Monocyte (MO) subpopulations display distinct phenotypes and functions which can drastically change during inflammatory states. We hypothesized that discrete MO subpopulations are induced during malaria infection and associated with anti-parasitic activity. We characterized the phenotype of blood MO from healthy malaria-exposed individuals and that of patients with acute uncomplicated malaria by flow cytometry. In addition, MO defense function was evaluated by an in vitro antibody dependent cellular inhibition (ADCI) assay. At the time of admission, the percentages and absolute numbers of CD16+ MO, and CCR2+CX3CR1+ MO, were high in a majority of patients. Remarkably, expression of CCR2 and CX3CR1 on the CD14high (hi) MO subset defined two subgroups of patients that also differed significantly in their functional ability to limit the parasite growth, through the ADCI mechanism. In the group of patients with the highest percentages and absolute numbers of CD14hiCCR2+CX3CR1+ MO and the highest mean levels of ADCI activity, blood parasitemias were lower (0.14±0.34%) than in the second group (1.30±3.34%; p = 0.0053). Data showed that, during a malaria attack, some patients' MO can exert a strong ADCI activity. These results bring new insight into the complex relationships between the phenotype and the functional activity of blood MO from patients and healthy malaria-exposed individuals and suggest discrete MO subpopulations are induced during malaria infection and are associated with anti-parasitic activity.
Intraerythrocytic maturation of the malaria parasite Plasmodium falciparum is associated with profound changes in the asymmetry of phospholipids in the lipid bilayer of the parasitized red blood cells (pRBCs). These changes may contribute to adherence of pRBCs to endothelial cells. This study investigates the effect of febrile temperature and proinflammatory cytokines on phosphatidylserine (PS) expression on the exofacial surface of pRBCs during parasite maturation. The expression of PS on the pRBCs was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS and a vital nucleic acid fluorochrome for parasite staining. The results showed that PS expression on the surface of pRBCs increased in association with parasite maturation, especially at the late parasite stage. Furthermore, the growth of P. falciparum also accelerated senescence of the uninfected RBCs in parasite cultures. Exposure to febrile temperature led to significant increases in the expression of PS on the surface of pRBCs, particularly at the late parasite stage associated with the virulence strain of the parasite. In contrast, proinflammatory cytokines had no detectable effect on PS expression on pRBCs. These data suggest that PS molecule expression is more dependent on fever, parasitemia, parasite strain, and virulence than on cytokine exposure. These findings contribute to our understanding of the factors that are involved in malaria pathogenesis.
Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.
BackgroundHighly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary.MethodsA dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from Plasmodium falciparum cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates.ResultsIn HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures.ConclusionThe novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.
Background: CD4 1 T-lymphocyte count remains the most important surrogate marker for management of HIV patients in resource-poor countries. The standard single-platform (SP) bead-based flow cytometric method for CD4 1 testing is expensive; more affordable methods are needed. We evaluated the SP flowrate-based calibration method for determining CD4 1 counts, using three flow cytometers of varying ages.Methods: CD4 1 counts from 103 HIV-1 infected Thai patients were determined using a SP flow-rate method in flow cytometers with 2, 12, and 16 years of service. Results were compared to the bead-based method. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis.Results
BackgroundRelapse infections resulting from the activation hypnozoites produced by Plasmodium vivax and Plasmodium ovale represent an important obstacle to the successful control of these species. A single licensed drug, primaquine is available to eliminate these liver dormant forms. To date, investigations of vivax relapse infections have been few in number.ResultsGenotyping, based on polymorphic regions of two genes (Pvmsp1F3 and Pvcsp) and four microsatellite markers (MS3.27, MS3.502, MS6 and MS8), of 12 paired admission and relapse samples from P. vivax-infected patients were treated with primaquine, revealed that in eight of the parasite populations in the admission and relapse samples were homologous, and heterologous in the remaining four patients. The patients’ CYP2D6 genotypes did not suggest that any were poor metabolisers of primaquine. Parasitaemia tended to be higher in the heterologous as compared to the homologous relapse episodes as was the IgG3 response. For the twelve pro- and anti-inflammatory cytokine levels measured for all samples, only those of IL-6 and IL-10 tended to be higher in patients with heterologous as compared to homologous relapses in both admission and relapse episodes.ConclusionsThe data from this limited number of patients with confirmed relapse episodes mirror previous observations of a significant proportion of heterologous parasites in relapses of P. vivax infections in Thailand. Failure of the primaquine treatment that the patients received is unlikely to be due to poor drug metabolism, and could indicate the presence of P. vivax populations in Thailand with poor susceptibility to 8-aminoquinolines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1877-x) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.