Artemether-lumefantrine to treat malaria in pregnancy is associated with reduced placental haemozoin deposition compared to quinine in a randomized controlled trial

ABSTRACT: BACKGROUND: Data on efficacy of artemisinin-based combination therapy (ACT) to treat Plasmodium falciparum during pregnancy in sub-Saharan Africa is scarce. A recent open label, randomized controlled trial in Mbarara, Uganda demonstrated that artemether-lumefantrine (AL) is not inferior to quinine to treat uncomplicated malaria in pregnancy. Haemozoin can persist in the placenta following clearance of parasites, however there is no data whether ACT can influence the amount of haemozoin or the dynamics of haemozoin clearance. METHODS: Women attending antenatal clinics with weekly screening and positive blood smears by microscopy were eligible to participate in the trial and were followed to delivery. Placental haemozoin deposition and inflammation were assessed by histology. To determine whether AL was associated with increased haemozoin clearance, population haemozoin clearance curves were calculated based on the longitudinal data. RESULTS: Of 152 women enrolled in each arm, there were 97 and 98 placental biopsies obtained in the AL and quinine arms, respectively. AL was associated with decreased rates of moderate to high grade haemozoin deposition (13.3% versus 25.8%), which remained significant after correcting for gravidity, time of infection, re-infection, and parasitaemia. The amount of haemozoin proportionately decreased with the duration of time between treatment and delivery and this decline was greater in the AL arm. Haemozoin was not detected in one third of biopsies and the prevalence of inflammation was low, reflecting the efficacy of antenatal care with early detection and prompt treatment of malaria. CONCLUSIONS: Placental haemozoin deposition was decreased in the AL arm demonstrating a relationship between pharmacological properties of drug to treat antenatal malaria and placental pathology at delivery. Histology may be considered an informative outcome for clinical trials to evaluate malaria control in pregnancy. Trial registration REGISTRY: http://clinicaltrials.gov/ct2/show/NCT00495508.     

Synergistic and antagonistic interactions between haemozoin and bacterial endotoxin on human and mouse macrophages

Haemozoin (malaria pigment) is a birefringent crystalline material made of Fe (III) Protoporphyrin IX dimers that derives from the degradation of haemoglobin by intraerythrocytic Plasmodia. At schizont rupture, it accumulates indigested inside phagocytic cells altering their immunological properties. Both pro-inflammatory and immunosuppressive activities have been associated with pigment-fed monocyte-macrophages or dendritic cells. These conflicting results were attributed to the source of macrophages or the different preparations of pigment. However, the interactions of malaria pigment with other phagocytes stimuli, such as bacterial endotoxin (LPS) or interferon-gamma have not been fully analysed, yet. The purpose of this study was to compare the immunological properties of native haemozoin (HZ), freshly extracted from Plasmodium falciparum cultures, versus beta-haematin (BH), the synthetic crystals identical to native haemozoin, and to evaluate the relationship between haemozoin and endotoxin on the immune response of different macrophages populations. The results indicate that the iron-porphyrin moiety of both native and synthetic pigment can exert either a synergistic or antagonistic effect with LPS that is related to the length and sequence of treatment, the source of macrophages and is associated with the generation of oxidative stress. These data rise the question of whether and how in vivo concomitant gram(-) bacteremia may affect the pathogenesis and/or the immune response of malaria infections and vice versa.     

Laser-induced inactivation of Plasmodium falciparum

ABSTRACT: BACKGROUND: Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite's food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. METHODS: Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU). RESULTS: Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with ~4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (~2-log reduction). A ~1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 muM synthetic haemozoin and a ~2-log reduction in the presence of 10 muM haemozoin. Minimal ([less than or equal to]15%) cell kill was observed in the presence of 10 muM haemin. CONCLUSIONS: Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient's blood through the skin or through dialysis tubing with a NIR laser.     

