Cerebral malaria is associated with differential cytoadherence to brain endothelial cells
2019; Springer Nature; Volume: 11; Issue: 2 Linguagem: Inglês
10.15252/emmm.201809164
ISSN1757-4684
AutoresJanet Storm, Jakob S. Jespersen, Karl B. Seydel, Tadge Szestak, Maurice Mbewe, Ngawina V. Chisala, Patricia Phula, Christian W. Wang, Terrie E. Taylor, Christopher A. Moxon, Thomas Lavstsen, Alister Craig,
Tópico(s)Parasites and Host Interactions
ResumoReport4 January 2019Open Access Transparent process Cerebral malaria is associated with differential cytoadherence to brain endothelial cells Janet Storm Corresponding Author Janet Storm [email protected] orcid.org/0000-0001-7812-4220 Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi College of Medicine, University of Malawi, Blantyre, Malawi Search for more papers by this author Jakob S Jespersen Jakob S Jespersen orcid.org/0000-0002-4930-5900 Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Karl B Seydel Karl B Seydel College of Medicine, University of Malawi, Blantyre, Malawi Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Search for more papers by this author Tadge Szestak Tadge Szestak Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Search for more papers by this author Maurice Mbewe Maurice Mbewe Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Ngawina V Chisala Ngawina V Chisala Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Patricia Phula Patricia Phula Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Christian W Wang Christian W Wang Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Terrie E Taylor Terrie E Taylor Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Search for more papers by this author Christopher A Moxon Christopher A Moxon Institute of Infection and Global Health, University of Liverpool, Liverpool, UK Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Thomas Lavstsen Thomas Lavstsen orcid.org/0000-0002-3044-4249 Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Alister G Craig Corresponding Author Alister G Craig [email protected] orcid.org/0000-0003-0914-6164 Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Search for more papers by this author Janet Storm Corresponding Author Janet Storm [email protected] orcid.org/0000-0001-7812-4220 Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi College of Medicine, University of Malawi, Blantyre, Malawi Search for more papers by this author Jakob S Jespersen Jakob S Jespersen orcid.org/0000-0002-4930-5900 Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Karl B Seydel Karl B Seydel College of Medicine, University of Malawi, Blantyre, Malawi Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Search for more papers by this author Tadge Szestak Tadge Szestak Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Search for more papers by this author Maurice Mbewe Maurice Mbewe Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Ngawina V Chisala Ngawina V Chisala Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Patricia Phula Patricia Phula Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Search for more papers by this author Christian W Wang Christian W Wang Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Terrie E Taylor Terrie E Taylor Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Search for more papers by this author Christopher A Moxon Christopher A Moxon Institute of Infection and Global Health, University of Liverpool, Liverpool, UK Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Thomas Lavstsen Thomas Lavstsen orcid.org/0000-0002-3044-4249 Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark Search for more papers by this author Alister G Craig Corresponding Author Alister G Craig [email protected] orcid.org/0000-0003-0914-6164 Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK Search for more papers by this author Author Information Janet Storm *,1,2,3, Jakob S Jespersen4,5, Karl B Seydel3,6,7, Tadge Szestak1, Maurice Mbewe2, Ngawina V Chisala2, Patricia Phula2, Christian W Wang4,5, Terrie E Taylor6,7, Christopher A Moxon8,9, Thomas Lavstsen4,5 and Alister G Craig *,1 1Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK 2Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi 3College of Medicine, University of