TBF I buffer
5 R ESULTS
5.6 Cytoadhesion inhibition assay with sCSA
P. falciparum parasitized erythrocytes, enriched over human endothelial cell line 5i (HBEC-5i) at 40°C demonstrate a very high interaction affinity towards chondroitin sulfate A. These macromolecular structures are part of proteoglycans and omnipresent on most of human cell types. Also, it is found in high numbers on the placenta, giving rise to the pregnancy associated malaria (PAM), a very dangerous, severe and widespread form of the disease. Within the most experimental setups, CHO cells were used, as this cell line lacking CSA on the surface due to a mutation causing a defect in glycosaminoglycan biosynthesis.
During the static biding assays, it was observed that the P. falciparum IT4 isolate show
RESULTS
highly significant elevated binding capacities towards HBEC-5i, after repetitive enrichment under febrile conditions of 40°C. This phenotype was constant over various rounds of enrichment and is most likely due to the extensive expression of the var2csa-var gene within the IT4 population. The investigation of this phenotype and behavior were performed at the same time within the Bruchhaus-Laboratory, but were focus of a different project. The PfEMP1 'VAR2CSA' is very well known in connection with PAM and therefore studied in detail. So far, the fact that a similar phenotype can be observed during the contact of IE and human brain endothelial cells in conjunction with an enrichment at febrile temperature is barely described. To prove the general binding capability of the P. falciparum IT4 isolate towards this surface structure, the CSA expressing HBEC-5i was utilized within binding assays and electron microscopy. To investigate whether the resulting binding capacity of the IT4 isolate and CSA is of specific nature and related to the febrile enrichment procedure, a cytoadhesion inhibition assay was performed. Within this assay, it was tested if the binding capability of the IT4-St, IT4-37°C and IT4-40°C isolates was directed towards the CSA on the surface of the HBEC-5i cells and if this interaction could be inhibited by sCSA.
Furthermore, the concentration of sCSA was decreased stepwise, to determine the gradual inhibition limit. The assay was performed at least in triplicates and repeated at least twice, while the average number of firmly bound IE were calculated per 100 HBEC cells; the results are shown in Figure 17.
The 3 different IT4 isolates were assayed over either HBEC-5i cells (light green) or CHO-CD36 cells (dark green) as control. Prior to the assay, the P. falciparum IE were incubated for 30 min at 37°C, with respective amounts of sCSA, dissolved in D-PBS. As negative control, the IE were also incubated with sBSA, also dissolved in D-PBS at the highest amount used within the assay (10 µg/ml) to rule out unspecific inhibition of cytoadhesion by a non-relevant soluble protein. Furthermore, as a second negative control and to set a baseline to compare the subsequent results, the IE were incubated in D-PBS only. All samples were treated equally, independent from the combination of incubation.
The left section shows the results for the IT4-37°C isolate. The combination with the HBEC-5i sample, serving as negative control by the use of D-PBS only, shows a mean of 6.4 bound IE per 100 HBEC-5i cells. The results for the incubation of HBEC-5i cells in combination with sBSA were similar (9.2 IE / 100 HBEC-5i). The inhibition with 10 µg/ml sCSA (0.8 IE / 100 HBEC-5i; p ≤ 0.05) as well as with 1 µg/ml sCSA (0.6 IE / 100 HBEC-5i; p
≤ 0.05) significantly reduced the binding capability of the IT4-37°C isolate towards the HBEC-5i cell line. The observed findings for the IT4-37°C isolate in combination with CHO-CD36 and D-PBS only (21.6 IE / 100 HBEC-5i) were in line with previous results of static binding assays. Also, the combination with the additives in concentrations of 10 µg/ml sBSA (37.5 IE / 100 HBEC-5i) and 1 µg/ml sBSA (22.3 IE / 100 HBEC-5i) did not change the general range of the binding capability.
