Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.     

How specific is Plasmodium falciparum adherence to chondroitin 4-sulfate?

Plasmodium falciparum infection during pregnancy results in the sequestration of infected red blood cells (IRBCs) in the placenta, contributing to pregnancy associated malaria (PAM). IRBC adherence is mediated by the binding of a variant Plasmodium falciparum erythrocyte binding protein 1 named VAR2CSA to the low sulfated chondroitin 4-sulfate (C4S) proteoglycan (CSPG) present predominantly in the intervillous space of the placenta. IRBC binding is highly specific to the level and distribution of 4-sulfate groups in C4S. Given the strict specificity of IRBC-C4S interactions, it is better to use either placental CSPG or CSPGs bearing structurally similar C4S chains in defining VAR2CSA structural architecture that interact with C4S, evaluating VAR2CSA constructs for vaccine development or studying structure-based inhibitors as therapeutics for PAM.     

Molecular mechanistic insights into the endothelial receptor mediated cytoadherence of Plasmodium falciparum-infected erythrocytes

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the 'holder' for providing stable binding.     

Rodent Plasmodium-infected red blood cells: Imaging their fates and interactions within their hosts

Malaria, a disease caused by the Plasmodium parasite, remains one of the most deadly infectious diseases known to mankind. The parasite has a complex life cycle, of which only the erythrocytic stage is responsible for the diverse pathologies induced during infection. To date, the disease mechanisms that underlie these pathologies are still poorly understood. In the case of infections caused by Plasmodium falciparum, the species responsible for most malaria related deaths, pathogenesis is thought to be due to the sequestration of infected red blood cells (IRBCs) in deep tissues. Other human and rodent malaria parasite species are also known to exhibit sequestration. Here, we review the different techniques that allow researchers to study how rodent malaria parasites modify their host cells, the distribution of IRBCs in vivo as well as the interactions between IRBCs and host tissues.     

Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to vascular endothelium and for platelet-mediated clumping

The ability of Plasmodium falciparum-infected red blood cells (IRBCs) to bind to vascular endothelium, thus enabling sequestration in vital host organs, is an important pathogenic mechanism in malaria. Adhesion of P. falciparum IRBCs to platelets, which results in the formation of IRBC clumps, is another cytoadherence phenomenon that is associated with severe disease. Here, we have used in vitro cytoadherence assays to demonstrate, to our knowledge for the first time, that P. falciparum IRBCs use the 32-kDa human protein gC1qR/HABP1/p32 as a receptor to bind to human brain microvascular endothelial cells. In addition, we show that P. falciparum IRBCs can also bind to gC1qR/HABP1/p32 on platelets to form clumps. Our study has thus identified a novel host receptor that is used for both adhesion to vascular endothelium and platelet-mediated clumping. Given the association of adhesion to vascular endothelium and platelet-mediated clumping with severe disease, adhesion to gC1qR/HABP1/p32 by P. falciparum IRBCs may play an important role in malaria pathogenesis.     

Plasmodium falciparum: the behavior of clinical isolates in an in vitro model of infected red blood cell sequestration

An in vitro model of Plasmodium falciparum-infected red blood cell sequestration which uses C32 amelanotic melanoma cells as targets has been used to examine the binding capacity of infected red blood cells from subjects with naturally acquired P. falciparum infections of varying severity. The binding of infected red blood cells (IRBCs) to melanoma cells was specific to cells containing mature parasites. Variations in target cell density and in conditions of growth had significant effects on binding. Binding was pH dependent, being maximum at a pH of 6.9. Using standardized conditions the binding capacity of individual isolates of P. falciparum could be measured with a high degree of reproducibility. Binding capacity of IRBCs from 51 subjects between the ages of 6 months and 15 years varied between 12 and 1254 IRBCs per 100 melanoma cells when RBC suspensions at a 1% parasitemia and 4% hematocrit were used. Variation in binding was not related to the level of peripheral parasitemia of the isolate or to differences in adaptation to culture conditions. The binding capacity of parasitized cells from subjects with cerebral malaria did not differ from that of IRBCs from subjects with less serious clinical manifestations.     

A Maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells

The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.     

Investigation of host factors possibly enhancing the emergence of the chondroitin sulfate A-binding phenotype in Plasmodium falciparum

In malaria endemic areas, regardless of immunity acquired during lifelong exposure to malaria, pregnant women become susceptible to Plasmodium falciparum infections. Malaria during pregnancy is associated with a massive sequestration of infected erythrocytes in the placenta and the emergence of a unique parasite-derived adhesive molecule (encoded by var2CSA) that binds to chondroitin sulfate A (CSA). How P. falciparum achieves the timely expression of the CSA ligand in pregnant women remains puzzling. We investigated whether host serum-specific factors present only during pregnancy may induce var2CSA expression. Our panel of experiments did not reveal significant changes in var2CSA levels and CSA-binding capacity.     

