- Email: [email protected]
Chapter 6 Malaria Pigment
CHAPTER
6 Malaria Pigment
I was born in a small private hospital on the eastside of lower Manhattan on 12 February 1933. My parents were immigrants from Russia who had lived in the same village (Okna) and met and married after coming to the United States. My father was a factory manager and my mother was a housewife. Our family was distinctly at the lower end of the middle class. I attended the local public schools (P.S. 93 and Hermann Ridder Junior High School and James Monroe High School). After graduating from high school, I enrolled at the City College of New York (CCNY, New York) where I majored in biology and education. CCNY was famous for its academic rigor and a student body composed of high achievers. As the time of graduation approached, several of my biology professors, especially James Dawson (the Biology Department’s chair and a protozoologist), William Tavolga (a behaviorist/histologist) and Herman Spieth (a Drosophila geneticist/behaviorist) encouraged me to go on to graduate school. Through personal contact between Tavolga and Spieth I was introduced to Libbie Hyman at the American Museum of Natural History and Dawson and Spieth arranged that W. C. Allee (who, because of his age, had been forced to retire from the University of Chicago and was now heading the Zoology Department at the University of Florida (Gainesville, Florida)) accept me as a graduate student; Allee in turn arranged a research assistantship for me with the protozoologist James B. Lackey in the Department of Sanitary Engineering. My first independent research project involved a survey of oligotrich ciliates (protozoans) in the Gulf of Mexico. Just as I was becoming proficient with ciliate taxonomy and behavior I received a letter from the Selective Service Board—I was drafted into the United States Army. This interruption of my graduate studies turned out to be an opportunity to learn some parasitology and to travel the world at Uncle Sam’s Advances in Parasitology, Volume 67 ISSN 0065-308X, DOI: 10.1016/S0065-308X(08)00406-5
#
2008 Elsevier Ltd. All rights reserved.
49
50
Irwin W. Sherman
expense. After infantry basic training (Fort Dix, NJ) I was sent to Fort Sam Houston in Texas and then to Valley Forge Army Hospital in Pennsylvania to be trained as a medical technologist. I was then shipped overseas, where, through good fortune, I was assigned to medical laboratories in Salzburg, Austria and Darmstadt and Heidelberg, Germany. In Germany and Austria, I worked in clinical laboratories doing hematology, parasitology, microbiology, phlebotomy and blood chemistry. Upon my discharge from the United States Army (where by an act of Congress I received the exalted rank of Private First Class) I decided not to return to Florida to continue my graduate studies; instead I began to teach science and mathematics in a junior high school in Yonkers, NY. At the same time I took a few graduate courses at CCNY and assisted in the teaching of the evening session introductory biology laboratories at CCNY. Knowing of my interests in research, William Etkin, an endocrinologist/behaviorist and a professor at CCNY, encouraged me to return to graduate school on a full-time basis. He said, ‘If you want to run with the hounds and do some original research get yourself an advanced degree.’ It was sage advice. During the summer of 1957, thanks to a CCNY Biology Club Scholarship, I took the invertebrate zoology course at the Marine Biological Laboratory (MBL) in Woods Hole (Massachusetts). At the MBL I was able to spend all day, every day, for 10 weeks studying live invertebrates. The field trips were extraordinary, especially for a South Bronx slum kid, and the faculty was exceptional: Clark Read (Rice University, Houston, Texas), Howard Schneiderman (Case Western Reserve University, Cleveland, Ohio), John Buck (National Institutes of Health (NIH), Bethesda, Maryland), Theodore Bullock (University of California, Los Angeles, UCLA), Grover Stephens (Minnesota) and Ralph Smith (University of California, Berkeley, California). At the end of that summer I was invited to be the laboratory assistant for the course. I readily accepted and for the next two summers shared the work with another young parasitologist, Frank Friedl, from the University of Minnesota. The experiences in the United States Army and the MBL crystallized my interests in combining protozoology with clinical disease. I scoured university catalogues for those offering the greatest number of courses in parasitology and eventually settled on Northwestern University (or more accurately, they settled on me). I was awarded an Abbott Laboratory Fellowship with Robert Hull (a student of the eminent protozoologist R. R. Kudo). Hull (who had little training either in parasitology or biochemistry) had an NIH grant and together with a graduate student (Father Truong, a Vietnamese priest) they had begun a malaria project with Plasmodium lophurae and chickens. When I was at the start of my graduate studies very few laboratories in the United States were working on malaria biochemistry and those who
Malaria Pigment
51
had done so previously (see p. 12 ) were carrying out biochemical research on other subjects. Hull’s laboratory had minimal instrumentation: there was an ancient Warburg manometer, an assortment of low-speed centrifuges, a Klett colorimeter, a DU spectrophotometer and a paper electrophoresis apparatus but there was no possibility for working with radioisotopes. Hull, who did no bench work, appeared not to be very knowledgeable about malaria and was more interested in his favorite organism the suctorian, Tokophrya, so no specific malaria project was assigned to me. It soon became apparent that if I wanted to study the biochemistry of malaria I would have to do it on my own by learning more about the parasite and then I would have to find mentors who could teach me biochemistry. My first year as a graduate student was spent reading the literature and taking notes. Hemozoin, the brown–black pigment found within organs such as the spleen and liver as well as in erythrocytes of those infected with malaria, has long been a source of fascination for students of the disease (Sullivan, 2002a). Laveran’s finding (1880) of pigmented bodies in the blood of a soldier suffering with fever led to his discovery of the malarial parasite (see p. 3) and when Ross examined the stomachs of mosquitoes that had fed on a malaria patient and found ‘each of these bodies contained . . . granules of black pigment absolutely identical in appearance . . . with the . . . characteristic pigment of