The right section gives the results for the IT4-40°C isolate, mimicking febrile conditions within the infected human host. The negative control, IE incubated with D-PBS only shows a mean of 332.3 bound IE per 100 HBEC-5i cells. The addition of 10 µg/ml sBSA (367.5 IE / 100 HBEC-5i) did not change the range of the binding potential. Both
86 approaches. The addition of either 10 µg/ml sCSA (0.2 IE / 100 HBEC-5i; p ≤ 0.0001), 1 µg/ml sCSA (64.4 IE / 100 HBEC-5i; p ≤ 0.0001) or even 0.1 µg /ml sCSA (187.5 IE / 100 HBEC-5i; p ≤ 0.0001) decreased the binding capability of the IT4-40°C isolate significantly. Furthermore, the results proved a stepwise inhibition in correlation with the added concentration of sCSA. Strikingly, the concentration dependent inhibition of firmly bound IE towards HBEC-5i could be proven by the use of sCSA up to 1 µg/ml.
Here, a nearly complete inhibition of 99.9% was observed for the highest concentration of 10 µg/ml sCSA, while the 1 µg/ml sCSA inhibited the binding of IE by 81.6% and the addition of 0.1 µg/ml still performed an inhibition of 46.4%. As expected and in line with previously results during static binding assays, the binding ability of the IT4-40°C isolate towards CHO-CD36 was in the range of 40 bound IE per 100 HBEC-5i and did not changed significantly with the addition of the sBSA or sCSA solution.
Also, as anticipated, the results for the IT4-ST isolate (data not shown) exhibit nearly no binding potential when assayed with HBEC-5i, independent of the incubation with D-PBS only (7.6 IE / 100 HBEC-5i), 10 µg/ml sBSA (0.5 IE / 100 HBEC-5i) or 10 µg/ml and 1 µg/ml sCSA (0.5 and 1.8 IE / 100 HBEC-5i), respectively. The range of the amounts of bound IE towards the CHO-CD36 cells in combination with D-PBS only (250.6 IE / 100 HBEC-5i) is in line with previously performed static binding assays and did not vary significantly by the use of 10 µg/ml sBSA (193.3 IE / 100 HBEC-5i) and 1 µg/ml sCSA (199.0 IE / 100 HBEC-5i).
> Taken together, the addition of various concentrations of sCSA inhibited the binding capability of the P. falciparum IT4-40°C and IT4-37°C isolates in a highly significant and concentration dependent manner.
Figure 17. Cytoadhesion inhibition assay of specific IE binding towards HBEC-5i with sCSA. The cytoadhesion inhibition assay by the use of HBEC-5i and CHO-CD36 in combination with D-PBS only, 10 µg/ml sBSA and 10, 1 and 0.1 µg/ml sCSA was performed to determine the general binding capability and the gradual inhibition limit of P. falciparum IT4-37°C, IT4-40°C and IT4-ST isolates towards the omnipresent expressed cell surface structure CSA. IT4-37°C and IT4-40°C isolates showed a highly significant decrease in the binding potential of up to 99,9%, when co-incubated with sCSA. The inhibition is graduated, depending on the added sCSA
HBEC + D-PBS only HBEC + sBSA 10µg/ml HBEC + sCSA 10µg/ml HBEC + sCSA 1µg/ml HBEC + sCSA 0,1µg/ml CHO-CD36 + D-PBS only CHO-CD36 + sBSA 10µg/ml CD36 + sCSA 10µg/ml
0 300
600 ****
****
****
****
****
****
***
**
IT4-40°C / HBEC-5i + D-PBS only IT4-40°C / HBEC-5i + sCSA (1 µg / ml)
IT4-40°C / HBEC-5i + sCSA (10µg / ml)
