Modulation of the Host's Immune Response to Plasmodium berghei by a Parasite-Derived Immunosuppressive Factor

ABSTRACT. It is well known that Plasmodium -infected hosts are immunosuppressed, as shown by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei -infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Modulation of the host's immune response to Plasmodium berghei by a parasite-derived immunosuppressive factor

It is well known that Plasmodium-infected hosts are immunosuppressed, as show by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei-infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Optimisation of flow cytometric measurement of parasitaemia in plasmodium-infected mice

Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei.     

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.     

Anemia and antibodies to the 19-kDa fragment of MSP1 during Plasmodium falciparum infection in Aotus monkeys

To determine whether antibodies to the 19-kDa fragment of merozoite surface protein 1 (MSP1(19)) help to control blood-stage Plasmodium falciparum infection, we performed a rechallenge experiment of previously infected Aotus monkeys. Monkeys previously exposed to the FVO strain of P. falciparum that did or did not develop high antibody titers to MSP1(19) and malaria-na?ve monkeys were challenged with erythrocytes infected with the same strain. Prepatent periods were prolonged in previously infected monkeys compared with malaria-na?ve monkeys. Previously infected monkeys with preexisting anti-MSP1(19) antibodies showed low peak parasitemias that cleared spontaneously. Previously infected monkeys that had no or low levels of pre-existing anti-MSP1(19) antibodies also showed low peak parasitemias, but because of low hematocrits, all of these animals required treatment with mefloquine. All previously malaria-na?ve animals were treated because of high parasitemias. The results of this study suggest that antibody to the 19-kDa carboxy-terminal fragment of MSP1 plays a role in preventing the development of anemia, an important complication often associated with malaria.     

Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes

Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. ? 2011 International Society for Advancement of Cytometry.     

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes.In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.     

Aotus infulatus monkey is susceptible to Plasmodium falciparum infection and may constitute an alternative experimental model for malaria

Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.     

Lead decreases parasitemia and enhances survival of Plasmodium berghei-infected mice

Malaria, a disease accounting for more than one million deaths per year, is caused by intraerythrocytic growth of Plasmodia. Parasitemia may be blunted by suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine exposure. Triggers of eryptosis include lead nitrate (Pb(NO3)2). As shown here, Pb(NO3)2 (> or = 10 microM) increased phosphatidylserine exposure of Plasmodium falciparum-infected human erythrocytes, an effect significantly more marked than in noninfected cells. Pb(NO3)2 treatment accelerated the clearance of erythrocytes from circulating blood. Parasitemia in Plasmodium berghei-infected mice was significantly decreased and mouse survival significantly enhanced by 100 microM Pb(NO3)2 (20 ppm) in drinking water. The treatment significantly increased reticulocyte number but did not significantly decrease erythrocyte number in noninfected mice and in infected animals mainly triggered the disappearance of P. berghei harbouring erythrocytes. In conclusion, Pb(NO3)2 accelerates eryptosis and subsequent clearance of infected erythrocytes and thus favourably influences the course of malaria.     

Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents.

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.     

TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation.     

Hematologic characterization of naturally occurring malaria (Plasmodium inui) in cynomolgus monkeys (Macaca fascicularis)

Twenty of 47 recently imported cynomolgus monkeys (Macaca fascicularis) were found to have malarial infections. The agent identified was Plasmodium inui. All infections were subclinical in nature. Parasitemias ranged from 10 to 900 parasites/mm3 of whole blood. Pre- and post-treatment hematologic values were evaluated following treatment with chloroquine. Treatment was effective in clearing parasitemias from 13 of 14 infected monkeys. Pretreatment values of hematocrit, hemoglobin, and mean corpuscular volume were significantly different in infected animals compared to noninfected animals. While post-treatment hemoglobin and hematocrit values returned to noninfected control levels, mean corpuscular volume values of infected animals remained significantly lower in the post-treatment period.     

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1.

