Experimental cerebral malaria pathogenesis--hemodynamics at the blood brain barrier.

Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8(+) T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45(hi) CD8(+) T cells, ICAM-1(+) macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8(+) T cells and ICAM(+) macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.

Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1(GFP/+) mice. CX3CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX3CR1-GFP⁺ Ly6C(low) monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX3CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX3CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C(high) and Ly6C(low) monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C(high) monocytes, but that these cells may transition through a Ly6C(low) state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C(high) monocytes via help from CD4⁺ T cells.

Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens.

The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high-throughput screening of inhibitory compounds.

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein.

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4(+) T cells in immunised volunteers. Both lytic and non-lytic CD4(+)T cell clones recognised a series of overlapping epitopes within a 'universal' T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4(+) T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4(+) cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4(+) CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8(+) CTLs, and these multifunctional sporozoite- and peptide-induced CD4(+) T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.

Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells.

Plasmodium sporozoites traverse Kupffer cells on their way into the liver. Sporozoite contact does not elicit a respiratory burst in these hepatic macrophages and blocks the formation of reactive oxygen species in response to secondary stimuli via elevation of the intracellular cAMP concentration. Here we show that increasing the cAMP level with dibutyryl cyclic adenosine monophosphate (db-cAMP) or isobutylmethylxanthine (IBMX) also modulates cytokine secretion in murine Kupffer cells towards an overall anti-inflammatory profile. Stimulation of Plasmodium yoelii sporozoite-exposed Kupffer cells with lipopolysaccharide or IFN-gamma reveals down-modulation of TNF-alpha, IL-6 and MCP-1, and up-regulation of IL-10. Prerequisite for this shift of the cytokine profile are parasite viability and contact with Kupffer cells, but not invasion. Whilst sporozoite-exposed Kupffer cells become TUNEL-positive and exhibit other signs of apoptotic death such as membrane blebbing, nuclear condensation and fragmentation, sporozoites remain intact and appear to transform to early exo-erythrocytic forms in Kupffer cell cultures. Together, the in vitro data indicate that Plasmodium possesses mechanisms to render Kupffer cells insensitive to pro-inflammatory stimuli and eventually eliminates these macrophages by forcing them into programmed cell death.

Exoerythrocytic development of Plasmodium gallinaceum in the White Leghorn chicken.

Plasmodium gallinaceum typically causes sub-clinical disease with low mortality in its primary host, the Indian jungle fowl Gallus sonnerati. Domestic chickens of European origin, however, are highly susceptible to this avian malaria parasite. Here we describe the development of P. gallinaceum in young White Leghorn chicks with emphasis on the primary exoerythrocytic phase of the infection. Using various regimens for infection, we found that P. gallinaceum induced a transient primary exoerythrocytic infection followed by a fulminant lethal erythrocytic phase. Prerequisite for the appearance of secondary exoerythrocytic stages was the development of a certain level of parasitaemia. Once established, secondary exoerythrocytic stages could be propagated from bird to bird for several generations without causing fatalities. Infected brains contained large secondary exoerythrocytic stages in capillary endothelia, while in the liver primary and secondary erythrocytic stages developed primarily in Kupffer cells and remained smaller. At later stages, livers exhibited focal hepatocyte necrosis, Kupffer cell hyperplasia, stellate cell proliferation, inflammatory cell infiltration and granuloma formation. Because P. gallinaceum selectively infected Kupffer cells in the liver and caused a histopathology strikingly similar to mammalian species, this avian Plasmodium species represents an evolutionarily closely related model for studies on the hepatic phase of mammalian malaria.

Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells.

After transmission by infected mosquitoes, malaria sporozoites rapidly travel to the liver. To infect hepatocytes, sporozoites traverse Kupffer cells, but surprisingly, the parasites are not killed by these resident macrophages of the liver. Here we show that Plasmodium sporozoites and recombinant circumsporozoite protein (CSP) suppress the respiratory burst in Kupffer cells. Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl mono-phosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the respiratory burst. Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP-1 and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defence mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighbouring hepatocytes.