Cell binding assays

The assays were extended using TNF-activated endothelial cells HUVEC and resting HDMEC under static and flow binding assays. Our finding supports our preliminary protein binding assays. Both ItG and A4 show slightly high binding to activated HUVEC at pH 7.2 (Figure 4.5 and 4.7) and C24 highly binding to HDMEC was at pH 7.1 (Figure 4.6 and 4.8).

Figure 4. 5: The effect of pH on binding of pRBC to HUVEC endothelial cells under static conditions. Plotted bars are the means ± standard deviation for triplicate or duplicate independent binding assays for two representative lab isolates.

0 200 400 600 800 1000 1200 1400 7 7.1 7.2 7.3 7.4 Bo un d p RB C/ mm 2 pH ItG A4

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Figure 4. 6: The effect of pH on binding of pRBC to HDMEC endothelial cells under static conditions. Plotted bars are the means ± standard deviation for triplicate or duplicate independent binding assays for the C24 lab isolate.

0 200 400 600 800 1000 1200 1400 7 7.1 7.2 7.3 7.4 Bo un ds p RB C/ mm 2 pH C24

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Figure 4. 7: The effect of pH on binding of pRBC to HUVEC endothelial cells under flow conditions (0.24 ml/min). Plotted bars are the means ± standard deviation for triplicate or duplicate independent binding assays for two representative lab isolates.

Figure 4. 8: The effect of pH on binding of pRBC to HDMEC cells under flow conditions (0.24 ml/min). Plotted bars are the means ± standard deviation for triplicate or duplicate assays for the C24 lab isolate.

0 100 200 300 400 500 600 7 7.1 7.2 7.3 7.4 bo un d p RB C/ mm2 pH A4 ItG 0 200 400 600 800 1000 1200 7 7.1 7.2 7.3 7.4 bo un d o f p RB C/ mm 2 pH C24

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4.1.4 Discussion

Metabolic acidosis is the best prognostic indiator for SM but there as yet no conclusive correlation of host pH environmental changes to pRBC binding. Moreover, there is still debate on what is the best optimum pH for pRBC cytoadherence. The dependence of cytoadherence on the pH of the surrounding environment was first observed by Marsh et al., (1988) by using resting C32 amelanotic melanoma cells towards testing binding of clinical parasite isolates from patients showing variety of malaria severity at pH ranges 6.6 to 7.8 (Marsh et al. 1988). The study was then confirmed by Sherman and Valdez (1989), who used resting C32 melanoma and HUVEC to investigate binding of parasite line FCR-3 (Sherman & Valdez 1989). Though these two observations do agree that pH may enhance parasite binding to endothelial receptors, unfortunately, they did not define what receptors the pRBC were binding to. However, we do know that parasite line used in that study is FCR-3 which is a rosette strain and binds to CD36 and HS, and C32 melanoma cells do express more CD36 along with other receptors such as TSP, but not ICAM-1.

Here in our study we extended these observations by looking at the optimal pH which mediates binding of lab-adapted P. falciparum _{with known binding} properties towards ICAM-1 and CD36. Adhesion was assessed using standard static and flow protein and cell binding assays. We found that lab-adapted parasites associated with ICAM-1 (ItG) optimally bound to ICAM-1 protein and ICAM-1 activated endothelial cell (HUVEC) at pH 7.2 when compared with normal blood pH 7.4 and a parasite line associated with CD36 (C24) optimally bound to CD36 protein and endothelial cells at pH 7.0 compared with normal blood pH 7.4. Meanwhile, A4 bound to ICAM-1 reference and CD36 at similar levels through all the pH conditions tested. From our observation, here we conclude that binding of pRBC to specific receptors especially ICAM-1 and CD36 are pH-dependent, but critically the pH dependency was variant specific, which has implications for the design of experiments to measure the adhesion characteristics of patient isolates.

In a healthy human the pH of the blood plasma is rigidly maintained at pH 7.4, a value distinctly higher than the cytoadherence pH optimum (pH ≤ 7.3); however individuals infected with P. falciparum _{frequently have abnormally low}

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levels of blood pH (Fisher 1983) as well as high lactate levels (Molyneux et al. 1989; Taylor et al. 1988; White et al. 1983; White et al. 1985). How might pH mediate malaria cytoadherence? Here we hypothesise that it may mediate it through conformational protein structure change. Physiological pH changes are known to affect secondary and tertiary protein structure through changes in protein energetic properties (Berg, Tymoczko & Stryer 2002; Branden & Tooze 1999; Dill et al. 1995; Han & Tamm 2000) such examples of pH driven conformational changes of hemagglutinin trimer, pH dependent on formation changes surface protein of influenza virus (Han & Tamm 2000). It also has been thought that the interaction of pRBC to binding receptors is mediated through protein-protein ionic interactions, and hence could be variably dependent on pH based on the contact residues employed by the variant PfEMP1-receptor combinations.

Crandall et al _{suggested that cytoadherence pH dependency of pRBC to EC} was also related to Calcium (Ca2+_{) content of binding buffer used during the} binding assays. Ca2+ _{affected cytoadherence at a much higher concentration (50} mM), which is well beyond any physiological levels (Crandall, Smith & Sherman 1991). The presence of high levels of Ca2+ _{also has been thought to enhance} binding by reducing the surface charge on the target cells (e.g high level of Ca2+ reduce the surface charge of HUVEC (Vargas et al. 1989). Here, we argue against Crandall’s findings because level of Ca2+ used in our binding buffer is fifty-fold lower (1 mM) than Crandall used. Moreover, 50 mM of Ca2+ _{is too high to be found} in humans. Therefore, from our observation, we suggested that Ca2+ does not mediate pRBC binding to ICAM-1 and CD36 receptors in a physiological context.

In this present study, we evaluated the effect of various pH (7.0, 7.1, 7.2, 7.3 and 7.4) on P. falciparum _{pRBC interaction to ICAM-1 and CD36 proteins. We found} that the optimal pH for pRBC binding to both receptors is in the range pH 7.0 to 7.2 and suggest that optimal pH of binding buffer is critical for successful pRBC binding assays. Adhesion of P. falciparum _{pRBC varies with pH and this variation is} different between the three parasite lines used in this study, probably due to the variant PfEMP1 proteins expressed on the surface of infected erythrocytes. These findings might reflect the difficulties in collating adhesion information from

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different studies and suggest how malaria pathogenesis might progress and how pH factors might influence pRBC sequestration.

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4.2 Effect of endothelial glycocalyx on P. falciparum