Lentinula edodes produces a multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and beta-glucosidase

Haemozoin, the malaria pigment, regulates the synthesis of several host cytokines and has been found to be associated with the disease severity. Here we describe that malarial patients produce a significant amount of anti-haemozoin IgM antibodies. Levels of these antibodies were higher among the complicated Plasmodium falciparum cases compared to the non-complicated P. falciparum group and Plasmodium vivax patients. The P. falciparum haemozoin also induced the synthesis of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) by the monocytes of the healthy individuals, but the production of these cytokines by the monocytes was inhibited in the presence of the anti-haemozoin IgM antibodies. Therefore, it seems that the host produces these antibodies (mainly IgM types) during malarial infection that can influence the progression of the disease by inhibiting the production of cytokines.     

Automated malaria diagnosis using pigment detection

Several new methods of malaria diagnosis have recently been developed, but these all rely on clinical suspicion and, consequently, an explicit clinical request. Although some methods lend themselves to automation (eg. PCR), no technique can yet be used for routine clinical automated screening. Detection of birefringent haemozoin has been used to diagnose malaria since the turn of the 20th century. A new generation of full blood count analysers, used widely in clinical laboratories, have the potential to detect haemozoin in white blood cells and probably erythrocytes. Thomas H?nscheid, Emilia Valadas and Martin Grobusch here describe this novel technique for malaria diagnosis and discuss its potential applications.     

Haemozoin (beta-haematin) biomineralization occurs by self-assembly near the lipid/water interface

Several blood-feeding organisms, including the malaria parasite detoxify haem released from host haemoglobin by conversion to the insoluble crystalline ferriprotoporphyrin IX dimer known as haemozoin. To date the mechanism of haemozoin formation has remained unknown, although lipids or proteins have been suggested to catalyse its formation. We have found that beta-haematin (synthetic haemozoin) forms rapidly under physiologically realistic conditions near octanol/water, pentanol/water and lipid/water interfaces. Molecular dynamics simulations show that a precursor of the haemozoin dimer forms spontaneously in the absence of the competing hydrogen bonds of water, demonstrating that this substance probably self-assembles near a lipid/water interface in vivo.     

Theories on malarial pigment formation and quinoline action

Haeme metabolism remains a vulnerable problem for the intraerythrocytic Plasmodium which catabolises haemoglobin as a source of amino acids in an acidic, oxygen-rich lysosome-like digestive vacuole. Haeme monomer, capable of generating oxygen radicals, transforms into an inert crystal named malarial pigment or haemozoin by forming unique dimers that then crystalise. Laveran first described pigmented bodies in humans to define a protozoan as the aetiologic agent of malaria. The trail of malaria pigment enabled Ross to implicate the mosquito in the life cycle of Plasmodium. In 1991, Slater and Cerami postulated a unique iron-carboxylate bond between two haemes in haemozoin crystals based on infrared and X-ray spectroscopy data. Additionally, parasite extracts were shown to possess a 'haeme polymerase' enzymatic activity as the process of crystal formation was then termed. Importantly, the quinolines, such as choloroquine, inhibit haemozoin formation. A Plasmodium falciparum derived histidine-rich protein II, which binds haeme and initiates haemozoin formation, is present in the digestive vacuole. Pfhistidine-rich protein II and Pfhistidine-rich protein III are sufficient, but not necessary for haemozoin formation as a laboratory clone lacking both still makes the haeme crystals. The reduvid bug, and the Schistosoma and Haemoproteus genera also make haemozoin. Recently, Bohle and coworkers used X-ray diffraction to document the iron-carboxylate bond in intact desiccated parasites and to show that a Fe1-O41 head to tail haeme dimer is the unit building block of haemozoin. The role of the Plasmodium histidine-rich protein family members, lipids or potential novel proteins in the exact molecular assembly of the large molecular weight haeme crystals in the protein rich digestive vacuole needs to be solved. Accurate experimental determination of the role of haemozoin formation and inhibition as the target of chloroquine is fundamental to determination of the mechanism of quinoline drug action and resistance. The enhanced understanding of the biosynthetic pathway leading to haemozoin formation using functional proteomic tools and the mechanisms through which existing antimalarial drugs affect Plasmodium haeme chemistry will help design improved chaemotherapeutic agents.     