Malawi, Blantyre, Malawi 4Department of International Health, Immunology & Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark 5Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark 6Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi 7Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA 8Institute of Infection and Global Health, University of Liverpool, Liverpool, UK 9Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow, UK *Corresponding author. Tel: +44 151 705 3297; E-mail: [email protected] *Corresponding author. Tel: +44 151 705 3161; E-mail: [email protected] EMBO Mol Med (2019)11:e9164https://doi.org/10.15252/emmm.201809164 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF-activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM-1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature. Synopsis Cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to the endothelial cells lining brain vessels is a hallmark of cerebral malaria (CM). This study shows that the ability of IE to cytoadhere in the brain of patients with CM and uncomplicated malaria is associated with the disease. IE from children with uncomplicated malaria do not bind well to brain endothelial cells, whereas IE from CM patients show high levels of binding. Significant associations in IE binding to brain endothelial cells were seen for both ICAM-1 and EPCR. PfEMP1 variants containing EPCR-binding motifs were associated with cerebral malaria. Introduction Despite the significant reductions in mortality and morbidity of malaria in the last decade, the percentage of patients infected with Plasmodium falciparum that succumb to severe malaria (SM) is not changing (WHO, 2017), with cerebral malaria (CM) contributing to much of the mortality. The overall mortality rate for CM in children is 15–25%, with a recent MRI study showing that brain swelling is strongly associated with fatal outcome in CM (Seydel et al, 2015). The pathology of CM has been studied extensively (Idro et al, 2005; Hawkes et al, 2013) but also debated for many decades, as discussed in numerous reviews (Shikani et al, 2012; Cunnington et al, 2013; Storm & Craig, 2014; Wassmer & Grau, 2017). What is clear is that the pathogenesis is multifactorial, with a role for the immune response to the Plasmodium infection (Hunt & Grau, 2003; Ioannidis et al, 2014; Dieye et al, 2016; Mandala et al, 2017; Wolf et al, 2017) and obstruction of the microvasculature by sequestration and rosetting (Rowe et al, 2009; Craig et al, 2012; Ponsford et al, 2012; White et al, 2013; Milner et al, 2015), leading to endothelial dysfunction. Sequestration of P. falciparum-infected erythrocytes (IE) in brain microvasculature is a hallmark of human CM as shown in post-mortem studies (Pongponratn et al, 1991; Taylor et al, 2004), but whether this sequestration is due to differential binding of IE to brain endothelium has been harder to demonstrate. The major mediator of parasite cytoadherence to endothelium is P. falciparum erythrocyte membrane protein 1 (PfEMP1), a variant surface antigen expressed on knobs on the IE surface and encoded by approximately 60 var genes per parasite genome, with only one PfEMP1 being expressed on the surface of any individual IE (Scherf et al, 2008; Pasternak & Dzikowski, 2009). PfEMP1 is composed of multiple Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR) domains and can be classified into four main groups A, B, C and E based on the 5′ upstream sequence of the encoding var gene (Fig 1; Smith, 2014). PfEMP1 binds to a range of receptors and includes the mutually exclusive CD36 and endothelial protein C receptor (EPCR)-binding phenotypes, mediated by N-terminal CIDR domains (Kraemer & Smith, 2006; Semblat et al, 2006, 2015; Rask et al, 2010; Hviid & Jensen, 2015). Approximately half of group A PfEMP1 and a subset of group B/A chimeric PfEMP1, also known as domain cassette 8 (DC8), bind to EPCR via CIDRα1 domains, whereas group B and C PfEMP1 bind CD36 via CIDRα2-CIDRα6 domains. In addition, binding to intercellular adhesion molecule 1 (ICAM-1) is mediated via DBLβ domains adjacent to the CIDR domains and in some cases has been associated with a dual-binding phenotype with EPCR (Lennartz et al, 2017). Figure 1. PfEMP1 domain structureA