Brain endothelial STING1 activation by Plasmodium-sequestered heme promotes cerebral malaria via type I IFN response.

Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnß1 transcription in the brain of mice infected with Plasmodium berghei ANKA (Pba). This STING1/IFNß-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFNß-reporter and STING1-deficient Pba-infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba-infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNß in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNß/CXCL10 axis crucial to CM pathogenesis and lethality.

Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Trypanosoma cruzi Exploits E- and P-Selectins To Migrate Across Endothelial Cells and Extracellular Matrix Proteins.

The Chagas disease parasite Trypanosoma cruzi must extravasate to home in on susceptible cells residing in most tissues. It remains unknown how T. cruzi undertakes this crucial step of its life cycle. We hypothesized that the pathogen exploits the endothelial cell programming leukocytes use to extravasate to sites of inflammation. Transendothelial migration (TEM) starts after inflammatory cytokines induce E-selectin expression and P-selectin translocation on endothelial cells (ECs), enabling recognition by leukocyte ligands that engender rolling cell adhesion. Here, we show that T. cruzi upregulates E- and P-selectins in cardiac ECs to which it binds in a ligand-receptor fashion, whether under static or shear flow conditions. Glycoproteins isolated from T. cruzi (TcEx) specifically recognize P-selectin in a ligand-receptor interaction. As with leukocytes, binding of P-selectin to T. cruzi or TcEx requires sialic acid and tyrosine sulfate, which are pivotal for downstream migration across ECs and extracellular matrix proteins. Additionally, soluble selectins, which bind T. cruzi, block transendothelial migration dose dependently, implying that the pathogen bears selectin-binding ligand(s) that start transmigration. Furthermore, function-blocking antibodies against E- and P-selectins, which act on endothelial cells and not T. cruzi, are exquisite in preventing TEM. Thus, our results show that selectins can function as mediators of T. cruzi transendothelial transmigration, suggesting a pathogenic mechanism that allows homing in of the parasite on targeted tissues. As selectin inhibitors are sought-after therapeutic targets for autoimmune diseases and cancer metastasis, they may similarly represent a novel strategy for Chagas disease therapy.

Toxoplasma gondii infection impairs radial glia differentiation and its potential to modulate brain microvascular endothelial cell function in the cerebral cortex.

Congenital toxoplasmosis is a parasitic disease that occurs due vertical transmission of the protozoan Toxoplasma gondii (T. gondii) during pregnancy. The parasite crosses the placental barrier and reaches the developing brain, infecting progenitor, glial, neuronal and vascular cell types. Although the role of Radial glia (RG) neural stem cells in the development of the brain vasculature has been recently investigated, the impact of T. gondii infection in these events is not yet understood. Herein, we studied the role of T. gondii infection on RG cell function and its interaction with endothelial cells. By infecting isolated RG cultures with T. gondii tachyzoites, we observed a cytotoxic effect with reduced numbers of RG populations together with decrease neuronal and oligodendrocyte progenitor populations. Conditioned medium (CM) from RG control cultures increased ZO-1 protein levels and organization on endothelial bEnd.3 cells membranes, which was impaired by CM from infected RG, accompanied by decreased trans-endothelial electrical resistance (TEER). ELISA assays revealed reduced levels of anti-inflammatory cytokine TGF-ß1 in CM from T. gondii-infected RG cells. Treatment with recombinant TGF-ß1 concomitantly with CM from infected RG cultures led to restoration of ZO-1 staining in bEnd.3 cells. Congenital infection in Swiss Webster mice led to abnormalities in the cortical microvasculature in comparison to uninfected embryos. Our results suggest that infection of RG cells by T. gondii negatively modulates cytokine secretion, which might contribute to endothelial loss of barrier properties, thus leading to impairment of neurovascular interaction establishment.

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers.

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses non-permissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immune-privileged sites such as the brain parenchyma, after crossing the blood-brain barrier. In the present study, we have established a protocol for high-purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance. In contrast, challenge with parasite lysate or LPS increased barrier permeability by destabilising intercellular tight junctions (TJs) and accentuated transmigration of T. gondii. Conversely, reduced phosphorylation of the TJ-regulator focal adhesion kinase (FAK) was observed dose-dependently upon challenge of monolayers with live T. gondii but not with parasite lysate or LPS. Pharmacological inhibition of FAK phosphorylation reversibly altered barrier integrity and facilitated T. gondii translocation. Finally, gene silencing of FAK by shRNA facilitated transmigration of T. gondii across epithelial and endothelial monolayers. Jointly, the data demonstrate that T. gondii infection transiently alters the TJ stability through FAK dysregulation to facilitate transmigration. This work identifies the implication of the TJ regulator FAK in the transmigration of T. gondii across polarised cellular monolayers and provides novel insights in how microbes overcome the restrictiveness of biological barriers.

