Rosette formation by Plasmodium coatneyi-infected red blood cells

Animal models are needed for the study of cytoadherence in falciparum malaria. Red blood cell (RBC) rosette formation is one type of cytoadherence and appears to be associated with knob formation, endothelial cell adhesion and sequestration of Plasmodium-infected RBCs. Since Plasmodium coatneyi-infected RBCs develop knobs and sequester, we hypothesized that they also form rosettes. RBCs from P. coatneyi-infected rhesus monkeys (Macaca-mulatta) were collected, allowed to mature overnight in vitro and found to form rosettes as hypothesized. This observation adds to the known falciparum-like characteristics of P. coatneyi, and suggests that the Macaca mulatta-P. coatneyi model may be appropriate for pathophysiologic studies of cytoadherence.     

Cytokine production in rhesus monkeys infected with Plasmodium coatneyi.

Plasmodium coatneyi infection in rhesus monkeys has been used as a model for studying human malaria. Cytokine production in this model, however, has so far not been examined. In this study, four rhesus monkeys were infected with P. coatneyi, with another four animals serving as uninfected controls. Blood samples were taken for the determination of daily parasitemia, and cytokine and prostaglandin E2 (PGE2) levels at days 0, 3, 5, 7, and 10. All inoculated animals became infected, with synchronized appearance of ring-stage parasites. Infected monkeys had increased plasma levels of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha) during the late stage of the infection. They also had increased production of ciliary neurotrophic factor. In conjunction with the production of proinflammatory cytokines, infected monkeys also had gradual increases in the production of PGE2. A continued definition of the P. coatneyi/rhesus monkey animal model should be useful for the elucidation of the immunopathogenesis of human malaria.     

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.     

Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin

Summary. Adhesion of parasitized erythrocytes to microvascular endothelium is a central event in the pathogenesis of severe falciparum malaria. We have characterized the adhesion of flowing parasitized red blood cells to three of the known endothelial receptors coated on plastic surfaces (CD36, intercellular adhesion molecule-1 (ICAM-1) and thrombospondin (TSP)), and also to cells bearing these receptors (human umbilical vein endothelial cells (HUVEC) and platelets). All of the surfaces could mediate adhesion at wall shear stress within the physiological range. The great majority of adherent parasitized cells formed rolling rather than static attachments to HUVEC and ICAM-1, whereas static attachments predominated for platelets, CD36 and TSP. Studies with monoclonal antibodies verified that binding the HUVEC was mainly via ICAM-1, and to platelets via CD36. Adhesion via ICAM-1 was least sensitive to increasing wall shear stress, but absolute efficiency of adhesion was greatest for CD36, followed by ICAM-1, and least for TSP. TSP did not give long-lasting adhesion under flow, whereas cells remained adherent to CD36 or ICAM-1. We propose that the different receptors may have complementary roles in modulating adhesion in microvessels. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM-1 or other receptors, with immobilization and stabilization occurring via CD36 and/or TSP.     

Cerebral metabolic reduction in severe malaria: fluorodeoxyglucose-positron emission tomography imaging in a primate model of severe human malaria with cerebral involvement

Cerebral metabolic changes in Japanese macaques (Macaca fuscata) infected with Plasmodium coatneyi, a primate model of severe human malaria with cerebral involvement, were directly evaluated by fluorodeoxyglucose-positron emission tomography (FDG-PET). We observed diffuse and heterogeneous reduction of metabolism in the cerebral cortex in the acute phase of malaria infection. Neuropathologic examination showed preferential sequestration of parasitized red blood cells in the cerebral microvasculature. However, hemorrhagic change or necrosis was not observed in hematoxylin and eosin-stained and Nissl-stained brain tissues. This suggests that reduction of cerebral metabolism occurs before parenchymal changes appear in the brain. This may be one reason why more than half of the patients with cerebral malaria have no neurologic sequelae after recovery.     

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.     