IT4-40°C / HBEC-5i + sBSA (10µg / ml) IT4-40°C / HBEC-5i + sCSA (0,1µg / ml) IT4-40°C / CHO-CD36 + sBSA (10µg / ml)
IT4-40°C / CHO-CD36 + D-PBS only
IE / 100 CHO
X
IT4-40°C / CHO-CD36 + sCSA (1µg / ml)
HBEC + D-PBS only HBEC + sBSA 10µg/ml HBEC + sCSA 10µg/ml HBEC + sCSA 1µg/ml (HBEC + sCSA 0,1µg/ml) - NA CHO-CD36 + D-PBS only CHO-CD36 + sBSA 10µg/ml CD36 + sCSA 10µg/ml 0
25 50
*
*
IT4-37°C / HBEC-5i + D-PBS only IT4-37°C / HBEC-5i + sCSA (1 µg / ml)
IT4-37°C / HBEC-5i + sCSA (10µg / ml)
IT4-37°C / HBEC-5i + sBSA (10µg / ml) IT4-37°C / HBEC-5i + sCSA (0,1 µg / ml) IT4-37°C / CHO-CD36 + sBSA (10µg / ml)
IT4-37°C / CHO-CD36 + D-PBS only
IE / 100 CHO
X
IT4-37°C / CHO-CD36 + sCSA (1 µg / ml)
only 10µg/ml 10µg/ml 1µg/ml l) - NA BS only 10µg/ml 10µg/ml
0 150 300
BEC-5i BEC-5i / ml)
BEC-5i g / ml)
BEC-5i g / ml) BEC-5i g / ml) O-CD36 l)
O-CD36 O-CD36 l)
IE / 100 CHO
X
HBEC + D-PBS only HBEC + sBSA 10µg/ml HBEC + sCSA 10µg/ml HBEC + sCSA 1µg/ml HBEC + sCSA 0,1µg/ml CHO-CD36 + D-PBS only CHO-CD36 + sBSA 10µg/ml CD36 + sCSA 10µg/ml
0 300
600 ****
****
****
****
****
****
***
**
IT4-40°C / HBEC-5i + D-PBS only IT4-40°C / HBEC-5i + sCSA (1µg / ml)
IT4-40°C / HBEC-5i + sCSA (10µg / ml)
IT4-40°C / HBEC-5i + sBSA (10µg / ml) IT4-40°C / HBEC-5i + sCSA (0,1µg / ml) IT4-40°C / CHO-CD36 + sBSA (10µg / ml)
IT4-40°C / CHO-CD36 + D-PBS only
IE / 100 CHO
X
IT4-40°C / CHO-CD36 + sCSA (1µg / ml)
HBEC + D-PBS only HBEC + sBSA 10µg/ml HBEC + sCSA 10µg/ml HBEC + sCSA 1µg/ml (HBEC + sCSA 0,1µg/ml) - NA CHO-CD36 + D-PBS only CHO-CD36 + sBSA 10µg/ml CD36 + sCSA 10µg/ml
0 25 50
*
*
IT4-37°C / HBEC-5i + D-PBS only IT4-37°C / HBEC-5i + sCSA (1µg / ml)
IT4-37°C / HBEC-5i + sCSA (10µg / ml)
IT4-37°C / HBEC-5i + sBSA (10µg / ml) IT4-37°C / HBEC-5i + sCSA (0,1µg / ml) IT4-37°C / CHO-CD36 + sBSA (10µg / ml)
IT4-37°C / CHO-CD36 + D-PBS only
IE / 100 CHO
X
IT4-37°C / CHO-CD36 + sCSA (1µg / ml)
HBEC + D-PBS only HBEC + sBSA 10µg/ml HBEC + sCSA 10µg/ml HBEC + sCSA 1µg/ml (HBEC + sCSA 0,1µg/ml) - NA CHO-CD36 + D-PBS only CHO-CD36 + sBSA 10µg/ml CD36 + sCSA 10µg/ml
0 150 300
IT4-Stamm / HBEC-5i + D-PBS only IT4-Stamm / HBEC-5i + sCSA (1µg / ml)
IT4-Stamm / HBEC-5i + sCSA (10µg / ml)
IT4-Stamm / HBEC-5i + sBSA (10µg / ml) IT4-Stamm / HBEC-5i + sCSA (0,1µg / ml) IT4-Stamm / CHO-CD36 + sBSA (10µg / ml)
IT4-Stamm / CHO-CD36 + D-PBS only IT4-Stamm / CHO-CD36 + sCSA (1µg / ml)
IE / 100 CHO
X
sCSA inhibition assay
IT4-40°C IT4-37°C
HBEC-5i CHO-CD36
RESULTS
concentration. The IT4-ST isolate did not show an obvious binding phenotype towards the HBEC-5i cell line. Furthermore, the assays in combination with CHO-CD36 were in line with previously findings and the range of bound IE did not changed when sBSA or sCSA were added. The assay was performed at least in triplicates and repeated at least twice, while the average number of firmly bound IE were calculated per 100 HBEC cells; the results are shown in Figure 17. For crossed samples, data are not available. Statistical significance was calculated by a one-way ANOVA with a Tukey correction for multiple comparisons and a 95% CI with
**** p ≤ 0,0001; *** p ≤ 0,005; ** p ≤ 0,01; * p ≤ 0,05 and compared as indicated within the graph.