAM-2, and CD36 (= glycoprotein IV) was determined for each of these cells by indirect immunofluorescence using the monoclonal antibodies RR1/1, 6D5, and OKM 5/OKM 8, respectively. It appeared that a major ligand for the FCR-3 line of P. falciparum with amelanotic melanoma cells and HBECs was CD36. Binding to HUVECs was very low, presumably due to their lack of expression of CD36. HBECs, because of their ease of in vitro propagation, long-term maintenance of cytoadherent properties, and their high degree of adhesiveness, will be useful for in vitro studies of adherence.

Cytoadherence of Plasmodium falciparum-infected erythrocytes to human umbilical vein and human dermal microvascular endothelial cells under shear conditions.

To investigate the role of hemodynamics in the adherence of Plasmodium falciparum-infected erythrocytes to cerebral endothelium in vivo, we investigated cytoadherence of parasitized erythrocytes to human umbilical vein endothelial cells (HUVEC) under shear conditions in vitro. At 1.0 dyne/cm2 shear stress, parasitized red blood cell (RBC) adherence to HUVEC ranged from 9.9 +/- 1.0 (+/- SEM) to 75.2 +/- 4.8 RBC/mm2 (mean +/- SEM: 35.1 +/- 2.8 RBC/mm2) and was 13-fold greater than uninfected erythrocyte adherence to HUVEC (range 0.1 +/- 0.1 to 6.7 +/- 1.6 RBC/mm2, mean +/- SEM 2.8 +/- 0.8 RBC/mm2). Only erythrocytes infected with trophozoites and schizonts adhered to HUVEC under shear conditions. Parasitized erythrocyte adherence to HUVEC decreased from 28.4 +/- 2.7 RBC/mm2 to 12.7 +/- 2.4 RBC/mm2 when shear stress was increased from 1.0 to 2.0 dynes/cm2. At 4.0 dynes/cm2, parasitized erythrocyte adherence decreased further to 2.0 +/- 1.3 RBC/mm2. In falciparum malaria patients, endothelial cytoadherence predominates in the microcirculation. Therefore, we also investigated adherence of parasitized erythrocytes to human dermal microvascular endothelial cells (MEC). At 1.0 dyne/cm2, cytoadherence of P. falciparum-infected erythrocytes to MEC ranged from 7.9 +/- 1.1 to 60.0 +/- 2.4 RBC/mm2 (mean +/- SEM: 23.0 +/- 1.7 RBC/mm2) and was 10-fold greater than uninfected erythrocyte cytoadherence to MEC (mean +/- SEM: 2.2 +/- 0.6 RBC/mm2). These data indicate that P. falciparum-infected erythrocytes adhere to human umbilical vein and microvascular endothelial cells under shear stress conditions typical of the postcapillary venules in vivo, and that cytoadherence is specific for parasitized erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)