schematic presentation of PfEMP1 domain structure comprising a N-terminal head structure, 2-6 subsequent C-terminal domains, a transmembrane domain (TM) and an intracellular acidic terminal segment (ATS) with known receptors indicated in bold. Receptor specificity is determined by combined DBL and CIDR domains with mutually exclusive binding to EPCR and CD36 by different CIDRα domains in the head structure. Part of group A PfEMP1 and a particular subset of group B/A chimeric PfEMP1 (DC8) bind to EPCR via CIDRα1 domains, whereas group B and C PfEMP1 bind CD36 via CIDRα2-6 domains. The atypical group E VAR2CSA PfEMP1 binds placental chondroitin sulphate A (CSA) via DBLpam1 and DBLpam2 domains. The binding phenotype of VAR 1, VAR3 and group A CIDRβ/γ/δ domains is unknown, but they do not bind EPCR or CD36. DBLβ domains can be involved in ICAM-1 binding and are from both groups A and B. Not much is known about the other DBL domains (γ/δ/ε/ζ), but the DBLε and DBLζ domains are implicated in IgM and α2-macroglobulin binding. Download figure Download PowerPoint In choosing which host receptors to study, we took into account the findings that categories of PfEMP1 types are also associated with in vivo expression in SM. A particularly strong example of this is where parasites expressing var genes encoding PfEMP1 containing EPCR-binding domains have shown a strong association with the development of SM, including CM (Avril et al, 2012; Claessens et al, 2012; Lavstsen et al, 2012; Bengtsson et al, 2013; Bertin et al, 2013; Jespersen et al, 2016; Kessler et al, 2017; Mkumbaye et al, 2017). In vitro, parasites expressing EPCR-binding PfEMP1 show greater degrees of binding to EPCR, as well as to ICAM-1 receptors, both of which are expressed on brain microvascular endothelium (Turner et al, 2013; Avril et al, 2016; Lennartz et al, 2017). ICAM-1 binding has been mapped to some, but not all, DBLβ domains found adjacent to the N-terminal CIDR domains in about one-third of all PfEMP1. A subset of ICAM-1-binding DBLβ domains were recently shown to be specific for group A EPCR-binding PfEMP1 and found to be expressed at higher levels in parasites from CM patients than in parasites from non-CM patients (Lennartz et al, 2017). Parasites expressing CD36-binding PfEMP1 are found in many patient isolates regardless of symptoms, although some data suggest that they may constitute a smaller proportion of parasites in SM patients (Heddini et al, 2001; Ndam et al, 2017), and are not seen in parasite isolates taken from women with placental malaria (Smith et al, 2013). Several other host receptors for PfEMP1 have been described, however, while PfEMP1 proteins that bind these receptors have been identified (Berger et al, 2013), links between cytoadherence and paediatric CM have not been established, and these were not tested in our study. Infected erythrocytes binding to specific receptors during an infection may have different functional consequences on the endothelium and hence on disease severity. One clear example is that by binding to EPCR, the IE interfere with production of activated protein C thereby launching the coagulation cascade, leading to increased thrombin production (Moxon et al, 2013) and the potential to cause pro-inflammatory PAR1-mediated endothelial activation (Petersen et al, 2015; Gillrie et al, 2016). Other PfEMP1-receptor interactions have been shown to activate signalling pathways in endothelial cells (Wu et al, 2011; Gillrie & Ho, 2017), but the effect of these events on pathology is unclear. More recent work has also suggested that as well as cytoadherence-mediated events, the accumulation of sequestered IE in vessels may facilitate endothelial dysfunction caused by the local release of soluble mediators following schizont rupture (Gallego-Delgado & Rodriguez, 2017). Thus, it remains unclear why a particular child develops CM at a particular time, as the vast majority of P. falciparum infections do not lead to CM. In addition, most African children who develop CM have had malaria previously without developing CM. One possible mechanism to explain why a child develops CM at a particular time is that they have been infected with a P. falciparum variant that facilitates recruitment of IE to endothelium in