Chondroitin sulphate A as an adherence receptor for Plasmodium falciparum-infected erythrocytes

Until recently, the sequestration of erythrocytes infected with Plasmodium falciparum has been thought to be due to one of a number of protein-protein interactions. In this article, Stephen Rogerson and Graham Brown summarize the emerging evidence that, in vitro, infected erythrocytes can also adhere to the glycosaminoglycan chondroitin sulphate A (CSA) expressed on the surface of cells and immobilized on plastic. In vivo, binding of infected erythrocytes to CSA could be crucial to the development of malarial infection of the placenta, and possibly to sequestration in the lung and brain. The consequences of this may include maternal morbidity and mortality, low birth weight in the infant, pulmonary oedema and cerebral malaria. They discuss the need to characterize the molecular basis of this interaction, and to investigate the possible therapeutic role of CSA in malaria. Chondroitin sulphates are nontoxic compounds already in use for other diseases in humans. Vaccines based on inhibiting this receptor-ligand interaction could also be appropriate.     

Plasmodium falciparum cytoadherence to human placenta: evaluation of hyaluronic acid and chondroitin 4-sulfate for binding of infected erythrocytes.

Chondroitin 4-sulfate (C4S) is known to mediate the adherence of Plasmodium falciparum infected red blood cells (IRBCs) to human placenta. Recently, hyaluronic acid (HA) has also been reported to bind IRBCs, and HA has been suggested as an additional receptor for the sequestration of IRBCs in the placenta. In this study, we assessed the adherence of 3D7 parasite strain, which has been reported to bind both C4S and HA, using highly purified clinical grade rooster comb HA, Streptococcus HA, several preparations of human umbilical cord HA (hucHA), and bovine vitreous humor HA (bvhHA). While all hucHA preparations and bvhHA bound with moderate to high density to IRBCs, the rooster comb and bacterial HAs did not bind IRBCs. IRBCs binding to the hucHA and bvhHA could be abolished by pretreatment with testicular hyaluronidase but not with Streptomyces hyalurolyticus hyaluronidase, suggesting that IRBC binding to hucHA and bvhHA was due to chondroitin sulfate (CS) contaminants in HAs. Compositional analysis confirmed the presence of CS in both hucHA and bvhHA. The CSs present in these commercial hucHA and bvhHA samples were isolated, characterized, and studied for their ability to bind IRBCs. The data suggested that IRBC adherence to hucHA and bvhHA was mediated by the CS present in these samples. However, our data did not exclude the possibility of a minor population of distinct parasite subtype adhering to HA and further studies using pure HA conjugated to proteins or lipids and placental parasite isolates should clarify whether HA is an in vivo receptor for IRBC adherence.     

The structural motif in chondroitin sulfate for adhesion of Plasmodium falciparum-infected erythrocytes comprises disaccharide units of 4-O-sulfated and non-sulfated N-acetylgalactosamine linked to glucuronic acid

An important characteristic of malaria parasite Plasmodium falciparum-infected red blood cells (IRBCs) is their ability to adhere to host endothelial cells and accumulate in various organs. Sequestration of IRBCs in the placenta, associated with excess perinatal and maternal mortality, is mediated in part by adhesion of parasites to the glycosaminoglycan chondroitin sulfate A (CSA) present on syncytiotrophoblasts lining the placental blood spaces. To define key structural features for parasite interactions, we isolated from CSA oligosaccharide fractions and established by electrospray mass spectrometry and high performance liquid chromatography disaccharide composition analysis their differing chain length, sulfate content, and sulfation pattern. Testing these defined oligosaccharide fragments for their ability to inhibit IRBC adhesion to immobilized CSA revealed the importance of non-sulfated disaccharide units in combination with 4-O-sulfated disaccharides for interaction with IRBCs. Selective removal of 6-O-sulfates from oligo- and polysaccharides to increase the proportion of non-sulfated disaccharides enhanced activity, indicating that 6-O-sulfation interferes with the interaction of CSA with IRBCs. Dodecasaccharides with four or five 4-O-sulfated and two or one non-sulfated disaccharide units, respectively, comprise the minimum chain length for effective interaction with IRBCs. Comparison of the activities of CSA and CSB oligo- and polysaccharides with a similar sulfation pattern and content achieved from partial desulfation demonstrated that glucuronic acid rather than iduronic acid residues are important for IRBC binding.     

