Browsing by Author "Pooe, Ofentse Jacob"

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    The detection of Plasmodium falciparum in human saliva samples
    (2011) Pooe, Ofentse Jacob; Shonhai, A.; Mharakurwa, S.
    Malaria still persists as a major public health scourge, claiming a global toll of up to 500 million clinical cases, mostly on African children. Current effective malaria diagnostic methods necessitate blood withdrawal from patients for accurate P. falciparum identification. Risks introduced by blood withdrawal can causes communities to be less cooperative to donate blood during malaria surveillance studies. Previous studies showed that saliva could be used to detect malaria by molecular techniques. This study, therefore, sought to establish the constituents of human saliva that harbours parasite DNA in malaria infected subjects. Furthermore the study optimised the use of Saliva as an alternative malaria DNA source in malaria infected patients. A total of 88 subjects were enrolled in this study, 35 (40, 7 %) were males and 51 (59, 3 %) were females. The age range was from 3 months to 99 years old (mean = 29.6 years; median = 18 years). Blood was drawn from each subject for subsequent use in microscopic examination and PCR tests. Saliva samples were also collected for PCR on Saliva derived parasite DNA. DNA was extracted using commercial kit on different saliva fractions from 46 malaria positive (thick film positive & blood PCR positives) individuals and 45 malaria negative individuals. Nested PCR was used to amplify malaria the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene. Generally PCR conducted on DNA purified from both blood and saliva was more sensitive than conventional microscopy, as previously reported. The pellet fraction of saliva was a more reliable and sensitive source of amplifiable parasite DNA compared to the soluble saliva fraction. After PCR optimisation amplification was enhanced 94.1% (sensitivity) and 97% (specificity) against DNA derived from blood as the gold standard. This study further confirms that saliva samples are a reliable non-invasive alternative to blood for the PCR detection of malaria, as previously reported. The source of DNA was primarily in the pellet fraction though; however, refinement is still needed to identify the exact source of DNA in that fraction.
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    Evaluation of the immunomodulatory activity of Plasmodium falciparum hsp70-1
    (University of Zululand, 2014) Pooe, Ofentse Jacob; Prof.A. Shonhai
    Heat shock proteins (Hsps) are conserved molecules that constitute a major part of the cell’s molecular chaperone system (protein folding machinery). Plasmodium falciparum Hsps play an important cytoprotective role ensuring that the malaria parasite survives under the harsh conditions that prevail in the host environment. P. falciparum Hsp70-1 (PfHsp70-1) is a ubiquitous, cytosol-localised Hsp70 that is essential for parasite survival. Apart from their role as molecular chaperones, it is believed that some Hsps of parasitic origin are capable of modulating host immunity through signal transduction (chaperokine role). Most investigations focusing on the chaperokine functions of Hsps use recombinant forms of the proteins produced in E. coli. The main drawback is that the recombinant proteins co-purify with lipopolysaccharides (LPS). Although LPS removal techniques have been developed, they do not completely remove these contaminants, leading to confounding data as LPS are active immune modulants. The current study sought to investigate the immunomodulatory role of PfHsp70-1. A recombinant form of the protein was produced in three bacterial expression hosts (E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis). The protein was expressed attached to an N-terminal polyhistidine tag to facilitate purification by nickel affinity chromatography. PfHsp70-1 produced using the E. coli ClearColi BL21 and Brevibacillus expression systems was associated with no detectable traces of LPS. The protein exhibited no immunomodulatory function when it was exposed to macrophage cells cultured in vitro. However, PfHsp70-1 expressed in E. coli XL1 Blue was tainted with LPS contaminants and exhibited apparent immunomodulatory function suggesting that the LPS background was responsible for the signal. Findings, from this study, suggest that endotoxin-free PfHsp70-1 does not possess immunomdulatory function. Furthermore, this study confirms that E. coli ClearColi BL21 and Brevibacillus expression are Evaluation of the immunomodulatory activity of Plasmodium falciparum Hsp70-1 reliable expression hosts for the production of recombinant protein for use in immunomodulatory studies. Furthermore, cytokine production was induced on PMN cells that were exposed to a protein preparation consisting of the N-terminal ATPase subdomain of PfHsp70-1. However, the ATPase subdomain is known to be aggregation prone. thus, this may explain its apparent immune modulatory function. Polymyxin-B has routinely been used to neutralise the adverse effects of LPS from recombinant Hsps produced using traditional E. coli. Polymyxin-B is a cationic cyclic antibiotic that binds and aggregates LPS. However, the effects of polymyxin-B treatment on the integrity of the chaperones during LPS removal is still unclear. This study, therefore, sought to investigate the effect of polymyxin-B on PfHsp70-1’s chaperone role, by investigating its effect on the thermal stability and structural conformation of PfHsp70-1. Results from this study clearly indicate that polymyxin-B interacts and interferes with the chaperone function of PfHsp70-1. Thus polymixin-B could potentially inhibit PfHsp70-1 function, thus, interfering with parasite growth. In, Conclusion this report demonstrates that recombinant forms of PfHsp70-1 can easily be produced without LPS contamination using E. coli ClearColi BL21 and Brevibacillus expression systems. Furthermore this study demonstrates that PfHsp70-1 proteins do not have immunomodulatory activity and that previously reported activity could have been due to the presence of co-purified LPS. Lastly this report discusses the prospects of using polymyxin-B’s as a potential antimalarial therapeutic drug.