Plasmodium products contribute to severe malarial anemia by inhibiting erythropoietin-induced proliferation of erythroid precursors.

Low reticulocytosis, indicating reduced red blood cell (RBC) output, is an important feature of severe malarial anemia. Evidence supports a role for Plasmodium products, especially hemozoin (Hz), in suppressed erythropoiesis during malaria, but the mechanism(s) involved remains unclear. Here, we demonstrated that low reticulocytosis and suppressed erythropoietin (Epo)-induced erythropoiesis are features of malarial anemia in Plasmodium yoelii- and Plasmodium berghei ANKA-infected mice, similar to our previous observations in Plasmodium chabaudi AS-infected mice. The magnitude of decreases in RBC was a reflection of parasitemia level, but low reticulocytosis was evident despite differences in parasitemia, clinical manifestation, and infection outcome. Schizont extracts and Hz from P. falciparum and P. yoelii and synthetic Hz suppressed Epo-induced proliferation of erythroid precursors in vitro but did not inhibit RBC maturation. To determine whether Hz contributes to malarial anemia, P. yoelii-derived or synthetic Hz was administered to naive mice, and the development of anemia, reticulocytosis, and RBC turnover was determined. Parasite-derived Hz induced significant decreases in RBC and increased RBC turnover with compensatory reticulocytosis, but anemia was not as severe as that in infected mice. Our findings suggest that parasite factors, including Hz, contribute to severe malarial anemia by suppressing Epo-induced proliferation of erythroid precursors.

Optimization of malaria detection based on third harmonic generation imaging of hemozoin.

The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin.

Malarial hemozoin activates the NLRP3 inflammasome through Lyn and Syk kinases.

The intraerythrocytic parasite Plasmodium -- the causative agent of malaria -- produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1beta (IL-1beta). However, the mechanism regulating Hz recognition and IL-1beta maturation has not been identified. Here, we show that Hz induces IL-1beta production. Using knockout mice, we showed that Hz-induced IL-1beta and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1beta augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.

Autofluorescence of condensed heme aggregates in malaria pigment and its synthetic equivalent hematin anhydride (beta-hematin).

The condensed crystalline phase of iron(III) protoporphyrin IX either isolated from parasite culture as malaria pigment (hemozoin) or synthetic equivalent hematin anhydride exhibits a solid-state autofluorescence characterized by an excitation maximum of 555 nm and an emission maximum of 577 nm. The excitation spectrum maximum at 555 nm corresponds to the Q(0,0) band in the absorption spectrum which represents the lowest singlet of the material. This suggests that the fluorescent emission is due to the heme condensed phase. The photoluminescence lifetime of tau(f) = 2.7 +/- 0.8 ns as measured at four wavelengths between 550 and 600 nm is in the range of Frankel exciton in porphyrinic condensed phases. The material is shown to have an optical band gap of 2.04 eV characteristic of a semiconductor. Luminescence is markedly dependent upon the degree of hydration and the emission does not seem to be caused by presence of zinc(II) protoporphyrin IX or free-base protoporphyrin IX in the lattice. The autofluorescence can be used for in vivo tracking of hemozoin, for determination of parasitemia levels, and for infection monitoring and possibly for drug screening studies.

Sensitive detection of malaria infection by third harmonic generation imaging.

Malaria remains a major health concern worldwide, with 350-500 million cases reported annually in endemic countries. In this study, we report a novel and highly sensitive optical-based detection of malaria-infected blood cells by third harmonic generation (THG) imaging of hemozoin pigment that is naturally deposited by the parasite during its lifecycle. The THG signal from the hemozoin was greater than we have observed in any cell type with signal/noise ratios that reach 1000:1. This method allows a rapid and robust detection of early stage infections of blood cells. The immense nonlinear response of the intrinsic parasitic by-product pigments suggests that automated optical detection by THG could be used for sensitive and rapid screening of parasite infection in blood samples.

Micro-Raman high-pressure investigation on the malaria pigment hematin anhydride (ß-hematin).

The effect of pressure on the Raman and fluorescence spectra of hematin anhydride (ß-hematin) is reported. In a diamond-anvil cell, DAC, with applied pressures up to 41 kbar, the Raman spectrum undergoes a series of intensity enhancements and increases in energy for many of the Raman-active bands up to a pressure of ~27 kbar. At higher pressures, there is either a leveling out or a decrease in the energies of these vibrational modes. The fluorescence bands also undergo a series of pressure- sensitive changes where, up to 10 kbar, there is a marked quenching of the intensity of the emissive bands, which is accompanied by a net increase in energy of the vibrational bands. The results are interpreted in terms of a high-pressure phase change, to account for the Raman shifts, and a separate defect or surface site of the emissive state, which is more efficiently quenched at higher pressure.