the parasite of malaria’ it provided the critical clue to mosquitoes as vectors of the disease (see p. 5). At Northwestern University, during my time as a graduate student, P. lophurae was maintained in chickens by intravenous inoculation. The method is rather simple, however, the amount of blood obtained from chicks (relative to ducklings) is small (5–10 ml), and because of age immunity the animals would not provide sufficient numbers of parasites when they were older than 5–6 weeks and weighed more than 200 g. As a result, most of my time during the first year involved passing the infection by blood inoculation every 3–4 days, measuring blood volumes, determining whether there were statistically significant differences between blood smears and trying to block natural immunity using carbon ink. By taking courses in biochemistry (with Lazlo Lorand) and biophysics (with Irving Klotz) I became more familiar with biochemical techniques and approaches. I first became curious about the composition and mode of formation of hemozoin when I peered through a microscope to examine a blood film taken from a chicken infected with P. lophurae and saw the parasites filled with refractile golden-brown granules. As a consequence, one of my doctoral thesis projects became a characterization of P. lophurae hemozoin. In 1891, Carbone reported that hemozoin had spectral properties similar to the iron-containing heme of hemoglobin and not melanin as had been proposed by Meckel (1847). Brown (1911) rediscovered this
52
Irwin W. Sherman
work and confirmed that it was hematin or ferriprotoporphyrin (FP). When Brown found intravenous injections of hematin produced a malaria-like paroxysm he suggested the pigment was involved in the production of the febrile attack. Later studies were consistent with FP being a constituent of hemozoin, however, there was other work— particularly that of Deegan and Maegraith (1956 a,b) at the Liverpool School of Tropical Medicine (LSTM, Liverpool, United Kingdom)—that indicated malaria pigment was more than monomers of FP. Deegan and Maegraith’s reasoning was that hemozoin could not be hematin since the latter would inhibit parasite succinoxidase, an enzyme vital to parasite survival. They proposed that vigorous extraction procedures (used by others) had cleaved the heme from its associated protein and by using milder extraction procedures found evidence for hemozoin being heme plus a proteinaceous moiety. Enlisting the support of Professors Lorand and Klotz, I was able to characterize malaria pigment spectrophotometrically and was able to carry out studies employing analytical ultracentrifugation (Sherman and Hull, 1960). The conclusion from this work with P. lophurae reaffirmed the work of Deegan and Maegraith. Years later (when newer biochemical/immunological methods were available), I once again became interested in malaria pigment (Yamada and Sherman, 1979). Together with a graduate student, Kenneth Yamada, we isolated hemozoin: erythrocyte-free parasites were homogenized using a French pressure cell; the suspension was centrifuged, the pellet recovered, sonicated and ultracentrifuged through a 1.7-M sucrose cushion. Using the techniques of peptide mapping, polyacrylamide gel electrophoresis (PAGE), gel (Sephadex) chromatography, as well as immuno-double diffusion we found hemozoin to consist of a complex of monomers and dimers of hematin, a 14-kDa protein, a 21-kDa protein (presumed to be of parasite origin coupled to heme) and methemoglobin. Electron microscopy of the purified hemozoin showed identity to that seen in sections of intact parasites. Supporting such work were reports by Ashong et al. (1989) who claimed protein was associated with FP, and the findings of Goldie et al. (1990) that hemozoin was a mixture of native and denatured globin. However, others claimed the non-covalently bound proteins resulted from contaminants (Fitch and Kanjananggulpan, 1987; Homewood et al., 1975) and when these were removed the pigment was identical to b-hematin. Even analyses by sophisticated biophysical methods did not completely clarify the nature of hemozoin: Morselt et al. (1973) using microspectrophotometry on intact cells found the pigment to differ from pure hematin, whereas the work of Bohle et al. (1997), Wood et al. (2003) and Pagola et al. (2000) found hemozoin to be identical with a cyclic dimer of b-hematin. Once hemozoin was equated with b-hematin subsequent investigations concerned themselves with its mode of formation.
Malaria Pigment
53
The literature is now replete with methods to synthesize b-hematin in the laboratory (Fitch, 2004; Sullivan, 2002a). Slater et al. (1991) postulated that hemozoin formation involved the ‘enzymatic polymerization of heme into a non-toxic crystal’ (i.e. b-hematin), however, later studies showed that a ‘heme polymerase’ did not exist and FP conversion to b-hematin could occur spontaneously in the absence of biological materials with solutions high in acetate concentrations and low in pH, but to increase the rate of formation the temperature had to be raised to 60 C (Egan et al., 2000, 2001). However, formation of b-hematin did not occur under physiological conditions or at pH 5 with preparations from uninfected red cells; it did form at pH 5 with preparations from infected erythrocytes. A more recent study has shown that under acid conditions b-hematin assembles rapidly and spontaneously near long-chain alcohol/ water and lipid/water interfaces (Egan et al., 2006). However, the morphology of the dimers is not identical to natural hemozoin. Although a considerable body of work describing the role of b-hematin in immunological and pathological events now exists (see below) I have continued to believe hemozoin is not identical to b-hematin. Indeed, many of the discrepancies regarding the chemical nature of hemozoin do not reflect the analytical techniques used, but largely depend on the manner by which the pigment is isolated and purified. For example, if hemozoin is isolated by physical means (mechanical breakage of cells or isolated food vacuoles (FVs) containing the pigment granules followed by density-gradient centrifugation) and the colored material is solubilized in alkali (0.1 N NaOH) the absorption spectrum is consistent with heme coupled to protein; further, as noted above immunological and chemical analysis of the associated protein (proteins?) has shown it to be different from native globin. However, when hemozoin is isolated by extensive proteolytic digestion and lipid extraction then the resultant product consists of crystals of b-hematin. Hemozoins from various plasmodia, when examined by electron microscopy (see Noland et al., 2003; Scheibel and Sherman, 1988) are so variable in morphology it is difficult to conceive of hemozoin as being pure crystals of b-hematin. This notion of lack of equivalence of b-hematin and hemozoin has received support from two recent studies. Parroche et al. (2007) found malarial deoxyribonucleic acid (DNA) to be a component of hemozoin when isolated using a strong magnetic field; in addition there was a ‘ladder of Coomasie -stained proteins.’ In another study when hemozoin, prepared from disrupted P. falciparum, was ultracentrifuged through a 1.7-M sucrose cushion (as we had done previously with P. lophurae) a paucity of proteins was found, however, the pigment granules were enveloped by neutral lipid (NL) nanospheres (Pisciotta et al., 2007). What roles does hemozoin play and how is it made? Coy Fitch began to study hemozoin in the late 1960s when he was at Walter Reed Army
