Malarial Pigment Research Articles

A luminescene method for the quantitative determination of phagocytosis of erythrocytes, of malaria‐parasitized eryithrocytes and of malarial pigment

A method is described for the quantitative measurement of phagocytosis of human erythrocytes, malaria-parasitized erythrocytes and isolated malarial pigment by adherent human monocytes. The method utilizes measurement of haem-elicited luminescence both for the assay of ingested haemoglobin or malarial pigment haem and for the quantification of adherent monocytes. The latter is based on assay of luminescence elicited by cytochrome haem. The method utilizes the same low-cost reagents and equipment for assay of ingested haem and for quantification of adherent monocytes. The method is fast and extremely sensitive. The lower sensitivity limit is 500 monocytes and 10 RBC, or RBC equivalents in the case of malarial pigment, per assay. A detailed protocol with full calculations of a typical phagocytosis experiment of oxidatively damaged RBC, malarial pigment and control RBS is presented.

The Diagnostic Histopathologic Features of Ocular Malaria

The ocular complications in patients with malaria have been studied clinically by many investigators, but the histopathologic changes were rarely described and generally regarded as nonspecific. The eye of a 53-year-old man who died of chloroquine-resistant Plasmodium falciparum malaria was studied by brightfield and polarized light microscopy. An epibulbar hemorrhage that involved the conjunctiva, episclera, and tendinous insertion of the medial rectus muscle was present. Cytoadherence and rosetting of the parasitized erythrocytes were observed within the partially occluded lumens of small retinal and uveal blood vessels. The birefringence of hemozoin (malarial pigment) within the lumens of small ocular blood vessels and in the hemorrhagic epibulbar area was demonstrated by polarized light. Birefringent hemozoinemia in vascular lumens of ocular tissues indicates systemic malarial infestation by any of the four species of malaria. Cytoadherence and rosetting of the parasitized erythrocytes inside ocular capillaries and venules is diagnostic of P. falciparum and is an important cause of ocular hemorrhage.

Phagocytosis of P. falciparum malarial pigment hemozoin by human monocytes inactivates monocyte protein kinase C

Hemozoin (malarial pigment) is a ferriprotoporphyrin IX-rich hemoglobin degradation product present in parasitized RBC. Avidly phagocytosed hemozoin abolishes phagocyte TPA-induced oxidative burst. Membrane-associated PKC increased transiently in hemozoin-fed monocytes by 50% after 30 min and decreased irreversibly to 20% of initial value within 5 h after phagocytosis. Control RBC-fed monocytes showed transient decay of membrane-associated PKC followed by complete recovery 12 h after phagocytosis. Cytosolic PKC was not impaired within 12 h and diminished drastically 24 h after phagocytosis of hemozoin. Results are compatible with increased degradation of membrane-translocated PKC, possibly by iron/H 2O 2-mediated damage of cysteine-rich regulatory domains of PKC.

Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment.

Human monocyte-derived macrophages ingest diamide-treated red blood cells (RBC), anti-D immunoglobulin (Ig)G-opsonized RBC, or Plasmodium falciparum ring-stage parasitized RBC (RPRBC), degrade ingested hemoglobin rapidly, and can repeat the phagocytic cycle. Monocytes fed with trophozoite-parasitized RBC (TPRBC), which contain malarial pigment, or fed with isolated pigment are virtually unable to degrade the ingested material and to repeat the phagocytic cycle. Monocytes fed with pigment display a long-lasting oxidative burst that does not occur when they phagocytose diamide-treated RBC or RPRBC. The phorbol myristate acetate-elicited oxidative burst is irreversibly suppressed in monocytes fed with TPRBC or pigment, but not in monocytes fed with diamide-treated or IgG-opsonized RBC. This pattern of inhibition of phagocytosis and oxidative burst suggests that malarial pigment is responsible for the toxic effects. Pigment iron released in the monocyte phagolysosome may be the responsible element. 3% of total pigment iron is labile and easily detached under conditions simulating the internal environment of the phagolysosome, i.e., pH 5.5 and 10 microM H2O2. Iron liberated from pigment could account for the lipid peroxidation and increased production of malondialdehyde observed in monocytes fed with pigment or in RBC ghosts and liposomes incubated at pH 6.5 in presence of pigment and low amounts of H2O2. Removal of the labile iron fraction from pigment by repeated treatments with 0.1 mM H2O2 at pH 5.5 reduces pigment toxicity. It is suggested that iron released from ingested pigment is responsible for the intoxication of monocytes. In acute and chronic falciparum infections, circulating and tissue-resident phagocytes are seen filled with TPRBC and pigment particles over long periods of time. Moreover, human monocytes previously fed with TPRBC are unable to neutralize pathogenic bacteria, fungi, and tumor cells, and macrophage responses decline during the course of human and animal malaria. The present results may offer a mechanistic explanation for depression of cellular immunity in malaria.