Human cerebral malaria

Possible factors contributing to the development of cerebral malaria were discussed based on pathological changes in Burmese patients who died of cerebral malaria. Blockage of cerebral capillaries by Plasmodium falciparum infected erythrocytes appeared to be the principal cause of cerebral malaria. From electron microscopic results, it was concluded that knobs on infected erythrocytes acted as focal junctions which mediated adhesion to endothelial cells. The knobs are, therefore, important contributors to the blockage of the capillary lumen and ensuing pathological changes in cerebral tissues. Host cell molecules such as OKM5 and thrombospondin may function as endothelial cell surface receptors for the attachment of knobs of P. falciparum infected erythrocytes. Immunological events might also play a role in the pathogenesis of cerebral malaria. This was suggested by the presence of IgG, IgM, P. falciparum antigens, and knob proteins in the cerebral capillaries of the people with cerebral malaria. It will be important to assess the candidate malaria vaccines now in development not only for their efficacy in reducing parasitemia but for effects they may have on the sequestration of infected erythrocytes in the brain.     

Alterations in the profile of blood cell types during malaria in previously unexposed primigravid monkeys

Malaria in nonimmune, primigravid women threatens both mother and fetus. We used the Plasmodium coatneyi/rhesus monkey model to examine factors associated with this. Clinical and immunologic responses during the blood stage of chronic malaria (4 months) were evaluated in 8 malaria-naive primigravid (PMI) and 8 age-matched nulligravid (NMI) infected monkeys, compared with those in 8 primigravid, noninfected control monkeys. Although parasitemia levels were similar, recrudescence was more frequent and prolonged, and anemia was more severe in PMI than in NMI monkeys. During infection, CD2+, CD4+, and CD8+ lymphocyte levels were higher in NMI than in PMI monkeys. Monocyte and neutrophil levels were lower in PMI than in NMI monkeys. During chronic, untreated malaria, NMI monkeys had a B lymphocyte count 23 times greater than that of PMI monkeys. Pregnancy-induced immunomodulation, defined as a lack of appropriate cellular responses to malaria, was indiscernible until the immune system was challenged by a pathogen.     

Pathology of falciparum malaria in Vietnam.

Autopsy samples from the brains of 20 patients who died of falciparum malaria were examined by light microscopy and by an immunohistologic method. Particular attention was paid to a comparison of the pathologic features of the white matter and the cortex. In the high-sequestration (greater than 50%) group (n = 8), the mean +/- SD percentage of cerebral microvessels that showed parasitized red blood cell (PRBC) sequestration was 71.2 +/- 8.1% in the cortex and 84.0 +/- 6.7% in the white matter. The difference in the PRBC sequestration rate between cortex and white matter was statistically significant (P less than 0.01). Perivascular and ring hemorrhages were seen more frequently in the white matter than in the cortex. Deposition of IgG and Plasmodium falciparum antigen in the cerebral microvessels was more highly significant in the white matter than in the cortex (P less than 0.01). Our study demonstrated that the localized concentration of PRBC sequestration in the brain correlated with the marked immunohistologic differences in the microvessels of cortex and white matter.     

脑型疟患者红细胞膜蛋白分子的表达

目的： 为研究脑型疟发病机制及防治提供理论依据。方法： 应用十二烷基硫酸钠－聚丙烯酰胺凝胶电泳 （ＳＤＳ－ＰＡＧＥ） 技术, 对云南省１９ 例脑型疟患者感染红细胞 （ＰＥ） 表达的红细胞膜蛋白１（ＰｆＥＭＰ－１） 进行分析, 并分别与４３ 例恶性疟、９ 例间日疟患者ＰＥ表达的ＰｆＥＭＰ－１和ＰｖＥＭＰ－１及６ 例健康人红细胞膜蛋白（ＥＭＰ） 进行比较。结果： 脑型疟患者ＰＥ存在高表达的高分子量ＰｆＥＭＰ－１, 分子量为２６０～３２０ ｋＤａ。恶性疟及间日疟患者ＰＥ不表达２６０ ｋＤａ 以上的高分子量ＰｆＥＭＰ－１,分别测到分子量最大为２４０ｋＤａ 的ＰｆＥＭＰ－１和１８０ ｋＤａ的ＰｖＥＭＰ－１。健康人对照组ＥＭＰ分子量为１４０ ｋＤａ。结论： 脑型疟患者ＰＥ高表达的不同高分子量ＰｆＥＭＰ－１ ２６０～３２０ ｋＤａ, 与脑血管内皮细胞（ＥＣ） 不同受体蛋白ＣＤ３６、血小板反应蛋白 （ＴＳＰ）、细胞间粘附分子１ （ＩＣＡＭ－１）、血管细胞粘附分子１ （ＶＣＡＭ－１） 和内皮白细胞粘附分子１（ＥＬＡＭ－１） 及硫酸软骨素Ａ （ＣＳＡ） 的结合是脑型疟发病的分子基础, 可导致脑型疟患者发生昏迷。     

A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.     

Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study

Thirty-nine falciparum malaria autopsy cases from the Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand were divided into two groups that had had either cerebral malaria (CM) or non-cerebral malaria (NCM). We then studied significant pathological differences between these groups in order to investigate the correlation between parasitized erythrocyte (PRBC) sequestration in small blood vessels in the brain, heart, lungs and small intestines. We found that the percentage of PRBC sequestration in the organs which we studied was higher in the CM patients than in the NCM patients. The difference of PRBC sequestration among the organs of two groups was significant (P less than 0.05). In the CM group, the average percentage of PRBC sequestration in the brain was significantly higher than in the heart, lungs and small intestines (P less than 0.05). No statistically significant difference was found between PRBC sequestration in the brains, hearts, lungs and small intestines in the NCM group (P greater than 0.05). Our study indicates that severity of malaria in the CM patients depends on PRBC sequestration, especially in the brain. A combination of functional disturbances of the other organs, in addition to the cerebral pathology, may augment the severity of the disease.     

Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1

The pathogenicity of Plasmodium falciparum is due largely to the parasite's unique ability to adhere to capillary and postcapillary venular endothelium during the second-half of the 48-hour life cycle. The resulting sequestration of infected erythrocytes (IRBC) in deep vascular beds leads to tissue hypoxia, metabolic disturbances, and organ dysfunction which characterize severe falciparum malaria. Several endothelial receptors of cytoadherence have been identified, but their clinical relevance remains controversial. In the present report, the receptor specificity of 60 clinical P falciparum isolates was determined using transfectants each expressing one of CD36, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). All isolates tested adhered to CD36 and ICAM-1, but the adherence to CD36 was at least 10-fold higher. Seven isolates adhered to E-selectin whereas none of 19 isolates adhered to VCAM-1. From a population standpoint, about 30% of IRBC in each isolate adhered to CD36, and 2% to 3% adhered to ICAM-1. The percentage adherent to E-selectin and VCAM-1 was negligible. IRBC selected on CD36 adhered almost exclusively to CD36 whereas 80% to 90% of IRBC selected on ICAM-1 could also adhere to CD36. Selected IRBC did not adhere to E-selectin or VCAM-1. These findings indicate that cytoadherence to multiple endothelial receptors is a rare occurrence with natural P falciparum isolates, but do not exclude a role for the adhesion molecules in promoting other IRBC-endothelial interactions such as rolling under flow conditions. Receptor specificity in vivo may be dictated by the ligand-receptor combination which provides the best survival potential for the parasite.     

Imaging analysis of the brain in a primate model of cerebral malaria

This paper reviews our studies concerning imaging analysis of the brain in a primate model of cerebral malaria. To elucidate the clinical features of cerebral malaria, we performed positron emission tomography with ¹⁸F-fluorodeoxyglucose (FDG-PET) scanning and magnetic resonance imaging (MRI) of the brain in Japanese macaques (Macaca fuscata) infected with Plasmodium coatneyi, a primate model of severe human malaria with cerebral involvement. On FDG-PET scanning, we observed diffuse and heterogeneous reduction of metabolism in the cerebral cortex in the acute phase of malaria infection. Although the monkey exhibited severe clinical signs, MR imaging did not reveal any significant changes during the course of infection. Histopathologic examination frequently revealed preferential sequestration of PRBCs in the cerebral and cerebellum capillaries, but neither parenchymal injury nor neuronal necrosis was found in the tissues. These results suggest that heterogeneous metabolic reduction and lack of abnormalities on MRI in the acute phase of CM may be due to any avoidance mechanisms from ischemia caused by sequestration. This may be one reason why more than half of CM patients have no neurological sequelae following recovery.     

Simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)-infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8(+) CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.     