54
Irwin W. Sherman
Institute of Research (WRAIR, Washington, DC). After finding that a chloroquine-resistant strain of P. berghei was deficient in a high-affinity receptor for the drug (Fitch, 1969, 1970), he went on to identify FP as the receptor (Chou et al., 1980; Fitch et al., 1974); later he found hemozoin to be dimerized FP similar to b-hematin (Fitch and Kanjananggulpan, 1987). Recently, he speculated that unsaturated fatty acids (FAs) and their mono- and diglycerides in the FV serve to concentrate the monomeric FP and keep it in a state favourable for dimerization (Fitch et al., 2003; Fitch and Russell, 2006). Coy Fitch (1934– ) as a medical student at the University of Arkansas School of Medicine (Little Rock, Arkansas), became interested in biochemistry and was provided research space and other resources. He earned an master of science (MS) in biochemistry as well as a doctor of medicine (MD) in 1958 and remained at the School of Medicine as a resident in medicine and Russell M. Wilder-National Vitamin Foundation Fellow in biochemistry (1958–1962). By the time the residency was completed he had developed an interest in membrane transport processes, and he remained on the faculty of the University of Arkansas as an assistant professor of medicine and biochemistry studying such. Five years later, he moved to the Saint Louis University School of Medicine (Saint Louis, Missouri) as Associate Professor of Internal Medicine and Biochemistry. That move made him vulnerable to military service. As a consequence, he was promptly drafted into the Army and assigned to the Division of Biochemistry of the WRAIR. The director of the Division of Biochemistry at WRAIR allowed Coy to have an independent laboratory but stipulated that it had to be devoted to malaria research. He decided to study the biochemistry of chloroquine (CQ) accumulation in erythrocytes infected with malaria parasites. This decision was based on a recent report that mouse erythrocytes infected with a CQ-susceptible strain of Plasmodium berghei accumulated more CQ than erythrocytes infected with a CQresistant strain. He thought he would be studying a membrane transport process; however, he soon realized that the movement of chloroquine across biological membranes was far too fast to measure with the centrifugation techniques available for his use. Nevertheless, by the end of his Army career (1967–1969), he had learned that CQ-resistant malaria parasites are deficient in a high-affinity receptor (target) for the drug. Returning to Saint Louis, Coy continued to study CQ accumulation by malaria parasites, focusing on the receptor. In 1974, he and his coworkers were able to describe the specificity and affinity of the binding site, so the receptor could be recognized when isolated. After
Malaria Pigment
55
describing the high-affinity binding site, several years were spent searching for a high-affinity protein receptor for CQ in malaria parasites, only to find none. In 1980, his group discovered that ferriprotoporphyrin IX (FP) had the specificity and affinity characteristics of the CQ receptor as had been suspected earlier by Macomber, Sprinz and Tousimis (Macomber and Sprinz, 1967). His group also found that FP and its complex with CQ are toxic for erythrocytes and malaria parasites (Banyal and Fitch, 1982; Fitch et al., 1982). These observations led Fitch and co-workers to propose that FP mediates the chemotherapeutic action of CQ and to ask how malaria parasites escape FP toxicity while ingesting and metabolizing hemoglobin. Believing the lack of toxicity to result from FP sequestration in hemozoin, they decided to characterize the pigment of hemozoin better. This pigment appeared to be an aggregate of FP that was similar, perhaps identical, to b-hematin (Fitch and Kanjananggulpan, 1987). Several years later, bhematin was found to be aggregated dimers of FP (Pagola et al., 2000). Since FP is rendered non-toxic by converting it to b-hematin, it was now logical to study this process. In 1992, Chou and Fitch discovered that FP dimerization could be measured easily and that CQ treatment reduces FP dimerization in vivo. At first, Chou and Fitch and others (Slater and Cerami, 1992) assumed that an enzyme catalysed the reaction; however, that assumption was wrong. Instead, it was found that the reaction is catalysed in malaria parasites by unsaturated FAs, probably predominantly linoleic acid. Furthermore, Fitch and co-workers (2003) obtained evidence that CQ treatment causes this catalyst to be masked (i.e. unavailable to catalyse FP dimerization) in CQ-susceptible malaria parasites, thus explaining the accumulation of toxic, undimerized FP in response to CQ treatment. The next task was to explain why unsaturated FAs are masked after CQ treatment. Based on their findings, Fitch and Russell (2006) proposed that the CQ–FP complex inhibits endosomal maturation and, thereby, reduces the release of unsaturated FAs from the membranes of erythrocytoid bodies. These bodies are found in endosomes early in the feeding process of malaria parasites. This explains, Coy believes, how CQ treatment results in masking of unsaturated FAs, reduces FP dimerization, causes toxic undimerized FP to accumulate and be available to bind CQ, and also causes denatured hemoglobin to accumulate in vesicles in malaria parasites (Fitch et al., 2003; Macomber and Sprinz, 1967; Warhurst and Hockley, 1967b). In this scenario, the toxic, undimerized FP that binds CQ and mediates its chemotherapeutic effect is derived from hemoglobin as it is denatured or hydrolyzed in immature endosomes. Currently, Coy is devoting his time to determining how the CQ–FP complex inhibits endosomal maturation.