the brain. While multiple lines of evidence indicate that specific PfEMP1 variants are associated with severe malaria, that association has not been substantiated by directly measuring the binding of IE to endothelial cells (EC). Thus, the question as to whether IE from children with CM have cytoadherence properties that enable them to bind to brain endothelium and thus enhancing their sequestration in that site has not been tested. The extent of sequestration in the brain is unknown for non-CM cases, although post-mortem observations of brain vessels from malaria-infected children dying from other causes of coma (not CM) show much lower levels of IE sequestration than CM cases (Milner et al, 2015). Therefore, as a comparison, isolates from children with uncomplicated malaria (UM) have also been tested for their binding phenotype in the present study. A number of studies have investigated cytoadherence of specific PfEMP1 variants to human microvascular endothelial cells, but these have been with laboratory strains or PfEMP1-modified parasites (Madkhali et al, 2014; Gillrie et al, 2015). Patient isolates have also been investigated for their binding phenotype, but mainly on purified protein and mostly under static conditions (Craig et al, 2012; Almelli et al, 2014; Mahamar et al, 2017; Ndam et al, 2017). While providing important evidence, these studies were unable, largely for technical reasons, to combine the most appropriate target (primary brain endothelium) and parasite isolates as close to the patient sample as possible, with a physiologically relevant assay. To address our hypothesis that CM is driven by increased binding of IE to brain endothelium, we assessed whether IE freshly isolated from circulating blood of children with CM preferentially bound TNF-activated primary brain microvascular endothelium, compared to IE isolated from UM children. We postulated that such a difference might be associated with the expression of particular PfEMP1 variants and with binding to specific endothelial receptors. We collected IE from carefully characterised paediatric CM and UM cases in Malawi and determined cytoadherence to primary human microvascular endothelial cells, with minimal in vitro expansion of the parasite population, using a microfluidic flow device, an experimental design reflecting in vivo physiology. Expression of PfEMP1 variants was investigated by qPCR using the most up-to-date set of var domain type-specific primers available to us. To our knowledge, this study is the first study to employ such a comprehensive approach to address the question of whether cytoadherence is involved in the pathogenesis of CM. Results Recruitment of study participants Children were recruited over three malaria seasons from 2013 to 2015 using the selection criteria described in the Materials and Methods section. Total CM cases admitted to the research ward have been decreasing since 2010, from 165 cases to 48 (18) cases in 2013, 78 (26) in 2014 and 43 (14) in 2015. Numbers in brackets are the recruited number of children for our cytoadherence study. To improve the specificity of the clinical diagnosis of CM, only children with at least one feature of malarial retinopathy (Maccormick et al, 2014) were included, resulting in the recruitment of a total of 58 cases. A total of 53 UM cases, matched on an annual basis to the number of CM cases, were included. Clinical characteristics of the total UM and CM cohorts and the cases used for experiments are summarised in Table 1. The median age of children with UM was higher than children with CM. Compared to children with UM, children with CM had significantly higher median pulse and respiratory rates, higher median lactate concentration and lower median haematocrit levels, indicators of severe disease (WHO, 2016). Ten of the children with CM (17%) died. To achieve 2% parasitaemia needed for the cytoadherence assays, only blood samples from children with at least 2% peripheral parasitaemia were utilised. The clinical characteristics of these selected cases were similar to the overall cohort of children with each of these clinical syndromes. Table 1. Clinical characteristics of the study participants Total cohort uncomplicated malaria (n = 53) Total cohort cerebral malaria (n = 60) P-value Used in assay uncomplicated malaria (n = 35) Used in assay cerebral malaria (n = 27) P-value Age, months 51 (30–74) 42 (24–59) 0.04 53 (38–89) 36 (23–50) 0.005 Gender, % female 53 47 51 48 Axillary temperature, °C 38.8 (38.2–39.4) 39.0 (38.1–40.0) 38.8 (381–39.4) 39.0 (37.9–40.0) Pulse rate, beats/min 124 (107–140) 157 (140–175) < 0.0001 124 (114–146) 156 (133–175) 0.0001 Systolic blood pressure, mmHg 98 (91–102) 95 (86–104)a 97 (91–99) 94 (85–103)b Respiratory rate, breaths/min 30 (25–30) 40 (36–52) < 0.0001 28 (24–30) 41 (32–52) < 0.0001 Blood glucose, mmol/l 5.7 (5.1–6.4) 5.4 (4.4–6.8) 5.8 (5.1–6.5) 5.0 (4.5–6.7) Blood lactate, mmol/l 2.9 (2.0–3.4) 4.5 (2.4–8.8) < 0.0001 2.9 (1.9–3.5) 4.6 (2.3–8.2) 0.0006 Haematocrit, % 35.0 (30.0–38.0) 22.0 (18.0–25.6) < 0.0001 36.0 (29.5–38.0) 20.4 (16.8–25.1) < 0.0001 HRP2, μg/ml 7.1 (2.2–9.9)c 9.5 (3.1–11.0)d Parasitaemia, parasites/μl (×104) 11.8 (4.2–38.4) 29.8 (14.6–59.9) Platelets/μl (×104) 4.9 (2.4–9.6)c 4.9 (2.4–9.8)d Shown are the median with the interquartile range in brackets for the total cohort and the cases used in the binding assays. For each variable, statistical differences between UM and CM cases were determined by Mann–Whitney U-test (continuous variables) or Fisher's exact test (categorical variables), and P-values < 0.05 are indicated. HRP2 = histidine-rich protein 2. Group size in a = 50, b = 21, c = 55 and d = 24 children. Cytoadherence of clinical isolates to microvascular endothelial cells under flow Isolated IE were cultured until the parasites were at the trophozoite stage, when PfEMP1 is expressed on the surface of the IE, and a suspension of 2% parasitaemia and 5% haematocrit was prepared. Using the microfluidic device, cytoadherence to primary human microvascular endothelial cells, derived from brain (HBMEC) and dermis (HDMEC), was determined under flow conditions. Isolates from CM cases demonstrated an average binding of 110 IE/mm2 (95% CI: 37–182) to HBMEC which was significantly higher (P = 0.041) than HBMEC binding of UM cases at 43 IE/mm2 (95% CI: 28–57; Fig 2). In contrast, there was no difference in binding to HDMEC (P = 0.171) between IE from CM cases (average 165 IE/mm2, 95% CI: 81–250) and UM cases (average 110 IE/mm2, 95% CI: 71–149). Binding of UM isolates to HBMEC was significantly lower compared to HDMEC (P = 0.002), which was not the case for CM isolates. For isolates from CM patients, avid binding was a common feature; isolates that bound well to HBMEC also bound well to HDMEC with a Spearman's correlation coefficient of 0.83 (P < 0.0001). For UM isolates, however, there was no correlation between binding to HBMEC and HDMEC (r = 0.20, P = 0.28). A recent publication by Azasi et al (2018b) showed that DC8-PfEMP1 expressing IE do not bind EPCR in the presence of normal human serum. Therefore, we tested whether adding 10% human serum to the binding buffer would decrease the cytoadherence of selected patient isolates to HBMEC (Appendix Fig S2). Human serum did not change the binding of three patient isolates that showed significant EPCR binding to HBMEC. The binding of DC8 variant IT4var19 was also not affected by the addition of human serum in our flow assay system. Figure 2. Cytoadherence of IE from CM and UM cases to HBMEC and HDMECIE were isolated, and binding to HBMEC and HDMEC was determined under flow conditions. Number of IE bound per mm2 EC surface was calculated and shown is the mean ± 95% CI for 26 CM and 33 UM cases on HBMEC and 21 CM and 35 UM cases on HDMEC on a log scale. P-value was calculated by two-tailed unpaired t-test. The dotted line is 20 IE/mm2, the cut-off value for inclusion of the inhibition data. Download figure Download PowerPoint Inhibition of cytoadherence to microvascular endothelial cells under flow To assess the differential role of the endothelial receptors ICAM-1, EPCR and CD36, binding was determined in the presence of inhibitory antibodies, αICAM-1 and αCD36, or recombinant protein, rEPCR (Fig 3). Paired analysis of the inhibition binding data is shown in Fig 3A and significant inhibition was observed for all the EC-inhibitor combinations, except for inhibition of binding of UM isolates to HDMEC by rEPCR. The data are summarised as percentage inhibition in Fig 