Advances in the study of var gene expression in field isolates have been made due to the availability of robust molecular techniques which have allowed the determination of various surface antigens expressed on the surface of an infected erythrocyte in relation to cytoadherence and disease severity. A highly dynamic and variable picture of simultaneously expressed var transcripts has been observed in field isolate samples confirming the recombinogenic nature of var genes,with a correlation
81 between the number of var transcripts and the number of infecting strains (Kaestli, Cortes et al. 2004; Kaestli, Cockburn et al. 2006). This has raised a common challenge of identifying several non-identical sequences between different isolates. This complication poses challenges in how the enormous amount of data that has already been generated can be meticulously analysed and fully understood. This problem is well pointed out by (Barry, Leliwa-Sytek et al. 2007) who observed a vast diversity of DBL1α domains of var genes in the genome of parasites sampled from widespread geographic origins compared to parasites from a single malaria endemic area of Papua New Guinea (PNG).
The vast var gene diversity is largely responsible for the inherent difficulties in population genomic analysis of highly diverse multigene families of Plasmodium spp. Individual P. falciparum genomes have repertoires of var genes that can recombine with other repertoires during the sexual phase of the life cycle in the mosquito (Su, Ferdig et al. 1999). There is also circumstantial evidence for ectopic recombination among var genes within the same genome, possibly during both meiosis and mitosis, creating a possibility to generate diversity even among closely related genomes (Ward, Clottey et al. 1999; Freitas-Junior, Bottius et al. 2000; Taylor, Kyes et al. 2000). Additional complications in the P. falciparum genome are the several unusual features such as extreme AT bias, large tracts of non-unique sequences and several large families of polymorphic genes (Gardner, Hall et al. 2002).
Parasite populations are generally distinct based on geographical location. For example, South American and Asia-Pacific isolates commonly amplified identical DBL1α
82 sequences from multiple patients, whereas this is a rare occurrence in sub-Saharan African samplings of circulating populations (Nogueira, Wunderlich et al. 2001; Fowler, Peters et al. 2002; Bull, Berriman et al. 2005; Albrecht, Merino et al. 2006; Fowler, Chavchich et al. 2006; Barry, Leliwa-Sytek et al. 2007). Bull et al. have shown that var
genes from Kenyan field isolates and laboratory isolates can be classified into biologically meaningful subsets based on cysteine-containing small blocks of semi- conserved sequences, such as the DBL domain cassette classification described by Lavstsen et al (Rask, Hansen et al. 2010) thus providing some evidence of the existence of var gene semi-structuring (Bull, Berriman et al. 2005). However, there was variation in expression of these semi-conserved sequences in parasites. In most of the isolates there were clear dominant sequences present at high frequencies expressed by the parasite in different infections that were consistent with those found in field isolates of other studies. Further, Warimwe et al. from the same group showed that expression of a minor component of all genomic var repertoires of semi-conserved blocks with two cysteine residues, “cys2”, were associated with parasites from young children with severe malaria and low immunity against malaria (Warimwe, Keane et al. 2009). The
var genes with the 2 cysteine structure mostly belong to group A var genes. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organised such that PfEMP1 molecules that confer the most virulence to the parasite belong to group A vars (Kyriacou, Stone et al. 2006).
So far, var gene studies have employed the use of polymerase chain reaction (PCR) techniques for the amplification of parasite DNA. A conserved DBL1domain is
83 targeted using “universal” primers to amplify a tag of variable sequence and length with which to identify each transcript distinct. These primers were subsequently shown to exhibit bias for a subset of var genes, affecting sequence abundances by leaving other var sequences untargeted (Taylor, Kyes et al. 2000). A new set of universal primers designed by Kyes et al. in 1997, revised by Taylor in 2000 and again by Bull in 2005, are the most utilised for field studies of all the primer sets developed so far (Kyes, Taylor et al. 1997; Taylor, Kyes et al. 2000; Bull, Pain et al. 2005).