56
Irwin W. Sherman
Fitch’s work on b-hematin synthesis (Fitch, 2004; Fitch et al., 1999; 2000) has recently been extended by Pisciotta et al. (2007). Hemozoin, isolated from saponin-purified P. falciparum trophozoites, followed by osmotic lysis and sonication and sedimentation through a sucrose cushion, was associated with lipid nanospheres consisting mainly of saturated lipids monostearic glycerol (MSG) and monopalmitic glycerol (MPG) with the principal diacylglycerols (DAGs) being distearic glycerol and 1-stearic-3-palmitic glycerol; no monooleic glycerol (MOG) or monolinoleic glycerol (MLG) was found. The source of these may be the neutral lipid bodies (NLBs) containing triacylglycerol (TAG) and DAG, which are associated with the FVs ( Jackson et al., 2004). The scenario proposed is: the double-membrane vesicle produced during cytostomal feeding (see p. 171) has its outer membrane fused with the FV membrane while its inner membrane is degraded by phospholipase C and/or lysosomal acid lipase (and found in the falciparum genome); the breakdown products are assembled into TAG and its precursors serve as promoters of b-hematin formation. Although Fitch et al. (1999) found TAG to be inactive in b-hematin formation Jackson et al. (2004) found MOG as well as mono- and dimyristoyl glycerol to be effective. In contrast, Pisciotta et al. (2007) found MPG to be a potent promoter of heme crystallization as was the combination of 1-stearic3-palmitic glycerol. A NL blend of MPG/MSG/dipalmitic glycerol (DPG)/ dioleic glycerol (DOG)/dilinoleic glycerol (DLG) (2:4:1:1:1) produced heme crystals rapidly. Of some interest is their observation, ‘the lipid blend crystals did not exactly replicate hemozoin made by P. falciparum (and) may require the presence of non-specific proteins or other molecular species.’ What is the role of hemozoin? Due to the massive degradation of hemoglobin a large amount of free heme is produced. Because free heme may intercalate with plasmodial membranes to lyse parasites and plasmodia lack the enzymes necessary to degrade the porphyrin ring the incorporation of the heme moieties into insoluble hemozoin has been postulated to be a detoxification mechanism (Fitch and Chevli, 1981; Fitch et al., 1982). Other postulates are peroxidative degradation within the FV (Loria et al., 1999) and glutathione (GSH)-mediated heme degradation in the cytosol (Ginsburg et al., 1998). Hemozoin (in the form of a- and b-hematin) has been reported to suppress erythropoiesis (Casals-Pascual et al., 2006), and after endothelial cell ingestion suppresses ICAM-1 and PECAM-1 expression, as well as production of interleukin (IL)-6 (Taramelli et al., 1998), and it can lyse red blood cells by incorporation into the membrane (Omodeo-Sale et al., 2005). In its native form, hemozoin has been shown to inhibit the function and maturation of dendritic cells (Millington et al., 2006; Skorokhod et al., 2004; Urban and Todryk, 2006) and to enhance matrix metalloproteinase activity as well as tumor necrosis factor (TNF)-a production in monocytes (Prato
Malaria Pigment
57
et al., 2005). Murine phagocytes fed natural hemozoin or FP plus supplemental interferon (IFN)-g increased their expression of nitric oxide (NO) synthase and NO (Jaramillo et al., 2003), however, human monocytes were neither able to generate NO nor to increase expression of NO synthase under the same conditions even with IFN-g (Skorokhod et al., 2007b). Further, there was little evidence of NO killing P. falciparum in vivo and in a Papua New Guinea study no correlation between NO levels and parasitemia was found in human subjects (Boutlis et al., 2004). Clearly, extrapolation of murine data to human malaria may not be justified and caution should be exercised when doing so. Others report that b-hematin does not promote such activities directly, but that it is the heme-catalysed peroxidation of cellular debris that results in a toxic gradient of primary and secondary FA oxidation products (15hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (HNE)) that not only activates the innate immune response (Carney et al., 2006) the HNE-protein adducts but also acts to decrease red cell deformability (Skorokhod et al., 2007a), promotes their removal and leads to anaemia. Coban et al. (2005) contend that b-hematin and hemozoin (prepared from a sonicated pellet of parasites, treated with proteinase K, washed in sodium dodecylsulphate (SDS), incubated with 6-M urea) on their own activate the toll-like receptor (TLR)-9 to mediate immune activation. By contrast, Parroche et al. (2007) report that b-hematin is immunologically inert, whereas natural hemozoin (isolated as described above) activates the TLR. Further, Parroche et al. have provided evidence that the natural pigment acts as a carrier for malarial DNA and that this results in it being targeted to TLR-9. (Malarial DNA on its own was found not to be immunostimulatory.) Although TLR-9 is known to recognize GC-rich areas that are typically found in bacterial DNA, and falciparum DNA is exceedingly AT-rich, there are small GC-rich regions in plasmodial DNA that are able to stimulate TLR-9. Clearly, the varied and inconsistent pathological and immunological findings result from a lack of equivalence of the ‘malaria pigment’ used. In my view, despite more than half a century of biochemical research on malaria pigment, much is yet to be learned about its natural composition and its varied roles. Sometimes there is a great temptation on the part of investigators to use a laboratory-synthesized compound that is easy to prepare and mimics the natural product, but extrapolation of the activities of a synthetic product such as b-hematin with natural hemozoin can be misleading. In addition, although b-hematin may have some utility in describing the relationships between different types of anti-malarials (Basilico et al., 1998; Egan and Ncokazi, 2005; Ncokazi and Egan, 2005; Solomonov et al., 2007; Trang et al., 2006) to conclude that the mimic and the authentic malaria pigment are identical is more often than not without adequate justification.
