Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen.

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

Ligation of B and T lymphocyte attenuator prevents the genesis of experimental cerebral malaria.

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.

Differential roles of CD36, ICAM-1, and P-selectin in Plasmodium falciparum cytoadherence in vivo.

Cytoadherence of Plasmodium falciparum-infected red blood cells (IRBCs) on human microvascular endothelium is mediated by synergistic adhesive interactions with different adhesion molecules in vitro. Here, the authors used a unique human/severe combined immunodeficient (SCID) mouse chimeric model to directly visualize IRBC-endothelial interactions in an intact human microvasculature in vivo. Stimulation of human skin grafts with 100 ng TNF-alpha for 4 h led to a dramatic reduction in the distance rolled by IRBCs before arrest, so that the majority of IRBCs adhered directly to the endothelium with a 1.8-fold increase in the number of adherent cells. The decrease in rolling distance and increase in adhesion could be reversed by anti-ICAM-1. More importantly, the effect of TNF-alpha could be seen only in the presence of CD36. A further increase in adhesion by 4.9-fold was observed after 24 h of TNF-alpha stimulation. The increase could be reversed by anti-ICAM-1, but not anti-VCAM-1. In histamine-stimulated grafts, the rolling flux fraction and adhesion increased by 2.8- and 1.6-fold, respectively. The increases were attributable to P-selectin as an inhibitory anti-P-selectin antibody abrogated both the increased rolling flux fraction and firm adhesion. These findings indicate that in addition to CD36, ICAM-1, and P-selectin are major contributors to the dynamic process of IRBC adhesion by different mechanisms in vivo.

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier.

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.

Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.

Brain lesions of cerebral malaria (CM) are characterized by a sequestration of Plasmodium falciparum-parasitized red blood cells (PRBC) and platelets within brain microvessels, as well as by blood-brain barrier (BBB) disruption. In the present study, we evaluated the possibility that PRBC and platelets induce functional alterations in brain endothelium. In a human brain endothelial cell line, named HBEC-5i, exhibiting most of the features demanded for a pathophysiological study of BBB, tumor necrosis factor (TNF) or lymphotoxin alpha (LT-alpha) reduced transendothelial electrical resistance (TEER), enhanced the permeability to 70-kDa dextran, and increased the release of microparticles, a recently described indicator of disease severity in CM patients. In vitro cocultures showed that platelets or PRBC can have a direct cytotoxic effect on activated, but not on resting, HBEC-5i cells. Platelet binding was required, as platelet supernatant had no effect. Furthermore, platelets potentiated the cytotoxicity of PRBC for TNF- or LT-alpha-activated HBEC-5i cells when they were added prior to these cells on the endothelial monolayers. This effect was not observed when platelets were added after PRBC. Both permeability and TEER were strongly affected, and the apoptosis rate of HBEC-5i cells was dramatically increased. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM.

Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism.

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase.

The microcirculation in severe malaria.

Severe malaria in humans and animals is initiated by interactions between malaria-infected cells, host blood cells (including monocytes, T cells and platelets) and endothelial cells of the microcirculation. Adhesion to vascular cells, and possible vascular obstruction in severe human disease, involves interaction between host receptors and parasite-derived proteins, such as the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Our understanding of how different PfEMP1 variants may target infected erythrocytes to specific sites, such as the placenta, is rapidly increasing. However, in most instances downstream immune-mediated inflammatory processes appear more central than parasite accumulation to development of severe malaria. Using genetically-manipulated animal models of severe malaria, key roles for CD8 T cells and mediators such as lymphotoxin in the pathogenesis of murine disease have been established. Experimental and human studies suggest vascular deposition of activated platelets may have a central role. Here, we review some recent advances in the understanding of severe malaria pathogenesis from human and animal studies, focusing on events at the level of the microcirculation, and highlight the role for activated host cells in initiating the pathology of the disease.

Myocardial contrast echocardiography in assessing microcirculation in baboons with chagas disease.