Rat spongiotrophoblast-specific protein is predominantly a unique low sulfated chondroitin sulfate proteoglycan

We have previously demonstrated that the human placenta contains a uniquely low sulfated extracellular aggrecan family chondroitin sulfate proteoglycan (CSPG). This CSPG is a major receptor for the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in placentas, causing pregnancy-specific malaria. However, it is not known whether such low sulfated CSPGs occur in placentas of other animals and, if so, whether IRBCs bind to those CSPGs. In this study, we show that rat placenta contains a uniquely low sulfated extracellular CSPG bearing chondroitin sulfate (CS) chains, which comprise only approximately 2% 4-sulfated and the remainder nonsulfated disaccharides. Surprisingly, the core protein of the rat placental CSPG, unlike that of the human placental CSPG, is a spongiotrophoblast-specific protein (SSP), which is expressed in a pregnancy stage-dependent manner. The majority of rat placental SSP is present in the CSPG form, and only approximately 10% occurs without CS chain substitution. Of the total SSP-CSPG in rat placenta, approximately 57% is modified with a single CS chain, and approximately 43% carries two CS chains. These data together with the previous finding on human placental CSPG suggest that the expression of low sulfated CSPG is a common feature of animal placentas. Our data also show that the unique species-specific difference in the biology of the rat and human placentas is reflected in the occurrence of completely different CSPG core protein types. Furthermore, the rat SSP-CSPG binds P. falciparum IRBCs in a CS chain-dependent manner. Since IRBCs have been reported to accumulate in the placentas of malaria parasite-infected rodents, our results have important implications for exploiting pregnant rats as a model for studying chondroitin 4-sulfate-based therapeutics for human placental malaria.     

Malaria parasite-infected erythrocytes inhibit glucose utilization in uninfected red cells

The erythrocytic stages of the malaria parasite depend on anaerobic glycolysis for energy. Using [2-13C]glucose and nuclear magnetic resonance, the glucose utilization rate and 2,3-diphosphoglycerate (2,3-DPG) level produced in normal RBCs and Plasmodium falciparum infected red blood cell populations (IRBCs, with <4% parasite infected red cells), were measured. The glucose flux in IRBCs was several-folds greater, was proportional to parasitemia, and maximal at trophozoite stage. The 2,3-DPG levels were disproportionately lower in IRBCs, indicating a downregulation of 2,3-DPG flux in non-parasitized RBCs. This may be due to lowered pH leading to selective differential inhibition of the regulatory glycolytic enzyme phosphofructokinase. This downregulation of the glucose utilization rate in the majority (>96%) of uninfected RBCs in an IRBC population may have physiological implications in malaria patients.     

CD36 Contributes to Malaria Parasite-Induced Pro-Inflammatory Cytokine Production and NK and T Cell Activation by Dendritic Cells

The scavenger receptor CD36 plays important roles in malaria, including the sequestration of parasite-infected erythrocytes in microvascular capillaries, control of parasitemia through phagocytic clearance by macrophages, and immunity. Although the role of CD36 in the parasite sequestration and clearance has been extensively studied, how and to what extent CD36 contributes to malaria immunity remains poorly understood. In this study, to determine the role of CD36 in malaria immunity, we assessed the internalization of CD36-adherent and CD36-nonadherent Plasmodium falciparum-infected red blood cells (IRBCs) and production of pro-inflammatory cytokines by DCs, and the ability of DCs to activate NK, and T cells. Human DCs treated with anti-CD36 antibody and CD36 deficient murine DCs internalized lower levels of CD36-adherent IRBCs and produced significantly decreased levels of pro-inflammatory cytokines compared to untreated human DCs and wild type mouse DCs, respectively. Consistent with these results, wild type murine DCs internalized lower levels of CD36-nonadherent IRBCs and produced decreased levels of pro-inflammatory cytokines than wild type DCs treated with CD36-adherent IRBCs. Further, the cytokine production by NK and T cells activated by IRBC-internalized DCs was significantly dependent on CD36. Thus, our results demonstrate that CD36 contributes significantly to the uptake of IRBCs and pro-inflammatory cytokine responses by DCs, and the ability of DCs to activate NK and T cells to produce IFN-γ. Given that DCs respond to malaria parasites very early during infection and influence development of immunity, and that CD36 contributes substantially to the cytokine production by DCs, NK and T cells, our results suggest that CD36 plays an important role in immunity to malaria. Furthermore, since the contribution of CD36 is particularly evident at low doses of infected erythrocytes, the results imply that the effect of CD36 on malaria immunity is imprinted early during infection when parasite load is low.     