6 Malaria Pigment
I was born in a small private hospital on the eastside of lower Manhattan on 12 February 1933. My parents were immigrants from Russia who had lived in the same village (Okna) and met and married after coming to the United States. My father was a factory manager and my mother was a housewife. Our family was distinctly at the lower end of the middle class. I attended the local public schools (P.S. 93 and Hermann Ridder Junior High School and James Monroe High School). After graduating from high school, I enrolled at the City College of New York (CCNY, New York) where I majored in biology and education. CCNY was famous for its academic rigor and a student body composed of high achievers. As the time of graduation approached, several of my biology professors, especially James Dawson (the Biology Department’s chair and a protozoologist), William Tavolga (a behaviorist/histologist) and Herman Spieth (a Drosophila geneticist/behaviorist) encouraged me to go on to graduate school. Through personal contact between Tavolga and Spieth I was introduced to Libbie Hyman at the American Museum of Natural History and Dawson and Spieth arranged that W. C. Allee (who, because of his age, had been forced to retire from the University of Chicago and was now heading the Zoology Department at the University of Florida (Gainesville, Florida)) accept me as a graduate student; Allee in turn arranged a research assistantship for me with the protozoologist James B. Lackey in the Department of Sanitary Engineering. My first independent research project involved a survey of oligotrich ciliates (protozoans) in the Gulf of Mexico. Just as I was becoming proficient with ciliate taxonomy and behavior I received a letter from the Selective Service Board—I was drafted into the United States Army. This interruption of my graduate studies turned out to be an opportunity to learn some parasitology and to travel the world at Uncle Sam’s Advances in Parasitology, Volume 67 ISSN 0065-308X, DOI: 10.1016/S0065-308X(08)00406-5
#
2008 Elsevier Ltd. All rights reserved.
49
50
Irwin W. Sherman
expense. After infantry basic training (Fort Dix, NJ) I was sent to Fort Sam Houston in Texas and then to Valley Forge Army Hospital in Pennsylvania to be trained as a medical technologist. I was then shipped overseas, where, through good fortune, I was assigned to medical laboratories in Salzburg, Austria and Darmstadt and Heidelberg, Germany. In Germany and Austria, I worked in clinical laboratories doing hematology, parasitology, microbiology, phlebotomy and blood chemistry. Upon my discharge from the United States Army (where by an act of Congress I received the exalted rank of Private First Class) I decided not to return to Florida to continue my graduate studies; instead I began to teach science and mathematics in a junior high school in Yonkers, NY. At the same time I took a few graduate courses at CCNY and assisted in the teaching of the evening session introductory biology laboratories at CCNY. Knowing of my interests in research, William Etkin, an endocrinologist/behaviorist and a professor at CCNY, encouraged me to return to graduate school on a full-time basis. He said, ‘If you want to run with the hounds and do some original research get yourself an advanced degree.’ It was sage advice. During the summer of 1957, thanks to a CCNY Biology Club Scholarship, I took the invertebrate zoology course at the Marine Biological Laboratory (MBL) in Woods Hole (Massachusetts). At the MBL I was able to spend all day, every day, for 10 weeks studying live invertebrates. The field trips were extraordinary, especially for a South Bronx slum kid, and the faculty was exceptional: Clark Read (Rice University, Houston, Texas), Howard Schneiderman (Case Western Reserve University, Cleveland, Ohio), John Buck (National Institutes of Health (NIH), Bethesda, Maryland), Theodore Bullock (University of California, Los Angeles, UCLA), Grover Stephens (Minnesota) and Ralph Smith (University of California, Berkeley, California). At the end of that summer I was invited to be the laboratory assistant for the course. I readily accepted and for the next two summers shared the work with another young parasitologist, Frank Friedl, from the University of Minnesota. The experiences in the United States Army and the MBL crystallized my interests in combining protozoology with clinical disease. I scoured university catalogues for those offering the greatest number of courses in parasitology and eventually settled on Northwestern University (or more accurately, they settled on me). I was awarded an Abbott Laboratory Fellowship with Robert Hull (a student of the eminent protozoologist R. R. Kudo). Hull (who had little training either in parasitology or biochemistry) had an NIH grant and together with a graduate student (Father Truong, a Vietnamese priest) they had begun a malaria project with Plasmodium lophurae and chickens. When I was at the start of my graduate studies very few laboratories in the United States were working on malaria biochemistry and those who
Malaria Pigment
51
had done so previously (see p. 12 ) were carrying out biochemical research on other subjects. Hull’s laboratory had minimal instrumentation: there was an ancient Warburg manometer, an assortment of low-speed centrifuges, a Klett colorimeter, a DU spectrophotometer and a paper electrophoresis apparatus but there was no possibility for working with radioisotopes. Hull, who did no bench work, appeared not to be very knowledgeable about malaria and was more interested in his favorite organism the suctorian, Tokophrya, so no specific malaria project was assigned to me. It soon became apparent that if I wanted to study the biochemistry of malaria I would have to do it on my own by learning more about the parasite and then I would have to find mentors who could teach me biochemistry. My first year as a graduate student was spent reading the literature and taking notes. Hemozoin, the brown–black pigment found within organs such as the spleen and liver as well as in erythrocytes of those infected with malaria, has long been a source of fascination for students of the disease (Sullivan, 2002a). Laveran’s finding (1880) of pigmented bodies in the blood of a soldier suffering with fever led to his discovery of the malarial parasite (see p. 3) and when Ross examined the stomachs of mosquitoes that had fed on a malaria patient and found ‘each of these bodies contained . . . granules of black pigment absolutely identical in appearance . . . with the . . . characteristic pigment of the parasite of malaria’ it provided the