OBJECTIVE: Microvascular abnormalities have been postulated in the pathogenesis of chagasic cardiomyopathy. The objective of this study was to evaluate the relationship between coronary microcirculation and systolic function impairment in baboons with Chagas disease using myocardial contrast echocardiography (MCE). METHODS: Seventeen seropositive (5 males, 12 females; mean age 20 years) and 13 age- and gender-matched seronegative baboons underwent MCE using intravenous octafluoropropane human albumin microspheres. Color-coding was used to enhance tissue contrast in assessing regional myocardium uniformity and texture. Dipyridamole (0.54 mg/kg) was given to a subset of 4 animals to challenge coronary flow reserve. Systolic indices included left ventricular fractional shortening, velocity of circumferential fiber shortening, and left and right ventricular ejection fractions. RESULTS: Four of the 17 (24%) seropositive primates had decreased fractional shortening (25 +/- 8% vs. 40 +/-5%, p <.005), velocity of circumferential fiber shortening (1.05 +/- 0.36 circ/s vs. 1.84 +/- 0.23 circ/s, p <.0001), and reduced right ventricular ejection fraction (44 +/- 9% vs. 54 +/- 4%, p <.05) compared to other seropositive animals. Seropositive and seronegative groups showed no significant differences on the coronary microcirculation pattern as evaluated by MCE, including the 4 baboons with systolic function impairment. Moreover, coronary flow vasoreactivity resulted in a significant increase in myocardial flow as detected by color-coding masking. CONCLUSIONS: Chagasic heart disease is present in 24% of seropositive baboons spontaneously infected with Trypanosoma cruzi. MCE reveals a discrepancy between coronary microcirculation at rest and alterations in myocardial contractility, suggesting preservation of the microvascular integrity in this unique animal model.

Trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model.

African trypanosomiasis, sleeping sickness in humans, is caused by the systemic infection of the host by the extracellular parasite, the African trypanosome. The pathogenetic mechanisms of the severe symptoms of central nervous system involvement are still not well understood. The present study examined the routes of haematogenous spread of Trypanosoma brucei brucei (Tbb) to the brain, in particular on the question whether parasites can cross the blood-brain barrier, as well as their effect on tight junction proteins. Rats were infected with Tbb and at various times post-infection, the location of the parasite in the central nervous system was examined in relation to the brain vascular endothelium, visualized with an anti-glucose transporter-1 antibody. The tight junction-specific proteins occludin and zonula occludens 1, and the possible activation of the endothelial cell adhesion molecules ICAM-1 and VCAM-1 were also studied. At 12 and 22 days post-infection, the large majority of parasites were confined within blood vessels. At this stage, however, some parasites were also clearly observed in the brain parenchyma. This was accompanied by an upregulation of ICAM-1/VCAM-1. At later stages, 42, 45 and 55 days post-infection, parasites could still be detected within or in association with blood vessels. In addition, the parasite was now frequently found in the brain parenchyma and the extravasation of parasites was more prominent in the white matter than the cerebral cortex. A marked penetration of parasites was seen in the septal nuclei. In spite of this, occludin and zonula occludens 1 staining of the vessels was preserved. The results indicate that the Tbb parasite is able to cross the blood-brain barrier in vivo, without a generalized loss of tight junction proteins.

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature.

A murine model that closely resembles human cerebral malaria is presented, in which characteristic features of parasite sequestration and inflammation in the brain are clearly demonstrable. "Young" (BALB/c x C57BL/6)F(1) mice infected with Plasmodium berghei (ANKA) developed typical neurological symptoms 7 to 8 days later and then died, although their parasitemias were below 20%. Older animals were less susceptible. Immunohistopathology and ultrastructure demonstrated that neurological symptoms were associated with sequestration of both parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions. Increases in tumor necrosis factor alpha and CD54 expression were also present. Similar phenomena were absent or substantially reduced in older infected but asymptomatic animals. These findings suggest that this murine model is suitable both for determining precise pathogenetic features of the cerebral form of the disease and for evaluating circumventive interventions.