Disseminated intravascular coagulation complicated by peripheral gangrene in a rhesus macaque (Macaca mulatta) experimentally infected with Plasmodium coatneyi

We report the first case of disseminated intravascular coagulation (DIC) complicated by peripheral gangrene induced by Plasmodium coatneyi in rhesus monkeys. Ten days after experimental challenge, numerous petechiae were noted over the trunk and extremities, with polychromasia, severe anemia, thrombocytopenia, and moderate parasitemia. These changes were accompanied by elevated serum activity of blood urea nitrogen, creatinine, transaminases, and creatinine phosphokinase. The animal received intravenous fluid support, artemether, and blood transfusion. Three days after treatment, the platelet counts returned to normal, and parasitemia was abated. However, several areas of skin discoloration with gangrenous tissue in the hands and the tail were observed. Coagulation profile showed elevated D-dimers and elevated levels of fibrinogen/fibrin degradation products with low levels of protein S functional activity. DIC with peripheral gangrene is very rare in Plasmodium-infected individuals. Our results indicate that the experimental model of P. coatneyi infection of rhesus monkeys is important for studies of malarial anemia and coagulopathy.     

Sulfated glycoconjugates enhance CD36-dependent adhesion of Plasmodium falciparum-infected erythrocytes to human microvascular endothelial cells

A novel adhesive pathway that enhances the adhesion of Plasmodium falciparum-infected erythrocytes (IEs) to endothelial cells has been identified. The sulfated glycoconjugates heparin, fucoidan, dextran sulfate 5000, and dextran sulfate 500 000 caused a dramatic increase in adhesion of IEs to human dermal microvascular endothelial cells. The same sulfated glycoconjugates had little effect on IE adhesion to human umbilical vein endothelial cells, a CD36-negative cell line. The effect was abolished by a monoclonal antibody directed against CD36, suggesting that enhanced adhesion to endothelium is dependent on CD36. No effect was observed on adhesion to purified platelet CD36 cells immobilized on plastic. The same sulfated glycoconjugates enhanced adhesion of infected erythrocytes to COS cells transfected with CD36, and this was inhibited by the CD36 monoclonal antibody. These findings demonstrate a role for sulfated glycoconjugates in endothelial adherence that may be important in determining the location and magnitude of sequestration through endogenous carbohydrates. In addition, they highlight possible difficulties that may be encountered from the proposed use of sulfated glycoconjugates as antiadhesive agents in patients with severe malaria.     

Effects of hydroxyurea on malaria, parasite growth and adhesion in experimental models

We recently raised concern over using hydroxyurea (HU) in the treatment of sickle cell disease in areas endemic for malaria, becauseit up-regulates the endothelial surface expression of ICAM-1, a major receptor for Plasmodium falciparum-infected erythrocytes in the brain. Using human in vitro models of cerebral malaria, we evaluated the interaction of HU with parasites and demonstrated that HU pretreatment increased the number of infected red blood cells adhering to the endothelium, but did not increase endothelial apoptosis. Moreover, using an experimental cerebral malaria model, HU pretreatment was found to prevent significantly mice from developing neurological syndrome by inhibiting parasite growth, opening potential therapeutic avenues.     

Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1.

The CD36 and ICAM-1 glycoproteins on vascular endothelial cells have been implicated as cytoadherence receptors for Plasmodium falciparum-infected erythrocytes (IRBC). Adhesion of IRBC from Thai patients with uncomplicated and severe falciparum malaria to purified CD36 or ICAM-1 and to C32 melanoma cells was compared. All malaria isolates bound to solid phase-adsorbed CD36 and to fluid-phase 125I-labeled CD36. IRBC adhesion to purified ICAM-1 varied widely, and no correlation with clinical severity of disease was observed. The cytoadherent phenotype of IRBC was modulated by selective panning on plates coated with purified CD36 or ICAM-1. IRBC selected by panning on CD36+, ICAM-1+ melanoma cells bound to cells that express surface CD36 but not to CD36-deficient cells, indicating that CD36 exerts a strong selective pressure on the IRBC cytoadherent phenotype. IRBC adhesion to CD36 and ICAM-1 suggests that P. falciparum parasites may use these receptors in vivo to promote parasite survival and immune evasion.