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus

BACKGROUND: The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. METHODS: The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. RESULTS: Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. CONCLUSIONS: These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.     

Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A

In areas of intense Plasmodium falciparum transmission, clinical immunity is acquired during childhood, and adults enjoy substantial protection against malaria. An exception to this rule is pregnant women, in whom malaria is both more prevalent and severe than in nonpregnant women. Pregnancy-associated malaria (PAM) in endemic areas is concentrated in the first few pregnancies, indicating that protective immunity to PAM is a function of parity. The placenta is often heavily infected in PAM, and placental parasites show a striking preference for chondroitin sulfate A (CSA) as an adhesion receptor. Plasma Abs from malaria-exposed multiparous women are able to interfere with binding of P. falciparum parasites to CSA in vitro, and acquisition of Abs interfering with CSA-specific parasite sequestration thus appears to be a critical element in acquired protection against PAM. Here we show that adults from an area of hyperendemic P. falciparum transmission generally possessed low levels of Abs specifically recognizing surface Ags expressed by a CSA-adhering parasite isolate, while unselected isolates were well recognized. In marked contrast, most third-trimester pregnant women from that area had very high plasma levels of such Abs. Plasma levels of Abs specifically recognizing the CSA-adhering isolate strongly depended on parity, whereas recognition of CSA-nonadhering isolates did not. Finally, we demonstrate a clear correlation between plasma levels of Abs recognizing the CSA-specific isolate and the ability to interfere with its sequestration to CSA in vitro. Our study supports the hypothesis that Abs inhibiting CSA-specific parasite sequestration are important in acquisition of protection against PAM.     

Malaria during pregnancy: parasites, antibodies and chondroitin sulphate A

CSA-binding forms of P. falciparum appear uncommonly in non-pregnant hosts but are selected by the human placenta for growth. Parasites are presumably selected by adherence to CSA within the vascular compartment of the placenta, allowing IRBCs to sequester and multiply to high density. Chondroitin sulphate appears on the surface of placental syncytiotrophoblasts, and CSA is a component of PGs found in the placenta [42], but the identification of the CSA-containing PG(s) mediating IRBC adhesion in vivo requires further study. Anti-adhesion antibodies against CSA-binding parasites are associated with protection from maternal malaria, but these antibodies develop only over successive pregnancies, accounting for the susceptibility of primigravidas to infection. PfCSA-L, the parasite ligand mediating adhesion to CSA, has not yet been identified but is known to be antigenically conserved among isolates from around the world. An anti-adhesion vaccine delivered to women before first pregnancy could confer protection from maternal malaria and might be globally effective.     

Mild inflammatory activation of mammary arteries in patients with acute coronary syndromes

Acute coronary syndromes (ACS) are characterized by multiple unstable coronary plaques and elevated circulating levels of inflammatory biomarkers. The endothelium of internal mammary arteries (IMA), which are atherosclerosis resistant, is exposed to proinflammatory stimuli as vessels that develop atherosclerosis. Our study investigated the IMA endothelial expression of inflammatory molecules in patients with ACS or chronic stable angina (CSA). IMA demonstrated normal morphology, intact endothelial lining, and strong immunoreactivity for glucose transporter 1. E-selectin expression was observed more frequently in IMA of ACS patiention than CSA patients (ACS 61% vs. CSA 14%, P = 0.01). High fluorescence for major histocompatibility complex (MHC) was significantly more frequent on the luminal endothelium (ACS 66.7% vs. CSA 17.6%, P = 0.001 for class I; and ACS 66.7% vs. CSA 6.2%, P = 0.0003 for class II-DR) and on the vasa vasorum (ACS 92.9% vs. CSA 33.3% and 7.7%, P = 0.0007 and P < 0.0001 for class I and class II-DR, respectively) of ACS patients than CSA patients. ICAM-1, VCAM-1, Toll-like receptor 4, tissue factor, IL-6, inducible nitric oxide synthase, and TNF-alpha expression were not significantly different in ACS and CSA. Circulating C-reactive protein [ACS 4.8 (2.6-7.3) mg/l vs. CSA 1.8 (0.6-3.5) mg/l, P = 0.01] and IL-6 [ACS 4.0 (2.6-5.5) pg/ml vs. CSA 1.7 (1.4-4.0) pg/ml, P = 0.02] were higher in ACS than CSA, without a correlation with IMA inflammation. The higher E-selectin, MHC class I and MHC class II-DR on the endothelium and vasa vasorum of IMA from ACS patients suggests a mild, endothelial inflammatory activation in ACS, which can be unrelated to the presence of atherosclerotic coronary lesions. These findings indicated IMA as active vessels in coronary syndromes.     

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.