Heme polymerase: Modulation by chloroquine treatment of a rodent malaria

The biosynthesis of the β-hematin of malarial pigment (hemozoin) is catalyzed by a newly discovered enzyme, heme polymerase, which is described for Plasmodium berghei in this report. This novel enzyme is present in the insoluble fraction of hemolysates of infected erythrocytes but is not present in normal erythrocytes. The substrate is ferriprotoporphyrin IX (FP) released from hemoglobin. At pH 5 and 37° C the enzyme is saturated by 100 μM FP. The pH optimum is between 5 and 6 and the reaction is linear for 6 hours. All heme polymerase activity is destroyed by heating at 100° C for 3 minutes. Chloroquine treatment of malarious mice reduces by 80 percent the activity of this enzyme, without inhibiting release of FP from hemoglobin, and thereby causes excess nonpolymerized, nonhemozoin FP to accumulate. Since the accumulated FP is accessible to bind chloroquine, we propose that it is the mediator of the antimalarial activity of chloroquine.

A new microwave-stimulated stain for melanin

A new and rapid staining procedure for melanin is described. Sections of paraffin-embedded tissues from 10 primary cutaneous melanomas, 5 metastatic melanomas, 10 compound naevl, 5 intradermal naevl, 5 Spitz naevi, 5 dysplastic naevl, 5 amelanotic melanomas, 3 non-leslonal skin biopsies and 1 penile condyloma accumlnatum were covered with colloidal silver nitrate (a mixture of aqueous silver nitrate and gelatin in a 2:1 ratio by volume) and irradiated in a domestic microwave oven for 45 sec at 350 W “defrost” setting with a 500 ml water ballast. The staining of melanocytes and melanoma cells was consistently crisp and intense, with no staining of retlculin or background. A wide range of normal tissues including sections containing bile, iron, calcium, fungi, spirochetes, bacteria, formalin pigment, malarial pigment, lipofuschln as well as argyrophil and argentaffin carcinoid tumors did not stain with the technique.

Histopathology of the liver and kidney during malaria: relation to malaria-induced dyslipoproteinemia

A kinetic study concerning histologic and cytologic alterations during Plasmodium chabaudi infection of Swiss mice has been carried out. In liver, a reversible focal and non ischemic necrosis and a vascular congestion were observed together with an accumulation of malarial pigment. The endoplasmic reticulum cisternae and Golgi saccules of hepatocytes were highly distended. Hepatocyte microvilli in biliary canaliculi and in Disse' spaces were markedly less developed and less numerous than in normal liver. Intracytoplasmic lipid globules were found in large amount in hepatocytes before the peak of parasitaemia. Their number and size gradually diminished thereafter. Hepatocytic mitochondria showed important unspecific modifications probably in relation, at last partly, to the tissue anoxia. Some hepatocytic changes (intracytoplasmic lipid globules, enlargement of endoplasmic reticulum cisternae and Golgi saccules) were consistent with an increased synthesis of lipoproteins (VLDL). The kidney showed only minor histological and ultrastructural changes. However haemosiderin was observed in proximal tubules and in their bordering cells. The deposit of immune complex reported previously do not appear associated with tissular or cellular important alterations.

The effect of substance(s) associated with malarial parasites on C3b inactivator

The effects of a substance or substances associated with malarial parasites on the inhibitory role of C3b inactivator in immune adherence were investigated. The test system involved adherence of immune complexes of complement-bearing sheep erythrocytes and rabbit antibody, using human group O erythrocytes and human renal glomerular tissues as indicators. Malarial antigen and other soluble by-products of the malarial parasites, presumably present in spent culture medium, did not interfere with the inhibitory role of C3b inactivator. However, malarial pigment, both crude and purified, enhanced immune adherence in the presence of C3b inactivator. It is suggested that malarial pigment, by inhibiting the action of C3b inactivator, may play a significant role in the immunopathogenesis of renal lesions associated with malarial infections.