Dimethyl fumarate reduces TNF and Plasmodium falciparum induced brain endothelium activation in vitro.

BACKGROUND: Cerebral malaria (CM) is associated with morbidity and mortality despite the use of potent anti-malarial agents. Brain endothelial cell activation and dysfunction from oxidative and inflammatory host responses and products released by Plasmodium falciparum-infected erythrocytes (IE), are likely the major contributors to the encephalopathy, seizures, and brain swelling that are associated with CM. The development of adjunctive therapy to reduce the pathological consequences of host response pathways could improve outcomes. A potentially protective role of the nuclear factor E2-related factor 2 (NRF2) pathway, which serves as a therapeutic target in brain microvascular diseases and central nervous system (CNS) inflammatory diseases such as multiple sclerosis was tested to protect endothelial cells in an in vitro culture system subjected to tumour necrosis factor (TNF) or infected red blood cell exposure. NRF2 is a transcription factor that mediates anti-oxidant and anti-inflammatory responses. METHODS: To accurately reflect clinically relevant parasite biology a unique panel of parasite isolates derived from patients with stringently defined CM was developed. The effect of TNF and these parasite lines on primary human brain microvascular endothelial cell (HBMVEC) activation in an in vitro co-culture model was tested. HBMVEC activation was measured by cellular release of IL6 and nuclear translocation of NFκB. The transcriptional and functional effects of dimethyl fumarate (DMF), an FDA approved drug which induces the NRF2 pathway, on host and parasite induced HBMVEC activation was characterized. In addition, the effect of DMF on parasite binding to TNF stimulated HBMVEC in a semi-static binding assay was examined. RESULTS: Transcriptional profiling demonstrates that DMF upregulates the NRF2-Mediated Oxidative Stress Response, ErbB4 Signaling Pathway, Peroxisome Proliferator-activated Receptor (PPAR) Signaling and downregulates iNOS Signaling and the Neuroinflammation Signaling Pathway on TNF activated HBMVEC. The parasite lines derived from eight paediatric CM patients demonstrated increased binding to TNF activated HBMVEC and varied in their binding and activation of HBMVEC. Overall DMF reduced both TNF and CM derived parasite activation of HBMVEC. CONCLUSIONS: These findings provide evidence that targeting the NRF2 pathway in TNF and parasite activated HBMVEC mediates multiple protective pathways and may represent a novel adjunctive therapy to improve infection outcomes in CM.

Besnoitia besnoiti-driven endothelial host cell cycle alteration.

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24-30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that-in terms of host cell cycle modulation-infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti-infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii-related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti-infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.

Morphometric and molecular characterisation of Hepatozoon bashtari n. sp. in painted saw-scaled viper, Echis coloratus (Ophidia, Viperidae).

Hepatozoon species are the most widely known haemogregarines infecting a wide range of vertebrates, although predominately snakes. Herein, Hepatozoon bashtari n. sp., originally infecting the painted saw-scaled viper, Echis coloratus, in Saudi Arabia is described using both morphological features and molecular data from 18S rDNA sequences. The overall prevalence of infection was 60% (9/15) with parasitaemia ranging from 52 to 60%. Gamonts were entirely intraerythrocytic and were observed to cause considerable hypertrophy within the host cell. The mean size of mature gamonts was 15.4 × 3.3 µm. Merogonic stages were confined to the lung endothelial cells with monomorphic meronts. The average size of mature meronts was 32 × 12 µm and they were estimated to produce 13-16 merozoites each. The phylogenetic tree generated from SSU rDNA sequences revealed that Hepatozoon bashtari sp. n. clusters with the vast majority of other Hepatozoon species infecting snakes, lizards and geckos in various regions of the world, which would appear to support the hypothesis of prey-predator transmission of the genus Hepatozoon. Through a combination of morphological comparison with closely related Hepatozoon spp. and 18S rRNA gene sequence analysis, it is possible to confirm Hepatozoon bashtari sp. n. as a new species.

