Immune Mimicry Of Malarial Cells-Oetken

Outline for immune cells mimicry: P. falcipurum

Intro.
Malaria is a parasitic infection that infects and kills millions of people every year. It is estimated that 1.5 to 2 million children under the age of 5 die from malaria every year. Malaria is not bacteria, nor a virus, but a protozoa that is closely related to a single celled amoeba. This living organism is transferred primarily by mosquitoes in which they will immediately be cleared by the liver. It will only take roughly 48 hours for Malaria to multiply in these liver cells and then the liver will rupture spreading these organisms into the blood stream. This spreading of the infected cells to the red blood cells is what we consider the pathology of the disease malaria.

Basis: Researchers want to see the effects of TCTP on basophils and eosinophils and compare it to histamine to see if this immune mimicry makes it easier for malaria parasites to sequester themselves.
Researchers thoughts: Malarial TCTP is a homolog or related to the histamine releasing factor of immune cells. Researchers want to find out if malarial TCTP may affect host immune response in vivo as it showed in vitro. (graph: Histamine Releasing Factor effects basophils to release histamine and eosinophils to release IL-8. Then show related effects of TCTP in vitro with comparisons to histamine.)
Approach: The researchers want to test malarial TCTP by using newly infected patients and African children and test their basophil histamine release and eosinophils release of IL-8. They will compare these results to the basophil histamine release and eosinophil release of IL-8 from peripheral blood from normal allergic donors. This comparison will show if TCTP affects the host immune cells in ways similar to histamine.

Methods
A. Blood was taken from three volunteers with no history of malaria, 10 Malawaian children, and 3 allergic blood donors. The blood samples were then cleared of all contents except for basophils and eosinophils. Meanwhile, recombinant TCTP was cultured in E. Coli while HRF was cultured in a baculovirus vector. After the TCTP and histamine were extracted, a test was done to make sure no LPS was connected to the TCTP using the limulus amebocyte lysate kit.
Basophils extracted- dextran sedimentation
Eosinophils- lyse rbc’s then immunomagnetic bead technique

B. Once Eosinophils and Basophils were extracted= use HRF and TCTP to figure out their effects.

Results-
TCTP was found in 2 clinical settings with patients who were infected by P. falciparum. The three volunteers who were infected by P. falciparum showed TCTP to be present in their plasma (0.60 +/- .44 micrograms/mL of TCTP/ 10^9 infected cells). No TCTP was found in their plasma before being infected. Malawaian children showed 1.41 +/- 1.44 micrograms/mL of TCTP/ 10^9 infected cells.
Human basophils secreted histamine and Eosinophils secreted IL-8 in the presence of histamine and TCTP. Recombinant P. falcipurum TCTP caused basophils from allergic blood donors to secrete histamine as well as HRF. Malarial TCTP caused a response in histamine secretion at about 10 micrograms/mL, while HRF caused secretion with about 1 microgram/mL.
Eosinophils showed a similar effect in that malarial TCTP induced IL-8 protein production. Both recombinant molecules were able to induce IL-8 secretion over media alone from three separate donors. Again, malarial TCTP caused a response in IL-8 secretion at about 10 micrograms/mL and was less potent than HRF.

Discussion
1. TCTP concentrations in plasma approach levels that could effect the eosinophils and basophils responses to malaria.
2. Plasma concentrations of P. falcipurum TCTP do not reflect circulating parasitemias.
3. Patients with large # of sequestered parasites, the TCTP produced could have profound effects on the basophils/eosinophils of that area.

Weaknesses: E. coli vs. Baculovirus : need same vectors!

Endotoxin Removal-Since the TCTP was grown through the reproduction of E.coli, the E.coli needed to be killed in order to retrieve the TCTP, however, this left bits of LPS all over, so they needed to be removed in order to have pure TCTP in order for the study to be effective. So, they introduced lumulus amoebocytes before the removal of endotoxins and after (the amoebocytes release chemicals when they encounter LPS, so it is basically a measure of how much LPS is in the system). They removed the LPS with Detoxi Gel agar which results in a 1000 fold reduction in endotoxins. (BJK)

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