Expression of proinflammatory cytokines in four regions of the brain in Macaque mulatta (rhesus) monkeys infected with Plasmodium coatneyi.

We have characterized brain cytokine expression profiles in the Plasmodium coatneyi/rhesus (Macaque mulatta) malaria model. Eight rhesus monkeys were included in the study; four were infected with P. coatneyi, and four were used as uninfected controls. All inoculated animals became infected. Eleven days after parasite inoculation, the rhesus monkeys were killed and tissue samples from 4 regions of the brain (cortex and white matter of the cerebrum, cerebellum, and midbrain) were collected for quantitation of mRNA expression of cytokines, adhesion molecules, and inducible nitric oxide synthetase (iNOS) by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression levels of tumor necrosis actor-alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1-beta (IL-1beta), intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synethetase (iNOS) were highest in the cerebellum of infected animals, correlating well with pathologic observations of sequestration of parasitized erythrocytes in this region of the brain. Infected animals also had higher TNF-alpha expression levels in the cortex and IL-1beta expression levels in the cortex, white matter, and midbrain. Thus, the expression of pro-inflammatory and T helper-1 (TH-1) cytokines, adhesion molecules, and iNOS appears to predominate in the cerebellum of infected rhesus monkeys.

Glucose transport in cerebral microvessels during Plasmodium yoelii nigeriensis infection in mice.

Plasmodium yoelii infected cerebral micro vessels of mice registered a significant increase in D-[U-14C] Glucose transport as compared to normal microvessels which was found to be time, temperature and concentration dependent. Metabolic inhibitors galactose, manose, 2-deoxy glucose and D-glucose showed noticeable inhibition of the same.

IgE deposition in brain microvessels and on parasitized erythrocytes from cerebral malaria patients.

Postmortem brain tissues of 21 cerebral malaria cases were obtained in Myanmar and Vietnam. The tissues were examined by light microscopy and by an immunohistochemical method. Brain microvessels (capillaries and venules) were examined for the presence of immunoglobulins IgE and IgG, Plasmodium falciparum antigen, and parasitized erythrocytes (PRBC). Deposition of IgE, IgG, and P. falciparum antigen was observed in the microvessels from all specimens examined. Sequestered PRBC in the microvessels were positive for IgG in all 21 cases and for IgE in six cases. In the latter cases, the percentage of microvessels with sequestered PRBC was > 50%, with the frequency of IgE-positive cells ranging from 42% to 52%. In contrast, in five cases that were only weakly positive for IgE, the percentage of microvessels with sequestered PRBC was remarkably low (< 1%). These data indicate that the degree of deposition of IgE in microvessels and on PRBC from cerebral malaria patients correlated with that of PRBC sequestration. As IgE-containing immune complexes are known to induce local overproduction of tumor necrosis factor-alpha (TNF-alpha), a major pathogenic factor in cerebral malaria, IgE may contribute to the pathogenesis of this severe disease.

Can Babesia infections be used as a model for cerebral malaria?

Infections with certain species of Plasmodium and Babesia induce, among other symptoms, cerebral pathology. The finding of heavily parasitized cerebral capillaries upon postmortem examination has led to the assumption that blockage of capillaries with infected red blood cells caused the cerebral symptoms and subsequent death. As this type of cerebrovascular pathology is found both in humans dying from malaria and in cattle dying from babesiosis, the latter could possibly be used as an animal model for the study of human cerebral malaria. However, before such a model system is adopted, the experimental data concerning cerebral pathology of babesiosis needs critical evaluation. Here, Theo Schetters and Wijnand Eling review the pathological mechanisms in cerebral babesiosis and relate these to cerebral malaria. Finally, they discuss the use of animal model systems for specific aspects of the pathological picture.

Microcirculation and Chagas' disease: hypothesis an recent results: invited review

Esta revisao enfoca os estudos que levaram a formulacao da hipotese microvascular, alem da participacao de mecanismos imunologicos e neurogenicos e do papel do parasita, para explicar a patologia e curso clinico do envolvimento miocardico na cardiopatia chagasica. Sao discutidos alguns aspectos sobre microcirculacao e doenca de Chagas

Secondary collapsing glomerulopathy associated with Loa loa filariasis.