critical clue to mosquitoes as vectors of the disease (see p. 5). At Northwestern University, during my time as a graduate student, P. lophurae was maintained in chickens by intravenous inoculation. The method is rather simple, however, the amount of blood obtained from chicks (relative to ducklings) is small (5–10 ml), and because of age immunity the animals would not provide sufficient numbers of parasites when they were older than 5–6 weeks and weighed more than 200 g. As a result, most of my time during the first year involved passing the infection by blood inoculation every 3–4 days, measuring blood volumes, determining whether there were statistically significant differences between blood smears and trying to block natural immunity using carbon ink. By taking courses in biochemistry (with Lazlo Lorand) and biophysics (with Irving Klotz) I became more familiar with biochemical techniques and approaches. I first became curious about the composition and mode of formation of hemozoin when I peered through a microscope to examine a blood film taken from a chicken infected with P. lophurae and saw the parasites filled with refractile golden-brown granules. As a consequence, one of my doctoral thesis projects became a characterization of P. lophurae hemozoin. In 1891, Carbone reported that hemozoin had spectral properties similar to the iron-containing heme of hemoglobin and not melanin as had been proposed by Meckel (1847). Brown (1911) rediscovered this
52
Irwin W. Sherman
work and confirmed that it was hematin or ferriprotoporphyrin (FP). When Brown found intravenous injections of hematin produced a malaria-like paroxysm he suggested the pigment was involved in the production of the febrile attack. Later studies were consistent with FP being a constituent of hemozoin, however, there was other work— particularly that of Deegan and Maegraith (1956 a,b) at the Liverpool School of Tropical Medicine (LSTM, Liverpool, United Kingdom)—that indicated malaria pigment was more than monomers of FP. Deegan and Maegraith’s reasoning was that hemozoin could not be hematin since the latter would inhibit parasite succinoxidase, an enzyme vital to parasite survival. They proposed that vigorous extraction procedures (used by others) had cleaved the heme from its associated protein and by using milder extraction procedures found evidence for hemozoin being heme plus a proteinaceous moiety. Enlisting the support of Professors Lorand and Klotz, I was able to characterize malaria pigment spectrophotometrically and was able to carry out studies employing analytical ultracentrifugation (Sherman and Hull, 1960). The conclusion from this work with P. lophurae reaffirmed the work of Deegan and Maegraith. Years later (when newer biochemical/immunological methods were available), I once again became interested in malaria pigment (Yamada and Sherman, 1979). Together with a graduate student, Kenneth Yamada, we isolated hemozoin: erythrocyte-free parasites were homogenized using a French pressure cell; the suspension was centrifuged, the pellet recovered, sonicated and ultracentrifuged through a 1.7-M sucrose cushion. Using the techniques of peptide mapping, polyacrylamide gel electrophoresis (PAGE), gel (Sephadex) chromatography, as well as immuno-double diffusion we found hemozoin to consist of a complex of monomers and dimers of hematin, a 14-kDa protein, a 21-kDa protein (presumed to be of parasite origin coupled to heme) and methemoglobin. Electron microscopy of the purified hemozoin showed identity to that seen in sections of intact parasites. Supporting such work were reports by Ashong et al. (1989) who claimed protein was associated with FP, and the findings of Goldie et al. (1990) that hemozoin was a mixture of native and denatured globin. However, others claimed the non-covalently bound proteins resulted from contaminants (Fitch and Kanjananggulpan, 1987; Homewood et al., 1975) and when these were removed the pigment was identical to b-hematin. Even analyses by sophisticated biophysical methods did not completely clarify the nature of hemozoin: Morselt et al. (1973) using microspectrophotometry on intact cells found the pigment to differ from pure hematin, whereas the work of Bohle et al. (1997), Wood et al. (2003) and Pagola et al. (2000) found hemozoin to be identical with a cyclic dimer of b-hematin. Once hemozoin was equated with b-hematin subsequent investigations concerned themselves with its mode of formation.
Malaria Pigment
53
The literature is now replete with methods to synthesize b-hematin in the laboratory (Fitch, 2004; Sullivan, 2002a). Slater et al. (1991) postulated that hemozoin formation involved the ‘enzymatic polymerization of heme into a non-toxic crystal’ (i.e. b-hematin), however, later studies showed that a ‘heme polymerase’ did not exist and FP conversion to b-hematin could occur spontaneously in the absence of biological materials with solutions high in acetate concentrations and low in pH, but to increase the rate of formation the temperature had to be raised to 60 C (Egan et al., 2000, 2001). However, formation of b-hematin did not occur under physiological conditions or at pH 5 with preparations from uninfected red cells; it did form at pH 5 with preparations from infected erythrocytes. A more recent study has shown that under acid conditions b-hematin assembles rapidly and spontaneously near long-chain alcohol/ water and lipid/water interfaces (Egan et al., 2006). However, the morphology of the dimers is not identical to natural hemozoin. Although a considerable body of work describing the role of b-hematin in immunological and pathological events now exists (see below) I have continued to believe hemozoin is not identical to b-hematin. Indeed, many of the discrepancies regarding the chemical nature of hemozoin do not reflect the analytical techniques used, but largely depend on the manner by which the pigment is isolated and purified. For example, if hemozoin is isolated by physical means (mechanical breakage of cells or isolated food vacuoles (FVs) containing the pigment granules followed by density-gradient centrifugation) and the colored material is solubilized in alkali (0.1 N NaOH) the absorption spectrum is consistent with heme coupled to protein; further, as noted above immunological and chemical analysis of the associated protein (proteins?) has shown it to be different from native globin. However, when hemozoin is isolated by extensive proteolytic digestion and lipid extraction then the resultant product consists of crystals of b-hematin. Hemozoins from various plasmodia, when examined by electron microscopy (see Noland et al., 2003; Scheibel and Sherman, 1988) are so variable in morphology it is difficult to conceive of hemozoin as being pure crystals of b-hematin. This notion of lack of equivalence of b-hematin and hemozoin has received support from two recent studies. Parroche et al. (2007) found malarial deoxyribonucleic acid (DNA) to be a component of hemozoin when isolated using a strong magnetic field; in addition there was a ‘ladder of Coomasie -stained proteins.’ In another study when hemozoin, prepared from disrupted P. falciparum, was ultracentrifuged through a 1.7-M sucrose cushion (as we had done previously with P. lophurae) a paucity of proteins was found, however, the pigment granules were enveloped by neutral lipid (NL) nanospheres (Pisciotta et al., 2007). What roles does hemozoin play and how is it made? Coy Fitch began to study hemozoin in the late 1960s when he was at Walter Reed Army