Studies on circulating parasites have demonstrated that there is high turnover in the antigens expressed during infection with most studies linking high expression of group A var genes in peripheral blood to severe disease syndromes (Bull, Berriman et al. 2005; Kaestli, Cockburn et al. 2006; Kyriacou, Stone et al. 2006; Rottmann, Lavstsen et al. 2006; Warimwe, Keane et al. 2009). Examination of postmortem samples of severe infections has shown a reduced genetic diversity compared to mild and asymptomatic paediatric malaria (Montgomery, Milner Jr. et al. 2006; Milner, Valim et al. 2012). Their pattern of antigen expression is, however, largely unknown. Nonetheless, Dobano et al used immunofluorescence antibody typing for MSP 1 and 2 alleles to compare circulating parasites and sequestered parasites in the brain and other tissues in the same Malawian children with fatal malaria (Dobano, Rogerson et al. 2007). They found concordance between parasite serotypes in peripheral blood and parasite serotypes in tissues and no difference in the serotype distributions in the different tissues. Previous studies of sequestered parasites have utilised purified receptors or cultured endothelium from particular organs, plus clinical isolates and
84 laboratory-adapted lines selected for specific adhesive behaviour, to model and investigate potential host-parasite interactions (Gay, Robert et al. 1995; Prudhomme, Sherman et al. 1996; Xiao, Yang et al. 1996; Traore, Muanza et al. 2000).
There is evidence that the inflammatory responses seen at the blood-brain barrier in cases of CM involves the increased systematic production of pro- inflammatory cytokines such as TNF, lymphotoxin, IFN -γ and IL1β (Kwiatkowski, Hill et al. 1990; Newton and Krishna 1998; Brown, Turner et al. 1999)induced by the immune response to the malaria parasites. The overproduction of cytokines such as TNF results in up-regulation on cerebral EC of ICAM-1, VCAM-1 and E-selectin that facilitates the sticking of pRBC to host receptors when they are expressing the appropriate ligands (Ockenhouse, Ho et al. 1991; Turner, Morrison et al. 1994; Rogerson, Tembenu et al. 1999; Silamut, Phu et al. 1999). In accordance with this, a recent study on paediatric malaria in Ghana also showed staining for ICAM-1, VCAM-1 and E-selectin in association with pRBC in brain microvasculature of fatal CM patients (Armah, Dodoo et al. 2005). However, the specific ligands used by the pRBC during in vivo interactions are unknown, largely due to the immense diversity and complexity of these antigens, and the inaccessibility of sequestered parasites.
As previously mentioned, several adhesive phenotypes that do not utilise endothelia such as rosetting, sequestration of pRBC in the placenta (Miller, Baruch et al. 2002), formation of platelet-mediated clumps (Pain, Ferguson et al. 2001), and pRBC adhesion to vWF (Bridges, Bunn et al. 2010) are all thought to contribute to pathology. Such pRBC adhesion mechanisms are supported by post-mortem histological studies
85 that have shown pRBC accumulating in the microvasculature (Grau, Mackenzie et al. 2003; Wassmer, Combes et al. 2006).
Malawi has a high burden of malaria with no reduction in disease incidence observed over the past decade despite a change of first-line anti-malarial treatment in 2007 and intensification of vector control programmes (Roca-Feltrer, Kwizombe et al. 2012). As such, there is a local malaria surveillance at the QECH and the paediatric clinicopathological study of fatal malaria patients in Blantyre, Malawi, with more than 100 study cases of fatal malaria, including controls, from 1996-2011. The availability of such resources has allowed investigation of host-parasite clumping mechanism and examination of antigen properties of sequestered populations of pRBC in the brain, heart and gut and their interactions with the host.
86 PROJECT AIMS
The purpose of this work is to study tissue samples from a malaria clinicopathology study of fatal pediatric malaria patients. The main aims of the study are to:
1. Determine which var gene groups are expressed by parasites in severe malaria and sequestered in cerebral microvasculature
2. Determine the expression of putative sequestration receptors and cytokines in different paediatric malaria diagnostic groups and organs from fatal cases of P.
falciparum paediatric malaria
3. Determine which particular var/PfEMP1 subtypes and ABO blood groups mediate platelet-mediating clumping
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Chapter 2
2. STUDY PARTICIPANTS, MATERIALS AND METHODS