BACKGROUND: The need for improved malaria diagnostics has long been recognized. METHODS: Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of (3)H-hypoxanthine-labeled parasites. RESULTS: The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r(2) = 0.923-0.968) with the parasitemia measured by (3)H-hypoxanthine incorporation assay when parasites variants incubated with various antimalarial drugs. In addition, the low levels of parasitemia (7.9%-21.3%) detected by microscopy than by FCM may be related to a number of tiny schizonts externally attached to the erythrocyte membranes but were not definitely inside the erythrocyte that normally would never be included in microscopy counting. CONCLUSION: On the basis of data reported herein, a rapid, high sensitivity, lower sampling error and reliable identification of human parasitized erythrocytes by the principles of FCM have been established. Published 2007 Wiley-Liss, Inc.     

Effect of sequential infection with Plasmodium vivax and P. falciparum in the Aotus trivirgatus monkey.

Aotus trivirgatus monkeys with prior experience with Plasmodium vivax were inoculated with P. falciparum via the bites of infected mosquitoes. The animals with prior malaria had higher parasitemias and significantly higher levels of mosquito infectivity than monkeys with no prior P. vivax experience. Monkeys with a history of P. falciparum that were inoculated with P. vivax had essentially the same parasitemias as those with no prior malaria. However, levels of mosquito infectivity were markedly increased in those monkeys with a history of P. falciparum. The results imply that the introduction of another malaria species into a malarious area may result in higher levels of mosquito infection and more rapid establishment and distribution of that species.     

A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.     

The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15-26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II-IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.     

Pathogenicity of avian malaria in experimentally-infected Hawaii Amakihi

The introduction of avian malaria (Plasmodium relictum) and mosquitoes (Culex quinquefasciatus) to the Hawaiian Islands (USA) is believed to have played a major role in the decline and extinction of native Hawaiian honeycreepers (Drepanidinae). This introduced disease is thought to be one of the primary factors limiting recovery of honeycreepers at elevations below 1,200 m where native forest habitats are still relatively intact. One of the few remaining species of honeycreepers with a wide elevational distribution is the Hawaii Amakihi (Hernignathus virens). We measured morbidity and mortality in experimentally-infected Hawaii Amakihi that were captured in a high elevation, xeric habitat that is above the current range of the mosquito vector. Mortality among amakihi exposed to a single infective mosquito bite was 65% (13/20). All infected birds had significant declines in food consumption and a corresponding loss in body weight over the 60 day course of the experiment. Gross and microscopic lesions in birds that succumbed to malaria included enlargement and discoloration of the spleen and liver and parasitemias as high as 50% of circulating erythrocytes. Mortality in experimentally-infected amakihi was similar to that observed in Apapane (Himnatione sanguinea) and lower than that observed in Iiwi (Vestiaria coccinea) infected under similar conditions with the same parasite isolate. We conclude that the current elevational and geographic distribution of Hawaiian honeycreepers is determined by relative susceptibility to avian malaria.     

Octadecenoic Fattv Acids and their Association with Hemolysis in Malaria

SYNOPSIS. Octadecenoic fatty acids have been implicated in prehemolytic and hemolytic phenomena associated with malaria. Oleic [18:1 (n-9)] and cis-vaccenic [18:1 (n-7)] acids were found and quantified in the major neutral and phospholipids of the erythrocytes and plasmas of normal and Plasmodium lophurae-infected ducks, and in the parasite itself. The octadecenoic fatty acids were elevated over normal values in the major phospholipid classes of infected erythrocytes, in the erythrocyte-specific alkoxy phosphatidylethanolamine of infected erythrocytes, and in the plasma unesterified fatty acids, triacylglycerols, cholesterol esters and phosphatidylcholine of infected ducklings. Oleic acid was the major fatty acid of P. lophurae (33% total lipid fatty acids). Theoretical considerations of octadecenoic fatty acid modifications of erythrocyte membrane structure and function in malaria are discussed.     

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.     

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.