Erythrocytic Changes

Biology of the Cell, 64 (1988) 173-181 © Elsevier, Paris

173

Trypanosomes and Plasmodium

Morphological changes in erythrocytes induced by malarial parasites

Masamichi AIKAWA

Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA

Host cell alterations induced by Plasmodium falciparum, P. brasi;ianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoeleetrun microscopy. P. falciparum infection induces knobs, electron- dense material and clefts in the erythroeyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythroeyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasiUanum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasUianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces eaveola-vesiele complexes and clefts in the erythroeyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythroeyte membrane. P. malariae induces eaveolae, electrun-dense material, vesicles, clefts and knobs in the erythroeyte. Although vesicles and caveolae are seen in the erythroeyte cytoplasm, they do not form caveola-vesiele complexes as seen in P. vivax.infeeted erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host ceil changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not dear.

erythroeyte - - malarial parasites - - host cell alterations

INTRODUCTION

The erythrocytic stages of malarial parasites induce morphological and functional changes in infected erythrocytes. Such changes have been recognized by light microscopy for a long time and have been called by various names such as Schuffner's dots, Maurer's clefts. Ziemann's stipplings and Stinton's and Mulligan's stipplings. These host cell alterations appear to relate to the capability of malarial parasites to alter the properties of the erythrocyte and its membrane in order to export malarial proteins from the parasite to the host erythrocyte membrane. Although the significance of such changes is not clear, some of these alterations in host cells appear to be involved in the development of malarial-related complications in the host. For example, the development of cerebral malaria seems to relate to blockage of cerebral capillaries which is induced by the presence of knobs on P. falciparum-infected erythrocytes [I, 16].

In this chapter, we describe by electron microscopy the host cell alterations induced by P. falciparum, P. brasilianum, P. vivax and P. malariae infections; and by immunoelectron microscopy, the trafficking of these malarial proteins from the parasite to the erythrocyte membrane. Such studies may lead to a better understanding of the pathways whereby malarial proteins are export- ed from the parasite to the host erythrocyte membrane. Furthermore, some complications occurring in malaria infections may be better understood by understanding host cell changes induced by malarial parasites.

Erythrocyte changes induced by P. faleiparum

Piasmodium falciparum infection induces several morphological, antigenic and functional changes of the i,- fected erythrocyte and its membrane. These changes include: 1) knob-like protrusions of the host cell membrane; 2)clefts in the erythrocyte cytoplasm; and 3) electron-dense material (EDM) in the erythrocyte cytoplasm [4, 11].

The knobs consist of protrusions of the erythrocyte membrane with electron-dense material below the membrane, and measure 30 to 40 nm in height and 90 to 100 nm in width (Fig. 1). Scanning electron microscopy shows that knobs are distributed evenly over the erythrocyte membrane [4].

When brain tissues from patients with cerebral malaria were examined by light microscopy, cerebral capillaries were seen to be filled with parasitized erythrocytes mixed with uninfected erythrocytes (Fig. 2). Electron microscopy demonstrated multiple electron-dense knobs protruding from the membrane of the infected erythrocytes in capillaries. These electron-dense knobs form focal junctions with the endothelial cells and adjacent erythrocytes, causing blockage of cerebral capillaries (Fig. 3). Blockage of cerebral capillaries by P. falciparum-infected erythrocytes appears to be the principal causative agent of the pathology of cerebral malaria [16].

Electron-dense material (EDM) which appears within the infected cell seems to be associated with the formation of knobs [6]. EDM is associated with the trophozoite

174 M. Aikawa

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FIGURE 1, - Electron micrograph of knobs (K) protruding from the membrane of an erythrocyte infected with P. falciparum, x 75,000

FIGURE 2, - Light micrograph of cerebral capillaries blocked with parasitized erythrocytes. The brain tissue was from a patient with cerebral malaria.

FIGURE 3, - Electron micrograph of knobs (K) forming focal junctions with the endothelial cell (E) of a cerebral capillary. × 60,000.

membrane of several strains of P. falciparum cultured in vitro (Fig. 4). The EDM is also seen associated with unit membrane-bounded Maurer's clefts in K + P. falciparum- infected erythrocytes. This EDM has the same density and appearance as the material located under knobs at the erythrocyte membrane. Immunoelectron microscopy has shown the association of ankryin with the membrane of the clefts [8]. The presence of ankyrin on the membrane of clefts indicates that some clefts are associated with the erythrocyte membrane, since ankyrin is known to be present on the erythrocyte cytoskeleton. Thus, the parasite- derived EDM appears to be transported from the parasite

plasmalemma to the erythrocyte membrane via Maurer 's clefts in the erythrocyte cytoplasm.