This is the first case of nonprimary collapsing focal segmental glomerulosclerosis (FSGS) associated with Loa loa filariasis. Loa loa micofilariae were detected on a blood smear after a patient presented with nephrotic syndrome (NS), microhematuria, and renal failure. The renal biopsy showed a collapsing glomerulopathy variant of FSGS. Microfilariae also were identified in renal microvasculature, including the afferent arterioles and the glomerular and peritubular capillaries.

A correlation of the sequestration rate of Plasmodium coatneyi-infected erythrocytes in cerebral and subcutaneous tissues of a rhesus monkey.

Parasitized red blood cells (PRBCs) were sequestered in microvessels of cerebral and subcutaneous tissues of a rhesus monkey infected with Plasmodium coatneyi. A similar sequestration rate (approximately 80%) was observed in both cerebral and subcutaneous microvessels. Electron microscopy showed knobs of the sequestrated PRBCs cytoadhered to endothelial cells. These results are consistent with the finding of PRBC sequestration in subcutaneous tissues in a comatose patient with cerebral malaria. Biopsy specimens of subcutaneous tissue may be useful as indicators of PRBC sequestration in the brain of cerebral malaria patients.

Trypanosoma congolense in the microvasculature of the pituitary gland of experimentally infected Boran cattle (Bos indicus).

The pituitary glands of seven Boran cattle (Bos indicus), five infected with a clone of Trypanosoma congolense IL 1180 (ILNat 3.1) transmitted by Glossina morsitans centralis and two uninfected controls, were examined by light and electron microscopy 43 (experiment 2) or 56 (experiment 1) days after fly challenge. The three cattle used in the first experiment included a 15-month-old female (No. 1), a 24-month-old female (No. 2), and a 21-month-old male (No. 3) as a control. In the second experiment, four cattle were used: two females (Nos. 4, 5) and one male (No. 6), all between 15 and 24 months of age, and one female control (No. 7) of similar age. In all the infected animals, dilation of both the sinusoids and microvasculature was apparent, as was an increase in the thickness of the extracellular matrix between the pituitary lobules. Trypanosomes were found in the microvasculature of the adenohypophysis and neurohypophysis in all the infected animals. Focal degenerative changes were seen in the adenohypophyseal section of glands from the infected animals euthanatized 56 days post-infection. These degenerative structural changes were confined to the somatotrophic cells. The possible role that trypanosomes in the microvasculature may play in inducing pituitary damage and dysfunction is discussed.

Enhanced platelet adherence and aggregation in Chagas' disease: a potential pathogenic mechanism for cardiomyopathy.

Spasm and thrombosis of the coronary microcirculation has been implicated in the pathogenesis of the cardiomyopathy of Chagas' disease. We demonstrate that increases in platelet adherence and aggregation accompany Trypanosoma cruzi infection and may contribute to the observed microvascular pathology. Scanning electron microscopy and radiolabeled platelets studies revealed that platelet adherence to T. cruzi-infected human endothelial cells was significantly increased when compared to controls (P = 0.024). In in vitro experiments, we determined the influence of infection on prostacyclin production, a marker of endothelial cell perturbation. The basal levels of 6-keto-prostaglandin F1 alpha was significantly greater in the supernatant of infected endothelial cells than in those of uninfected endothelial cells (P less than 0.05). The influence of infection was assessed on platelet aggregation at days 5 and 12 post-infection in A/J mice. Platelets from T. cruzi-infected mice were 2-6-fold more sensitive to aggregation induced by adenosine diphosphate and sodium arachidonate than controls. Thromboxane B2 levels in the plasma of infected mice were greater than controls. These data support the hypothesis that heightened platelet reactivity and endothelial cell dysfunction are associated with acute Chagas' disease and may cause coronary microvascular spasm and/or occlusion.