3B–D, to compare receptor-dependent adherence between CM and UM. Approximately half of the IE displayed ICAM-1-dependent binding (> 50% inhibition) to both HBMEC and HDMEC, but there was no significant difference between CM and UM isolates nor between the dependency of ICAM-1 binding to HBMEC and HDMEC. CD36 expression is extremely low on primary HBMEC (Avril et al, 2016), so studies on binding to CD36 were only performed with HDMEC. Inhibition of cytoadherence by αCD36 antibody was variable and not significantly different between the CM and UM isolates (P = 0.23), although there was a trend for higher CD36-dependent binding in UM isolates. EPCR-dependent binding also varied, with a subset of isolates binding particularly well to EPCR. This was more pronounced for CM isolates binding to HBMEC, but not significantly different from UM isolates (P = 0.073). There was also no significant difference between rEPCR inhibition of binding to HBMEC and HDMEC. For a few isolates, there was more binding in the presence of αICAM-1 antibody or rEPCR compared to binding in the absence of inhibitor (Fig 3B and D), and this was more often the case for UM isolates. The reason for this is unclear; we were unable to collect the bound IE to investigate this phenomenon further. Figure 3. Inhibition of cytoadherence of IE from CM and UM cases to HDMEC and HBMEC by αICAM-1 and αCD36 antibody and rEPCR† A. IE were isolated, and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 μg/ml αICAM-1 or αCD36 antibody or 50 μg/ml rEPCR. Number of IE bound per mm2 EC surface was determined. B. Using the same data, percentage inhibition by αICAM-1 antibody was calculated relatively to binding in the absence of antibody. C. Using the same data, percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor. D. Using the same data, percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody. Data information: (A) shown is the paired analysis between the absence and presence of inhibitor, with number of cases (n) indicated. Statistical significance was determined by two-tailed paired t-test, and the P-value is shown; ns is not significant. (B–D) shown are the mean ± 95% CI, and no significant differences were determined with a two-tailed unpaired t-test. Each assay was only conducted once for each isolate. The number of isolates tested can be seen from the dot plot. Download figure Download PowerPoint Correlations between IE binding and clinical parameters We assessed the association between the in vitro cytoadherence properties of the IE and clinical parameters associated with sequestration. For the CM cases, the degree of IE binding to HDMEC was positively correlated with peripheral parasite density at recruitment (r = 0.56, P = 0.011). Binding of IE to HBMEC was less clearly associated with peripheral parasite density (r = 0.40, P = 0.056). Differences in levels of binding were not due to variation in parasitaemia of the cultured IE, as the binding assay was performed at a standardised 2% parasitaemia. Binding of CM isolates was negatively correlated with peripheral platelet levels for both binding to HDMEC (r = −0.66, P = 0.001) and HBMEC (r = −0.56, P = 0.005). We were unable to assess the association between IE adhesion and fatal outcome as only three children for whom we had binding data died. None of the other clinical characteristics, including histidine-rich protein 2 concentrations, showed any significant correlation with the cytoadherence of IE. For the UM cases, none of the parameters assessed were significantly correlated with cytoadherence. Peripheral parasite density (as per WHO standard) and platelet counts were not determined at admission in UM cases; however, after processing the UM blood samples, parasitaemia was determined by microscopy of Giemsa-stained smears and no correlation was found between parasitaemia and binding intensity. Analysis of var gene transcripts To analyse the PfEMP1 domain structure (Fig 1) of the patient isolates, transcript levels of the coding var genes were determined. qPCR was performed and var transcript values (Tu) were calculated and compared between CM and UM cases (Table 2). A detailed description of coverage, sensitivity and limitati
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