falciparum /em isolate, 3D7 and were cultured in O Rh+ RBCs as described previously [33]

falciparum /em isolate, 3D7 and were cultured in O Rh+ RBCs as described previously [33]. of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7SM parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 em var /em genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using em var /em -specific primers. Results A selection-induced increased adhesion of 3D7SM iRBC to CD36 resulted in a reduced em var4 /em transcription and VAR4 surface expression. Conclusion VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites. Background Variant surface antigens (VSA) on the em Plasmodium falciparum /em -infected red blood cells (iRBC) are involved in both cytoadherence contributing to disease pathology [1-8] and immune evasion which probably contributes to the severity and persistence of malaria infections [9-11]. Anti-VSA antibodies have been shown to contribute to protective immunity [12-16] and DNAJC15 data from several studies indicate that some important targets for immunity seem to have restricted heterogeneity. The em Plasmodium falciparum /em Erythrocyte Membrane Protein-1 (PfEMP1) is the most studied VSA. PfEMP1 molecules are encoded by the em var /em gene family comprising 50C60 highly diverse genes per haploid genome [17-22]. Any single parasite nuclei transcribes only one variant at a time, a phenomenon referred to as allelic exclusion or mutually exclusive expression [20,23-26]. Cultures of unselected 3D7 parasites predominantly express PfEMP1 mRNA species resembling those of parasites causing uncomplicated malaria (PfEMP1UM). The dominant serological phenotype (recognition by semi-immune and immune human plasma) changes to PfEMP1SM following selection of 3D7 using DynaBeads coated with IgG from semi-immune children [27]. This 3D7SM shows transcriptional upregulation and protein surface expression of one particular Parimifasor group A em var /em gene, em var4 /em ( em PFD1235w/MAL8P1.207 /em )[28]. More recently, group A em var /em genes, together with certain other B/A type were found to be transcribed in isolates from children with cerebral Parimifasor malaria, but not from isolates from equally highly parasitaemic patients without severe malaria syndromes [29]. Group A em var /em genes are one of three major groups (A, B and C) of 3D7 em var /em gene sequences categorized according to chromosomal location, gene orientation, domain structure of the encoded proteins and similarities in coding and non-coding regions. Group A contains seven genes encoding large PfEMP1s of about 400 kDa, with complex domain arrangements and three genes encoding PfEMP1s Parimifasor of about 150 kDa [30,31]. Unlike most other em var /em genes from group B and C, the Group A em var /em genes do not encode sequences thought to be consistent with binding to CD36, a Parimifasor major endothelial receptor for iRBC sequestration [31]. It should be noted however that the correlation between severity, binding phenotype and em var /em gene expression is not clear-cut [4,32]. In this study, changes in the transcription of all em var /em genes and surface expression levels of VAR4 as a result of drifting and changes following selection for binding of 3D7SM to CD36 was investigated. Methods Parasites All parasites used in this study were derived from the em P. falciparum /em isolate, 3D7 and were cultured in O Rh+ RBCs as described previously [33]. Three 3D7 sub-lines were used, namely: 3D7SM-CD36,3D7SM-drift and 3D7UM. 3D7SM-CD36 was obtained by bio-panning 3D7SM on Chinese hamster ovary (CHO) cells expressing CD36. 3D7SM has previously been selected from 3D7 using a pool of plasma from semi-immune Ghanaian children [27,28]. To select for a CD36-binding 3D7SM population, the method described in [27] was followed. Gelatine enriched late-stage 3D7SM was panned on a monolayer of wild-type CHO cells. Unbound iRBC and uninfected RBC (uRBC) were then panned on CHO cells transfected with human CD36. The monolayer was washed repeatedly to remove unbound iRBC and uRBC. To allow bound 3D7SM parasites to reinvade and grow, culture medium and Parimifasor uRBC were added to the monolayer.