54
Irwin W. Sherman
Institute of Research (WRAIR, Washington, DC). After finding that a chloroquine-resistant strain of P. berghei was deficient in a high-affinity receptor for the drug (Fitch, 1969, 1970), he went on to identify FP as the receptor (Chou et al., 1980; Fitch et al., 1974); later he found hemozoin to be dimerized FP similar to b-hematin (Fitch and Kanjananggulpan, 1987). Recently, he speculated that unsaturated fatty acids (FAs) and their mono- and diglycerides in the FV serve to concentrate the monomeric FP and keep it in a state favourable for dimerization (Fitch et al., 2003; Fitch and Russell, 2006). Coy Fitch (1934– ) as a medical student at the University of Arkansas School of Medicine (Little Rock, Arkansas), became interested in biochemistry and was provided research space and other resources. He earned an master of science (MS) in biochemistry as well as a doctor of medicine (MD) in 1958 and remained at the School of Medicine as a resident in medicine and Russell M. Wilder-National Vitamin Foundation Fellow in biochemistry (1958–1962). By the time the residency was completed he had developed an interest in membrane transport processes, and he remained on the faculty of the University of Arkansas as an assistant professor of medicine and biochemistry studying such. Five years later, he moved to the Saint Louis University School of Medicine (Saint Louis, Missouri) as Associate Professor of Internal Medicine and Biochemistry. That move made him vulnerable to military service. As a consequence, he was promptly drafted into the Army and assigned to the Division of Biochemistry of the WRAIR. The director of the Division of Biochemistry at WRAIR allowed Coy to have an independent laboratory but stipulated that it had to be devoted to malaria research. He decided to study the biochemistry of chloroquine (CQ) accumulation in erythrocytes infected with malaria parasites. This decision was based on a recent report that mouse erythrocytes infected with a CQ-susceptible strain of Plasmodium berghei accumulated more CQ than erythrocytes infected with a CQresistant strain. He thought he would be studying a membrane transport process; however, he soon realized that the movement of chloroquine across biological membranes was far too fast to measure with the centrifugation techniques available for his use. Nevertheless, by the end of his Army career (1967–1969), he had learned that CQ-resistant malaria parasites are deficient in a high-affinity receptor (target) for the drug. Returning to Saint Louis, Coy continued to study CQ accumulation by malaria parasites, focusing on the receptor. In 1974, he and his coworkers were able to describe the specificity and affinity of the binding site, so the receptor could be recognized when isolated. After
Malaria Pigment
55
describing the high-affinity binding site, several years were spent searching for a high-affinity protein receptor for CQ in malaria parasites, only to find none. In 1980, his group discovered that ferriprotoporphyrin IX (FP) had the specificity and affinity characteristics of the CQ receptor as had been suspected earlier by Macomber, Sprinz and Tousimis (Macomber and Sprinz, 1967). His group also found that FP and its complex with CQ are toxic for erythrocytes and malaria parasites (Banyal and Fitch, 1982; Fitch et al., 1982). These observations led Fitch and co-workers to propose that FP mediates the chemotherapeutic action of CQ and to ask how malaria parasites escape FP toxicity while ingesting and metabolizing hemoglobin. Believing the lack of toxicity to result from FP sequestration in hemozoin, they decided to characterize the pigment of hemozoin better. This pigment appeared to be an aggregate of FP that was similar, perhaps identical, to b-hematin (Fitch and Kanjananggulpan, 1987). Several years later, bhematin was found to be aggregated dimers of FP (Pagola et al., 2000). Since FP is rendered non-toxic by converting it to b-hematin, it was now logical to study this process. In 1992, Chou and Fitch discovered that FP dimerization could be measured easily and that CQ treatment reduces FP dimerization in vivo. At first, Chou and Fitch and others (Slater and Cerami, 1992) assumed that an enzyme catalysed the reaction; however, that assumption was wrong. Instead, it was found that the reaction is catalysed in malaria parasites by unsaturated FAs, probably predominantly linoleic acid. Furthermore, Fitch and co-workers (2003) obtained evidence that CQ treatment causes this catalyst to be masked (i.e. unavailable to catalyse FP dimerization) in CQ-susceptible malaria parasites, thus explaining the accumulation of toxic, undimerized FP in response to CQ treatment. The next task was to explain why unsaturated FAs are masked after CQ treatment. Based on their findings, Fitch and Russell (2006) proposed that the CQ–FP complex inhibits endosomal maturation and, thereby, reduces the release of unsaturated FAs from the membranes of erythrocytoid bodies. These bodies are found in endosomes early in the feeding process of malaria parasites. This explains, Coy believes, how CQ treatment results in masking of unsaturated FAs, reduces FP dimerization, causes toxic undimerized FP to accumulate and be available to bind CQ, and also causes denatured hemoglobin to accumulate in vesicles in malaria parasites (Fitch et al., 2003; Macomber and Sprinz, 1967; Warhurst and Hockley, 1967b). In this scenario, the toxic, undimerized FP that binds CQ and mediates its chemotherapeutic effect is derived from hemoglobin as it is denatured or hydrolyzed in immature endosomes. Currently, Coy is devoting his time to determining how the CQ–FP complex inhibits endosomal maturation.