New inflammation-related biomarkers during malaria infection.

Malaria is one of the most prevalent infectious diseases worldwide with more than 250 million cases and one million deaths each year. One of the well-characterized malarial-related molecules is hemozoin (HZ), which is a dark-brown crystal formed by the parasite and released into the host during the burst of infected red blood cells. HZ has a stimulatory effect on the host immune system such as its ability to induce pro-inflammatory mediators responsible for some of the malaria related clinical symptoms such as fever. However, the host serum proteins interacting with malarial HZ as well as how this interaction modifies its recognition by phagocytes remained elusive. In the actual study, using proteomic liquid chromatographic mass spectrometry (LC-MS/MS) and immunochemical approaches, we compared the serum protein profiles of malaria patients and healthy individuals. Particularly, we utilized the malarial HZ itself to capture serum proteins capable to bind to HZ, enabling us to identify several proteins such as apolipoprotein E (ApoE), serum amyloid A (SAA), gelsolin, complement factor H and fibrinogen that were found to differ among healthy and malaria individual. Of particular interest is LPS binding protein (LBP), which is reported herein for the first time in the context of malaria. LBP is usually produced during innate inflammatory response to gram-negative bacterial infections. The exact role of these biomarkers and acute phase responses in malaria in general and HZ in particular remains to be investigated. The identification of these inflammation-related biomarkers in malaria paves the way to potentially utilize them as diagnostic and therapeutic targets.

Toward understanding the chloroquine action at the molecular level in antimalarial therapy--X-ray absorption studies in acetic acid solution.

The local atomic structure around the central iron of the synthetic soluble analog of malarial pigment in acetic acid solution and with addition of chloroquine as found by X-ray absorption spectroscopy is reported. The special interest was drawn to the axial linkage between the central iron atom of the ferriprotoporphyrin IX (FePPIX) coordinated axially to the propionate group of the adjacent FePPIX. This kind of bonding is typical for hematin anhydride. Detailed analysis revealed differences in oxygen coordination sphere (part of dimer linkage bond) between synthetic equivalent of hemozoin in the powder state and dissolved in acetic acid and water at different concentrations mimicking the physiological condition of the parasite's food vacuole. The results of performed studies suggest that the molecular structure of synthetic analogue of hemozoin is no longer dimer-like in acidic solution. Further changes in atomic order around Fe are seen after addition of the antimalarial drug chloroquine.

Innate inflammatory response to the malarial pigment hemozoin.

Malaria is an infectious disease caused by parasites of the genus Plasmodium. This intraerythrocytic protozoan produces hemozoin (HZ), an insoluble crystalline metabolite resulting from the heme detoxification mechanism. This review will focus on HZ biosynthesis and synthetic preparation, but in particular on its effect on host's innate inflammatory responses.

Synthetic Plasmodium-like hemozoin activates the immune response: a morphology - function study.

Increasing evidence points to an important role for hemozoin (HZ), the malaria pigment, in the immunopathology related to this infection. However, there is no consensus as to whether HZ exerts its immunostimulatory activity in absence of other parasite or host components. Contamination of native HZ preparations and the lack of a unified protocol to produce crystals that mimic those of Plasmodium HZ (PHZ) are major technical limitants when performing functional studies with HZ. In fact, the most commonly used methods generate a heterogeneous nanocrystalline material. Thus, it is likely that such aggregates do not resemble to PHZ and differ in their inflammatory properties. To address this issue, the present study was designed to establish whether synthetic HZ (sHZ) crystals produced by different methods vary in their morphology and in their ability to activate immune responses. We report a new method of HZ synthesis (the precise aqueous acid-catalyzed method) that yields homogeneous sHZ crystals (Plasmodium-like HZ) which are very similar to PHZ in their size and physicochemical properties. Importantly, these crystals are devoid of protein and DNA contamination. Of interest, structure-function studies revealed that the size and shape of the synthetic crystals influences their ability to activate inflammatory responses (e.g. nitric oxide, chemokine and cytokine mRNA) in vitro and in vivo. In summary, our data confirm that sHZ possesses immunostimulatory properties and underline the importance of verifying by electron microscopy both the morphology and homogeneity of the synthetic crystals to ensure that they closely resemble those of the parasite. Periodic quality control experiments and unification of the method of HZ synthesis are key steps to unravel the role of HZ in malaria immunopathology.