Abstract
BackgroundParoxysms are recurrent febrile episodes, characteristic of Plasmodium vivax infections, which coincide with the rupture of schizont-infected erythrocytes in the patients' circulation. The present study describes the formation of prominent aggregates of leukocytes in vitro in the presence of parasite and host factors released during paroxysms.MethodsWhole blood cells from uninfected malaria-naïve donors were incubated with plasma taken during a paroxysm or normal human plasma as a control and cell smears were observed under the microscope for the presence of leukocyte aggregates. Plasma factors involved in mediating the leukocyte aggregation were identified using immune depletion and reconstitution experiments. Furthermore, biochemical characterization was carried out to determine the chemical nature of the active moieties in plasma present during paroxysms.ResultsLeukocyte aggregates were seen exclusively when cells were incubated in plasma collected during a paroxysm. Immune depletion and reconstitution experiments revealed that the host cytokines TNF-alpha, GM-CSF, IL-6 and IL-10 and two lipid fractions of paroxysm plasma comprise the necessary and sufficient mediators of this phenomenon. The two lipid components of the paroxysm plasmas speculated to be of putative parasite origin, were a phospholipid-containing fraction and another containing cholesterol and triglycerides. The phospholipid fraction was dependent upon the presence of cytokines for its activity unlike the cholesterol/triglyceride-containing fraction which in the absence of added cytokines was much more active than the phospholipids fraction. The biological activity of the paroxysm plasmas from non-immune patients who presented with acute P. vivax infections was neutralized by immune sera raised against schizont extracts of either P. vivax or Plasmodium falciparum. However, immune sera against P. vivax were more effective than that against P. falciparum indicating that the parasite activity involved may be antigenically at least partially parasite species-specific.ConclusionLeukocyte aggregation was identified as associated with paroxysms in P. vivax infections. This phenomenon is mediated by plasma factors including host-derived cytokines and lipids of putative parasite origin. The characteristics of the phospholipid fraction in paroxysm plasma are congruent with those of the parasite-derived, TNF-inducing GPI moieties described by others. The more active cholesterol/triglyceride(s), however, represent a novel malarial toxin, which is a new class of biologically active lipid associated with the paroxysm of P. vivax malaria.
Highlights
Paroxysms are recurrent febrile episodes, characteristic of Plasmodium vivax infections, which coincide with the rupture of schizont-infected erythrocytes in the patients' circulation
Aggregation of white blood cells from healthy donors in the presence of paroxysm plasma from acute P. vivax-infected patients Striking changes in the distribution of nucleated cells were observed between smears made from cultures in which whole peripheral blood cells from healthy individuals were incubated in the presence of paroxysm plasma from acute P. vivax-infected patients when compared with cultures incubated in the presence of normal human plasma
The absence of the leukocyte aggregation-inducing factors in paroxysm plasma from semi-immune P. vivax patients and evidence for serum-mediated immunity against parasite factors/"toxins" In contrast to the induction of leukocyte aggregation by paroxysm plasma from the clinically non-immune patients from Colombo, plasmas collected during paroxysm from age-matched, semi-immune, acutely infected P. vivax patients resident in Kataragama, an endemic area of Sri Lanka, had little such effect (Figure 1; EPP) (p < 0.001 relative to paroxysm plasmas from non-immune patients)
Summary
Paroxysms are recurrent febrile episodes, characteristic of Plasmodium vivax infections, which coincide with the rupture of schizont-infected erythrocytes in the patients' circulation. Previous investigations have been made on events associated with paroxysms in P. vivax infections including the inactivation of sexual stages of the parasites (gametocytes) in the presence of plasma taken at the time of a paroxysm [1,2]. These studies have demonstrated the roles in this process of the human cytokines TNF-α, GM-CSF and IL-2 together with parasite products. This assay enabled preliminary chemical and physical-chemical characterization of parasite-derived products present and active during a P. vivax paroxysm
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