Comparison of proteases from chloroquine-sensitive and chloroquine-resistant strains of plasmodium falciparum

An aminopeptidase and four hemoglobin-degrading acid proteases have been isolated from cloned strains of chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. Aminopeptidases from both strains showed similar properties including molecular weights of 63,000 and noncompetitive inhibition by chloroquine; K i = 535 and 410 μM for enzymes from the sensitive and resistant strains respectively. The acid proteases from the chloroquine-sensitive strain included a low molecular weight enzyme in the soluble fraction (protease S), an enzyme weakly associated with membrane (protease M 2), and two enzymes strongly associated with membrane (proteases M 3 and M 4). The acid proteases from the chloroquine-resistant strain included protease S, protease M 2, a second enzyme weakly associated with membrane (protease M 1), and protease M 3. All of the acid proteases were inhibited by ferriprotoporphyrin IX and by the chloroquine-ferriprotoporphyrin IX complex, I 50 = 5–25 μM. The data were consistent with a model for chloroquine action wherein chloroquine acts to divert ferriprotoporphyrin IX from sequestration into malarial pigment, leaving ferriprotoporphyrin IX (or its chloroquine complex) to interfere with digestion of host cytosol by inhibiting hemoglobin-degrading proteases. However, the similarities among the proteases from chloroquine-sensitive and chloroquine-resistant strains of parasites suggest that chloroquine resistance does not result from changes in parasite proteases.

Acute hepatic failure due to Plasmodium falciparum liver injury.

Nine patients with acute liver failure due to Plasmodium falciparum liver injury admitted to the Rajgarhia Liver Unit of the All-India Institute of Medical Sciences during 1982-84 are presented. The liver was palpable in all the patients, and eight had splenomegaly. Investigations revealed mild to moderate abnormality in liver function tests. All were negative for the markers of acute infection due to hepatitis A and B viruses. Blood film examination showed P. falciparum alone in seven and along with P. vivax in the remaining two patients. Liver histology, which was identical in all eight patients where liver biopsy was done, showed centrizonal necrosis and hyperplastic Kupffer cells loaded with malarial pigment. All the patients recovered with specific anti-malarial and supportive treatment. Our observations suggest that malaria due to P. falciparum may present as jaundice and encephalopathy which stimulates acute hepatic failure due to fulminant hepatitis.

Birefringent hemozoin identifies malaria.

The diagnosis of malaria is still sometimes difficult because of the insensitivity of microscopic screening at low levels of parasitemia. The malarial pigment, hemozoin, is a crystalline product of the digestion of hemoglobin by the parasites. Under polarized light at 500X magnification, brilliantly birefringent granules of the pigment were detected in Wright's stained smears, and the parasites easily localized, in 18 cases of malarial infection. Fresh, wet, coverslipped preparations of cultures of Plasmodium falciparum also were examined under polarized light. Serial dilutions of the cultures showed that, even at the very low level of 0.01% parasitemia, intracellular birefringent granules were detected in an average of 45 +/- 16 (SE) seconds at 500X magnification. Using polarized light is a simple, fast, sensitive, and specific method for localizing intracellular pigmented malaria parasites in wet preparations of blood.

The status of zinc in malaria ( Plasmodium falciparum) infected human red blood cells: stage dependent accumulation, compartmentation and effect of dipicolinate

The inhibitory effect of metal chelators on intraerythrocytic malarial parasites imply that trace metal have a vital role in the biology of these organisms. In the present work X-ray fluorometry was used to study the status of zinc and iron in human red blood cells infected with Plasmodium falciparum in culture conditions. It was found that while the iron level remains constant throughout the parasite cell cycle, that of zinc increases in parallel with parasite maturation to reach a 2.3-fold higher level than that of uninfected red blood cells. Compartment analysis of infected red blood cells indicated that most of this gain was associated with the parasite and some with the host-cell membrane. Analysis of the malarial pigment showed that the zinc/iron ratio was similar to that of red blood cells, implying the this compound, which results from the digestion of host-cell cytosol, sequesters the zinc of host metalloenzymes. Dipicolinic acid (DPA), like other chelators, was found to inhibit the intracellular development of the parasite with an ED 50 of 1 mM. DPA does not penetrate into normal red blood cells but readily permeates into infected cells, although it does not leach out their zinc. It is uncertain whether the inhibitory effect of DPA is exerted through alterations of host cell metabolism or by directly affecting that of the parasite. The putative receptors of zinc in the infected red blood cell are discussed.

Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum

A protease from Plasmodium falciparum was purified 150 fold by high performance liquid chromatography on a TSK-G-3000 SW exclusion column. The enzyme is not retained during pressure filtration with an Amicon PM10 membrane but is retained by a YM5 membrane. The molecular weight of the protease is less than 10 000, based upon mobility on a calibrated TSK column. The enzyme catalyzes the hydrolysis both of acid denatured hemoglobin and of albumin. The hydrolysis is optimal at pH 4.5, but considerable activity is seen at pH 6.0. Pepstatin strongly inhibits the protease ( I 50 = 70 nM) while bestatin, antipain and phosphoramidon produce moderate inhibition ( I 50 = 30, 30 and 3 μM, respectively). The protease is inhibited by ferriprotoporphyrin IX ( I 50 ca. 5 μM). This inhibition is insensitive to pH between pH 4.5 and 6. Although chloroquine does not strongly inhibit the protease, chloroquine-ferriprotoporphyrin IX complex produces inhibition similar to that of ferriprotoporphyrin IX. It is suggested that the antimalarial effect of chloroquine is due to the formation of ferriprotoporphyrin IX-chloroquine complex which prevents the sequestration of ferriprotoporphyrin IX into malarial pigment, thereby providing both ferriprotoporphyrin IX and its chloroquine complex as inhibitors of one of the proteases required for the degradation of hemoglobin.

Ingestion of erythrocytic stroma by Plasmodium chabaudi trophozoites: ultrastructural study by serial sectioning and 3-dimensional reconstruction.

An ultrastructural study of numerous serial sections of Plasmodium chabaudi trophozoites at various growth stages, followed by 3-dimensional reconstruction, allowed us to describe more precisely the internalization process of the erythrocytic stroma, both in space and in time. Two endocytotic processes are apparent. (1) Pinocytosis--as soon as the merozoite has become a young trophozoite (ring stage), small double membrane-bound vesicles can be seen budding off around the whole periphery of the parasite. After the inner membrane of the vesicle has disappeared, the contents alter and a pigment crystal appears. (2) Cytostomal system--this phenomenon coexists with, and eventually replaces, pinocytosis. It consists of invagination of the membrane of the parasitophorous vacuole and of the plasmalemma, through a typical cytostome, in order to form a cytostomal vacuole. This extends into a long tube, the cytostomal tube, which eventually digitates. When the tube reaches its maximal size, the cytostome disappears and the tube remains open to the erythrocytic stroma by a simple aperture. A new cytostome can form elsewhere on the parasite surface and another tube can extend. Two or three such tubes can coexist in a trophozoite although only one cytostome is functional at one time. At the end of the tubes vesicles bud off, the contents of which become modified as described previously. The residual product of haemoglobin degradation is the malarial pigment.

Lipoproteins and malaria. I. Immunogenicity of serum lipoproteins in mice infected with Plasmodium chabaudi

Anti-lipoprotein antibodies (anti-Lp Ab) have been investigated during the course of acute infection with P. chabaudi in Swiss mice using a radio-immunoprecipitation assay with (125)-iodine radiolabelled lipoproteins from normal or infected mice. Antibodies were detected 11 days after the beginning of infection; however, the highest lipoprotein precipitations were observed with purified (125)-I labelled lipoproteins from day-5 or day-7 infected mice. P. chabaudi infected mice were treated with chloroquine at various intervals after the beginning of infection and anti-Lp Ab were assayed on day 13. Anti-Lp Ab were not observed in mice treated before day 7 but were present in all mice treated after day 7. Anti-Lp Ab were not detected in mice infected with P. yoelii 17 X. Injection of purified lipoproteins from day-7 P. chabaudi infected mice (Lp day-7) to normal uninfected mice did not induce an antibody response to lipoproteins but anti-Lp Ab were observed when the same injection was performed in P. yoelii infected mice. Moreover, anti-Lp Ab were detected in uninfected mice injected concomitantly with Lp day-7 and hematin extracted from malarial pigment. Our results suggest that anti-Lp Ab observed in P. chabaudi infected mice are mainly against modified lipoproteins produced during infection and that the induction of the antibody response against lipoproteins requires an adjuvant effect such as the hematin which is released during infection.

