S5). 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 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. Ophiopogonin D’ may lead to severe disease as a result of infected erythrocyte (IE) binding in brain or placental microvascular blood vessels. erythrocyte membrane protein 1 (PfEMP1) (1C3). Clonal antigenic variation of genes modifies the antigenic and binding properties of IEs (2, 4, 5) and is believed to mediate IE tropism for different microvascular sites contributing to organ-specific pathology (6). Members Ophiopogonin D’ of the gene family are classified into three main subfamilies (groups A, B, and C) on the basis of chromosome location, direction of transcription, and upstream promoter sequence (UpsA, UpsB, and UpsC) (7C9). A small number of group B/A genes also have an UpsB promoter and group A coding sequence. This gene organization is conserved across parasite isolates (10, 11) and may contribute to gene recombination hierarchies that underlie the functional and antigenic specialization of groups for distinct binding niches (12). Whereas most PfEMP1 proteins bind CD36, group A and the related group B/A have a distinct protein head structure from other groups (8, 9) and do not bind CD36 (13, 14). Group A and B/A proteins also tend to be among the first PfEMP1 proteins expressed in early childhood infections (15C18) and have been associated with more severe infections (18C21), but whether they have affinity for brain endothelium is unknown. Studies of malaria during pregnancy have demonstrated how a single gene, VAR2CSA, is primarily responsible for placenta binding (22C24). However, attempts to associate specific PfEMP1 variants with cerebral binding (25) have been complicated by the extensive diversity of the gene family and the lack of animal models for studying binding of genotype can encode multiple ICAM-1Cbinding PfEMP1 variants (38), but whether all of these are involved in cerebral homing is unknown. Thus, the molecular mechanisms associated with cerebral recruitment remain poorly understood. In this study, we used several primary human brain microvascular endothelial cell (HBMEC) isolates (39C41) to assess the selectivity of different parasite subpopulations for brain endothelium and to characterize the gene repertoire and binding characteristics of Adhesion Types to Resting or Activated HBMECs. To investigate parasite specificity for primary HBMECs, binding was compared from five parasite lines that were derived from the IT4/FCR3 parasite genotype but expressed different genes (13). A CSA-binding parasite (FCR3-CSA) displayed the lowest binding level, a CD36 parasite line (A4long) had an intermediate binding level, and three ICAM-1 plus CD36-binding parasite lines (ItG-ICAM-1, 3G8, and A4ultra) bound at similar levels and only slightly higher than the CD36-binding parasite line (Fig. 1). Open in a separate window Fig. 1. Binding of 0.001). (are expressed as means SDs from two to three independent experiments. The inflammatory cytokine TNF- is produced during malaria infections and causes widespread endothelial activation (30). TNF- activated HBMECs displayed 10-fold higher ICAM-1 surface levels, but CD36 surface levels did not change (Fig. S2). TNF- activation did not enhance binding of CD36- or CSA-binding parasite lines, but binding increased 2-fold for two of the three ICAM-1Cbinding parasite lines (Fig. 1Genes. To investigate whether HBMEC panning selected for unique parasite adhesion traits, the parasite lines were characterized for gene transcription by quantitative (Q)-RT-PCR with specific primers to family members (13). Before selection, the starting ItG-ICAM-1 predominantly transcribed transcript Ophiopogonin D’ was strongly decreased and two new transcripts (and genes are up-regulated in parasite lines HTRA3 panned on primary HBMECs. (and genes was compared from ring-stage parasites before and after panning Ophiopogonin D’ on primary HBMECs (donor 13). Results were normalized to the housekeeping control gene adenylosuccinate lyase (genes that expressed onefold or more of are indicated. (products. Binding predictions are from repertoire-wide analysis of PfEMP1 recombinant proteins between CIDR-CD36 (14) and DBL-ICAM-1 (38). Two of the selected genes, IT4var6 and IT4var19, belong to the group B/A (Fig. 4gene, and transcript (Fig. 4transcription and gain of two non-CD36/nonCICAM-1 binding products. Moreover, both HBMEC panned parasite lines were less sensitive to inhibition with anti-CD36 or antiCICAM-1 mAbs than the starting populations (Fig. 2transcript (transcripts including (Fig. 5 and Fig. S5), which has also been observed to be a frequent switch event in previous limited dilution clonings (13). A clonal parasite line expressing was not.