Fenozyme Protects the Integrity of the Blood-Brain Barrier against Experimental Cerebral Malaria.

Cerebral malaria is a lethal complication of malaria infection characterized by central nervous system dysfunction and is often not effectively treated by antimalarial combination therapies. It has been shown that the sequestration of the parasite-infected red blood cells that interact with cerebral vessel endothelial cells and the damage of the blood-brain barrier (BBB) play critical roles in the pathogenesis. In this study, we developed a ferritin nanozyme (Fenozyme) composed of recombinant human ferritin (HFn) protein shells that specifically target BBB endothelial cells (BBB ECs) and the inner Fe3O4 nanozyme core that exhibits reactive oxygen species-scavenging catalase-like activity. In the experimental cerebral malaria (ECM) mouse model, administration of the Fenozyme, but not HFn, markedly ameliorated the damage of BBB induced by the parasite and improved the survival rate of infected mice significantly. Further investigations found that Fenozyme, as well as HFn, was able to polarize the macrophages in the liver to the M1 phenotype and promote the elimination of malaria in the blood. Thus, the catalase-like activity of the Fenozyme is required for its therapeutic effect in the mouse model. Moreover, the Fenozyme significantly alleviated the brain inflammation and memory impairment in ECM mice that had been treated with artemether, indicating that combining Fenozyme with an antimalarial drug is a novel strategy for the treatment of cerebral malaria.

Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.

Antiplasmodial Activity of p-Substituted Benzyl Thiazinoquinone Derivatives and Their Potential against Parasitic Infections.

Malaria is a life-threatening disease and, what is more, the resistance to available antimalarial drugs is a recurring problem. The resistance of Plasmodium falciparum malaria parasites to previous generations of medicines has undermined malaria control efforts and reversed gains in child survival. This paper describes a continuation of our ongoing efforts to investigate the effects against Plasmodium falciparum strains and human microvascular endothelial cells (HMEC-1) of a series of methoxy p-benzyl-substituted thiazinoquinones designed starting from a pointed antimalarial lead candidate. The data obtained from the newly tested compounds expanded the structure-activity relationships (SARs) of the thiazinoquinone scaffold, indicating that antiplasmodial activity is not affected by the inductive effect but rather by the resonance effect of the introduced group at the para position of the benzyl substituent. Indeed, the current survey was based on the evaluation of antiparasitic usefulness as well as the selectivity on mammalian cells of the tested p-benzyl-substituted thiazinoquinones, upgrading the knowledge about the active thiazinoquinone scaffold.

Blood-brain barrier disruption and angiogenesis in a rat model for neurocysticercosis.

Neurocysticercosis (NCC) is a helminth infection affecting the central nervous system caused by the larval stage (cysticercus) of Taenia solium. Since vascular alteration and blood-brain barrier (BBB) disruption contribute to NCC pathology, it is postulated that angiogenesis could contribute to the pathology of this disease. This study used a rat model for NCC and evaluated the expression of two angiogenic factors called vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF2). Also, two markers for BBB disruption, the endothelial barrier antigen and immunoglobulin G, were evaluated using immunohistochemical and immunofluorescence techniques. Brain vasculature changes, BBB disruption, and overexpression of angiogenesis markers surrounding viable cysts were observed. Both VEGF-A and FGF2 were overexpressed in the tissue surrounding the cysticerci, and VEGF-A was overexpressed in astrocytes. Vessels showed decreased immunoreactivity to endothelial barrier antigen marker and an extensive staining for IgG was found in the tissues surrounding the cysts. Additionally, an endothelial cell tube formation assay using human umbilical vein endothelial cells showed that excretory and secretory antigens of T. solium cysticerci induce the formation of these tubes. This in vitro model supports the hypothesis that angiogenesis in NCC might be caused by the parasite itself, as opposed to the host inflammatory responses alone. In conclusion, brain vasculature changes, BBB disruption, and overexpression of angiogenesis markers surrounding viable cysts were observed. This study also demonstrates that cysticerci excretory-secretory processes alone can stimulate angiogenesis.

Comparative 1D Blue-Native electrophoresis analysis of Plasmodium falciparum and human proteins associated with cytoadherence.