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.     

Oxidative stress and rheology in severe malaria

There is mounting evidence that the release of haemozoin (beta-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.     

Contribution of allergic inflammatory response to the pathogenesis of malaria disease

Plasmodium falciparum, the aetiological agent of human lethal malaria, is responsible for over 2 million deaths per year and malaria episodes may vary considerably in their severity and clinical manifestations. Dysregulated balance of the inflammatory response and a defect in the anti-Plasmodium parasite immune response represent the hallmarks of malaria disease. Among the many possible mechanisms, it is now widely recognized that the production of pro-inflammatory mediators and cytokines and upregulation of endothelial cell adhesion molecules play important roles in malaria pathogenesis. We and others provided evidence that some components of allergic inflammatory response to malaria parasites or elicited by by-products of parasite infection may contribute to malaria pathogenesis. This review provides some clue regarding these mechanisms where mast cells and histamine, an inflammatory mediator generated following IgE-independent or IgE-mediated immune response, were found to play a major role in parasite transmission and malaria pathogenesis, respectively. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Oxidative stress and rheology in severe malaria

Abstract

There is mounting evidence that the release of haemozoin (β-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.     

A fine balance between oxidised and reduced haem controls the survival of intraerythrocytic plasmodia

The polymerisation of haemoglobin-derived ferri-protoporphyrin IX (Fe(III)PPIX) to haemozoin (malaria pigment) is a crucial process for intraerythrocytic plasmodia to prevent haem toxicity. It is also the target of in-use antimalarial drugs and newer compounds. This reaction and the inhibition thereof can be reproduced and studied in vitro.     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

An unusual finding in a peripheral blood smear of Plasmodium falciparum malaria

An attack of hyperpyrexia, in a young Senegalese male resident in Rome on his return from a brief trip to Senegal, was recognized as Plasmodium falciparum malaria. In a Giemsa peripheral blood smear a merozoite cluster and haemozoin pigment were observed into a monocyte, as a phagocytosis outcome. This finding, unexpected in view of the relatively moderate severity of the disease, was likely to be expression of good immune response against the blood stages of P. falciparum.     

The Plasmodium falciparum-infected red blood cell

Plasmodium falciparum, the most virulent of the human malaria parasites, causes up to one million deaths per year. The parasite spends part of its lifecycle inside the red blood cells (RBCs) of its host. As it grows it ingests the RBC cytoplasm, digesting it in an acidic vacuole. Free haem released during haemoglobin digestion is detoxified by conversion to inert crystals of haemozoin. Malaria pathology is evident during the blood stage of the infection and is exacerbated by adhesion of infected RBCs to blood vessel walls, which prevents splenic clearance of the infected cells. Cytoadherence is mediated by surface-exposed virulence proteins that bind to endothelial cell receptors. These 'adhesins' are exported to the RBC surface via an exomembrane system that is established outside the parasite in the host cell cytoplasm. Antimalarial drugs that interfere with haem detoxification, or target other parasite-specific processes, have been effective in the treatment of malaria, but their use has been dogged by the development of resistance. Similarly, efforts to develop an effective blood vaccine are hindered by the variability of surface-exposed antigens.     

Ultrastructural characteristics,biological activity and pharmacological relevance of synthetic malaria pigment (beta-haematin)

Malaria pigment (haemozoin, HZ) is the detoxification product of haemoglobin-derived haem of intraerythrocytic malaria parasites. At schizont rupture, haemozoin accumulates inside host phagocytic cells. The chemical structure and the spectroscopic characteristics of haemozoin are identical to those of beta-haematin (BH), a synthetic pigment obtained from Ferriprotoporphyrin IX (Fe (III) PPIX) in acidic conditions. The process of BH formation is the target of quinoline antimalarials. Here, we summarise the results of our studies on the ultrastructural characteristics, biological and pharmacological relevance of synthetic vs. native haemozoin. 1) By electron microscopy, native HZ and synthetic BH appear as dark brown crystals, morphologically indistinguishable and are internalised by phagocytes at the same extent. 2) Both HZ and BH modulate the production of cytokines (TNF and NO) and increase the susceptibility to lipid peroxidation of mouse or human phagocytes. The antioxidant status of the phagocytes regulates the susceptibility to BH/HZ-mediated effects. 3) The process of BH formation from Fe(III)PPIX, hence haem detoxification, can be inhibited by electrochemically-reduced, Fe(II)PPIX molecules. Maintaining iron in the reduced state can thus be considered a new pharmacological target. This was confirmed by the observation that thiol-reducing agents (NAC, cystein) were able to inhibit BH formation and were toxic to parasites in vitro.     