Placental malaria and foetoplacental function: low plasma oestradiols associated with malarial pigmentation of the placenta

Placental biopsies were taken immediately post partum from 65 Gambian mothers who had not received anti-malaria chemoprophylaxis during pregnancy whilst living in an area hyperendemic for Plasmodium falciparum malaria. The biopsies were examined without knowledge of the mothers' health or the outcome of pregnancies. Histologically, they were divided into two groups: those with macrophages containing malarial pigment in the inter-villous spaces, and those without such pathology. Babies with pigmented placentae had a mean (SD) weight for gestational age of 83·3 (10·6)%, which was significantly lower (p<0·01) than that of 91·2 (7·7)% in the non-pigmented group. The plasma oestradiol concentrations in the mothers who later delivered pigmented placentae were significantly lower from 32 weeks of gestation onwards, and did not continue to rise in the last trimester as they did in the non-pigmented group. The last trimester appears to be the critical time for protection of the foeto-placental unit against malaria. Anti-malaria chemoprophylaxis should be given to all pregnant women.

The malarial pigment in rat infected erythrocytes and its interaction with chloroquine. A Mössbauer effect study.

Mössbauer studies of rat erythrocytes infected by Plasmodium berghei malaria parasites, using 57Fe-enriched rat red blood cells, were carried out in order to determine the physical parameters which characterize the malarial pigment iron and to test the effect of the widely used antimalaria drug, chloroquine, on these parameters. The iron in the malarial pigment which is derived from hemoglobin digestion by the intracellular parasite was found to be trivalent, high spin, with Mössbauer parameters which are significantly different from those of any known iron porphyrin containing compound. No difference was found between the parameters obtained in erythrocytes infected by drug-sensitive and drug-resistant strains of P. berghei, both before and after the treatment with chloroquine. The iron compound consists of microaggregates, about 30 A in diameter. These are somewhat larger in chloroquine-resistant strains and tend to increase in size in chloroquine-sensitive strains upon treatment with the drug. Mössbauer spectra of erythrocytes infected by a chloroquine-resistant strain revealed pigment iron in relative amounts invariable of those found in chloroquine-sensitive strains, demonstrating that drug-resistant parasites indeed digest hemoglobin.

A simple magnetic method for the purification of malarial pigment

Using a simple magnetic separation apparatus, Plasmodium berghei malarial pigment has been purified 200-fold in a single step. The technique exploits the paramagnetic properties of malarial pigment (hemozoin) and provides a simple and rapid method for the isolation of this material with high purification and yield.

Placental Pathologic Changes in Malaria

Department of Pathology and Parasitology, Centre International de Recherches Madicales de Franceville; and Department of Obstetrics and Gynaecology, Hospital de Franceville, Gabon, Africa

Inhibitory effects of immune monkey serum on synchronized Plasmodium falciparum cultures.

We studied the effects of heat-inactivated immune monkey serum on the growth of a partially synchronized culture of Plasmodium falciparum. By light microscopy, parasites within erythrocytes were morphologically indistinguishable from those cultured in normal serum. Immune serum reduced by 90% the number of erythrocytes containing newly invaded rings. Clusters of extracellular merozoites, usually around clumps of malarial pigment, were seen frequently in cultures grown with immune serum but rarely in cultures with normal serum. Electron microscopy confirmed the normal development of intraerythrocytic parasites. In immune serum cultures, electron-dense precipitates were found on the surface of schizonts, merozoites, and the excrescences on the plasma membrane of schizont-infected erythrocytes. Merozoites in immune serum culture appeared to aggregate by adherence between adjacent surface coats. These findings support the hypothesis that immune serum agglutinates merozoites and thereby inhibits their invasion into uninfected erythrocytes.