56
Irwin W. Sherman
Fitch’s work on b-hematin synthesis (Fitch, 2004; Fitch et al., 1999; 2000) has recently been extended by Pisciotta et al. (2007). Hemozoin, isolated from saponin-purified P. falciparum trophozoites, followed by osmotic lysis and sonication and sedimentation through a sucrose cushion, was associated with lipid nanospheres consisting mainly of saturated lipids monostearic glycerol (MSG) and monopalmitic glycerol (MPG) with the principal diacylglycerols (DAGs) being distearic glycerol and 1-stearic-3-palmitic glycerol; no monooleic glycerol (MOG) or monolinoleic glycerol (MLG) was found. The source of these may be the neutral lipid bodies (NLBs) containing triacylglycerol (TAG) and DAG, which are associated with the FVs ( Jackson et al., 2004). The scenario proposed is: the double-membrane vesicle produced during cytostomal feeding (see p. 171) has its outer membrane fused with the FV membrane while its inner membrane is degraded by phospholipase C and/or lysosomal acid lipase (and found in the falciparum genome); the breakdown products are assembled into TAG and its precursors serve as promoters of b-hematin formation. Although Fitch et al. (1999) found TAG to be inactive in b-hematin formation Jackson et al. (2004) found MOG as well as mono- and dimyristoyl glycerol to be effective. In contrast, Pisciotta et al. (2007) found MPG to be a potent promoter of heme crystallization as was the combination of 1-stearic3-palmitic glycerol. A NL blend of MPG/MSG/dipalmitic glycerol (DPG)/ dioleic glycerol (DOG)/dilinoleic glycerol (DLG) (2:4:1:1:1) produced heme crystals rapidly. Of some interest is their observation, ‘the lipid blend crystals did not exactly replicate hemozoin made by P. falciparum (and) may require the presence of non-specific proteins or other molecular species.’ What is the role of hemozoin? Due to the massive degradation of hemoglobin a large amount of free heme is produced. Because free heme may intercalate with plasmodial membranes to lyse parasites and plasmodia lack the enzymes necessary to degrade the porphyrin ring the incorporation of the heme moieties into insoluble hemozoin has been postulated to be a detoxification mechanism (Fitch and Chevli, 1981; Fitch et al., 1982). Other postulates are peroxidative degradation within the FV (Loria et al., 1999) and glutathione (GSH)-mediated heme degradation in the cytosol (Ginsburg et al., 1998). Hemozoin (in the form of a- and b-hematin) has been reported to suppress erythropoiesis (Casals-Pascual et al., 2006), and after endothelial cell ingestion suppresses ICAM-1 and PECAM-1 expression, as well as production of interleukin (IL)-6 (Taramelli et al., 1998), and it can lyse red blood cells by incorporation into the membrane (Omodeo-Sale et al., 2005). In its native form, hemozoin has been shown to inhibit the function and maturation of dendritic cells (Millington et al., 2006; Skorokhod et al., 2004; Urban and Todryk, 2006) and to enhance matrix metalloproteinase activity as well as tumor necrosis factor (TNF)-a production in monocytes (Prato
Malaria Pigment
57
et al., 2005). Murine phagocytes fed natural hemozoin or FP plus supplemental interferon (IFN)-g increased their expression of nitric oxide (NO) synthase and NO (Jaramillo et al., 2003), however, human monocytes were neither able to generate NO nor to increase expression of NO synthase under the same conditions even with IFN-g (Skorokhod et al., 2007b). Further, there was little evidence of NO killing P. falciparum in vivo and in a Papua New Guinea study no correlation between NO levels and parasitemia was found in human subjects (Boutlis et al., 2004). Clearly, extrapolation of murine data to human malaria may not be justified and caution should be exercised when doing so. Others report that b-hematin does not promote such activities directly, but that it is the heme-catalysed peroxidation of cellular debris that results in a toxic gradient of primary and secondary FA oxidation products (15hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (HNE)) that not only activates the innate immune response (Carney et al., 2006) the HNE-protein adducts but also acts to decrease red cell deformability (Skorokhod et al., 2007a), promotes their removal and leads to anaemia. Coban et al. (2005) contend that b-hematin and hemozoin (prepared from a sonicated pellet of parasites, treated with proteinase K, washed in sodium dodecylsulphate (SDS), incubated with 6-M urea) on their own activate the toll-like receptor (TLR)-9 to mediate immune activation. By contrast, Parroche et al. (2007) report that b-hematin is immunologically inert, whereas natural hemozoin (isolated as described above) activates the TLR. Further, Parroche et al. have provided evidence that the natural pigment acts as a carrier for malarial DNA and that this results in it being targeted to TLR-9. (Malarial DNA on its own was found not to be immunostimulatory.) Although TLR-9 is known to recognize GC-rich areas that are typically found in bacterial DNA, and falciparum DNA is exceedingly AT-rich, there are small GC-rich regions in plasmodial DNA that are able to stimulate TLR-9. Clearly, the varied and inconsistent pathological and immunological findings result from a lack of equivalence of the ‘malaria pigment’ used. In my view, despite more than half a century of biochemical research on malaria pigment, much is yet to be learned about its natural composition and its varied roles. Sometimes there is a great temptation on the part of investigators to use a laboratory-synthesized compound that is easy to prepare and mimics the natural product, but extrapolation of the activities of a synthetic product such as b-hematin with natural hemozoin can be misleading. In addition, although b-hematin may have some utility in describing the relationships between different types of anti-malarials (Basilico et al., 1998; Egan and Ncokazi, 2005; Ncokazi and Egan, 2005; Solomonov et al., 2007; Trang et al., 2006) to conclude that the mimic and the authentic malaria pigment are identical is more often than not without adequate justification.