Naturally occurring cobalamins have antimalarial activity

The acquisition of resistance by malaria parasites towards existing antimalarials has necessitated the development of new chemotherapeutic agents. The effect of vitamin B(12) derivatives on the formation of beta-haematin (synthetic haemozoin) was determined under conditions similar to those in the parasitic food vacuole (using chloroquine, a known inhibitor of haemozoin formation for comparison). Adenosylcobalamin (Ado-cbl), methylcobalamin (CH(3)-cbl) and aquocobalamin (H(2)O-cbl) were approximately forty times more effective inhibitors of beta-haematin formation than chloroquine, cyanocobalamin (CN-cbl) was slightly more inhibitory than chloroquine, while dicyanocobinamide had no effect. It is proposed that the cobalamins exert their inhibitory effect on beta-haematin formation by pi-interactions of their corrin ring with the Fe(III)-protoporphyrin ring and by hydrogen-bonding using their 5,6-dimethylbenzimidazole/ribose/sugar side-chain. The antimalarial activity for the cobalamins (Ado-cbl>CH(3)-cbl>H(2)O-cbl>CN-cbl) was found to be less than that for chloroquine or quinine. Ado-cbl, CH(3)-cbl and CN-cbl do not accumulate in the parasite food vacuole by pH trapping, but H(2)O-cbl does. Unlike humans, the malaria parasite has only one enzyme that uses cobalamin as a cofactor, namely methionine synthase, which is important for growth and metabolism. Thus cobalamins in very small amounts are necessary for Plasmodium falciparum growth but in larger amounts they display antimalarial properties.     

Natural haemozoin modulates matrix metalloproteinases and induces morphological changes in human microvascular endothelium

Severe malaria, including cerebral malaria (CM), is characterized by the sequestration of parasitized erythrocytes in the microvessels after cytoadherence to endothelial cells. Products of parasite origin, such as haemozoin (HZ), contribute to the pathogenesis of severe malaria by interfering with host inflammatory response. In human monocytes, HZ enhanced the levels of matrix metalloproteinase-9 (MMP-9), a protease involved in neuroinflammation. Here the effects of HZ on the regulation of MMPs by the human microvascular endothelial cell line HMEC-1 were investigated. Cells treated with natural (n)HZ appeared elongated instead of polygonal, and formed microtubule-like vessels on synthetic basement membrane. nHZ enhanced total gelatinolytic activity by inducing proMMP-9 and MMP-9 without affecting basal MMP-2. The level of the endogenous tissue inhibitor of MMP-9 (TIMP-1) was not altered by nHZ, while TIMP-2, the MMP-2 inhibitor, was enhanced. Additionally, nHZ induced MMP-1 and MMP-3, two enzymes sequentially involved in collagenolysis and proMMP-9 proteolytic activation. Lipid-free HZ did not reproduce nHZ effects. Present data suggest that the lipid moiety of HZ alters the MMP/TIMP balances and promotes the proteolytic activation of proMMP-9 in HMEC-1, thereby enhancing total gelatinolytic activity, cell activation and inflammation. These findings might help understanding the mechanisms of blood brain barrier damage during CM.     

Improved methods for haemozoin quantification in tissues yield organ- and parasite-specific information in malariainfected mice

ABSTRACT: BACKGROUND: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against lifethreatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intraerythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivoimmunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence. METHODS: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis. RESULTS: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas subnanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels. CONCLUSIONS: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.     

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.