BACKGROUND: To understand more about changes to the molecular components that occur when host endothelium interacts with Plasmodium falciparum-infected erythrocytes, a combined technique of protein separation (1D Blue-Native electrophoresis) and mass spectrometry of infected erythrocytes with endothelial cells (EC) in a co-culture system has been used. METHODS: Native proteins were extracted from co-cultures and identified by mass spectrometry. Proteomic data from different parasite strains, either adhesion proficient (to endothelial cells) or non-adherent, were analysed in parallel to reveal protein associations linked to cytoadherence. Informatic approaches were developed to facilitate this comparison. RESULTS: Blue-Native gel separation and LC/MS/MS identification revealed major differences in samples produced from endothelial cell co-culture with adherent and non-adherent parasite strains. This approach enabled us to identify protein associations seen only with the adhesion proficient parasite strain. CONCLUSIONS: The combination of proteomic and analytical approaches has identified differences between adherent and non-adherent parasite lines in co-culture with EC, providing potential candidates for complexes or associations formed during cytoadherence involved in cell structure, signalling and apoptosis.

Curcumin exerts anti-inflammatory and vasoprotective effects through amelioration of NFAT-dependent endothelin-1 production in mice with acute Chagas cardiomyopathy.

BACKGROUND: The anti-inflammatory and cardioprotective properties of curcumin (Cur), a natural polyphenolic flavonoid isolated from the rhizomes of Curcuma longa, are increasingly considered to have beneficial effects on the progression of Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi. OBJECTIVE: To evaluate the effects of oral therapy with Cur on T. cruzi-mediated cardiovasculopathy in acutely infected mice and analyse the in vitro response of parasite-infected human microvascular endothelial cells treated with this phytochemical. METHODS: Inflammation of heart vessels from Cur-treated and untreated infected mice were analysed by histology, with benznidazole (Bz) as the reference compound. Parasitaemia was monitored by the direct method. Capillary permeability was visualised by Evans-blue assay. Myocardial ET-1, IL-6, and TNF-α mRNA expressions were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Microvascular endothelial HMEC-1 cells were infected in vitro with or without addition of Cur or Bz. Induction of the Ca2+/NFAT pathway was assessed by fluorometry, immunoblotting, and reporter assay. FINDINGS: Oral Cur therapy of recently infected mice reduced inflammatory cell infiltration of myocardial arteries without lowering parasite levels. Compared to that of the phosphate-buffered saline-receiving group, hearts from Cur-treated mice showed significantly decreased vessel inflammation scores (p < 0.001), vascular permeabilities (p < 0.001), and levels of IL-6/TNF-α (p < 0.01) and ET-1 (p < 0.05) mRNA. Moreover, Cur significantly (p < 0.05 for transcript; p < 0.01 for peptide) downregulated ET-1 secretion from infected HMEC-1 cells. Remarkably, Cur addition significantly (p < 0.05 at 27.0 µM) interfered with T. cruzi-dependent activation of the Ca2+/NFATc1 signalling pathway that promotes generation of inflammatory agents in HMEC-1 cells. CONCLUSIONS: Oral treatment with Cur dampens cardiovasculopathy in acute Chagas mice. Cur impairs the Ca2+/NFATc1-regulated release of ET-1 from T. cruzi-infected vascular endothelium. These findings identify new perspectives for exploring the potential of Cur-based interventions to ameliorate Chagas heart disease.

Report: Effect of macrophage alone or primed with cytokines on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells in vitro.

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated <90% binding and <70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Tumor necrosis factor reduces Plasmodium falciparum growth and activates calcium signaling in human malaria parasites.

BACKGROUND: Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage. METHODS: Flow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining. RESULTS: TNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48 h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP (N(6)-benzoyladenosine-3',5'-cyclic monophosphate sodium salt). CONCLUSIONS: This is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF. GENERAL SIGNIFICANCE: The data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.

Gamma Interferon Mediates Experimental Cerebral Malaria by Signaling within Both the Hematopoietic and Nonhematopoietic Compartments.

Experimental cerebral malaria (ECM) is a gamma interferon (IFN-γ)-dependent syndrome. However, whether IFN-γ promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-γ receptor 2 (IFN-γR2)-deficient mice, we show that IFN-γ causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-γR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-γR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-γ and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN-γ causes ECM by acting redundantly or by targeting non-T cell or non-myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-γ leading to ECM development. Collectively, our data provide information on how IFN-γ mediates the development of cerebral pathology during malaria infection.

Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

BACKGROUND: Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. METHODS: The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. RESULTS: Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. CONCLUSION: These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

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.