Since Kilejian [15] first reported histidine-rich knob- associated protein in 1979, many investigators have described the expression and gene sequence of this molecule. Because the presence of histidine-rich proteins in knobs appears to be an important factor in knob formation, we investigated whether or not a histidine analogue, 2-fluoro-L-histidine, might affect the formation of knobs [7]. When the parasites were cultivated in the presence of 2-fluoro-L-histidine, the amount of EDM in the parasitophorous vacuole increased. We also noted that

Erythrocytes induced by malarial parasites 175

FIGURE 4. - Electron micrograph of EDM associated with a P. falciparum-infected erythrocyte, x 65,000.

FIGURE 5. - - Electron micrograph of EDM which shows increased density in the presence of 2-fluoro-L-histidine. x 60,000.

the density of this EDM increased with increasing levels of 2-fluoro-L-histidine (Fig. 5). 2-fluoro-L-histidine also increased the amount and density of EDM associated with Maurer's clefts in the host erythrocyte cytoplasm. As the concentration of 2-fiuoro-L-histidine increased, the number of infected cells having knobs and the number of knobs per cell both decreased. Thus, this histidine analogue blocks export of EDM from the parasite to knobs and thereby inhibits knob formation.

At least 4 malarial proteins (HRP 1, HRP2, EMP 1 and EMP2) have been identified in the surface of P. falciparum.infected erythrocytes (HRP: histidine-rich protein; EMP: erythrocyte membrane protein) [12]. Three of these proteins, HRPI, EMP1 and EMP2 are localized in knobs, as demonstrated by immunoelectron microscopy. Since knobs appear to play an important role in cerebral malaria, we performed immuno.cytochemistry on brain tissue from cerebral malaria patients using monoclonals specific to HRP1. Strong positive staining of HRPI was visible along cerebral capillaries packed with parasitized erythrocytes [12]. This finding may suggest that the knob proteins deposit in the brain of cerebral malaria patients. The mechanism of deposition of knob proteins in the vascular endothelium of the cerebral capillaries is unclear. One possibility is that knob proteins transfer to the cerebral capillary walls by endocytosis. Alternatively, since portions of the infected erythrocyte membrane containing knobs may bud off, the knob antigens may be present in the peripheral circulation and may be taken up by the capillary endothelium. The effects of the deposition of knob proteins in the cerebral capillaries of P. falciparum-infected patient are unknown. In addition, IgG, IgM and P. falciparum antigens are found along the walls of the capillaries [16]. The identification of the knob protein and immunoglobulins in the wall of the cerebral capillaries may indicate the involvement of immune mechanisms in cerebral malaria.

Recently, scientists in several laboratories have investigated host cell molecules such as OKM5 [10] and thrombospondin [17] that may function as the endothelial cell surface receptors for P. falciparum-infected erythrocytes. They could independently or interacting together, play a role in cytoadherence of knobs in vitro. However, there have been no reports as to whether these molecules actually play a role in cytoadherence of P. falciparum-infected erythrocytes in rive. In order to test this theory, we tried to demonstrate the presence of OKM5 antigen and thrombospondin in the endothelial cells of cerebral capillaries of uninfected autopsy specimens from the USA. Although we were able to demonstrate thrombospondin in the endothelial cells of human cerebral capillaries by a PAP method, no OKM5 antigen could be demonstrated in ~.he endothelial cells of human cerebral capillaries [1]. This indicates that, in man, OKM5 antigen may not be the cerebral receptor for erythrocytes infected with P. faiciparum. Alternatively, it is possible that some immunological or pathological factors influence the presence or absence of OKM5 antigen on the endothelial cells of cerebral capillaries in man. Patients who express OKM5 antigen on their cerebral capillaries may develop cerebral malaria and those who do not may not develop cerebral disease. Further studies are needed to clarify this hypothesis.

Erythrocyte changes induced by P. brasilianum infection

Erythrocyte changes include the formation of knobs on the infected erythrocyte membrane and the formation of cytoplasmic clefts [5, 18]. Knobs are cone-shaped and measure 45 nm in height and 100 nm in width (Fig. 6). They are bounded by the plasmalemma and possess and electron-dense matrix. The cytoplasmic clefts are

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membrane-bounded and can be divided into 3 types, namely short clefts, long clefts and circular clefts. The short clefts are slit-like structures with lengths of 0•3-0.8 ~m and are scattered randomly in the cytoplasm of the infected erythrocytes. Most long clefts are slighty curved or undulating and are 3-5/~m long• Some of the long clefts are connected with the parasitophorous vacuole membrane. The circular clefts form oblong loops in the erythrocyte cytoplasm and have a long axis of 1 •5-2.3 ~m.

Recently Cochrane et al. (unpublished) developed a series of monoclonal antibodies (MAbs) against blood stages of a Colombian strain of P. brasilianum. Im- munoelectron microscopy demonstrated that these MAbs react with knobs and clefts which appear in the infected erythrocytes.

MAbs which recognize an antigen with an apparent molecular weight of 38 kDa as determined by Western blot

of an extract of infected red blood cells (IRBCs) reacted with short clefts (Fig. 7). Gold particles appear over short clefts located in the cytoplasm of RBCs infected with asex- ual stages as well as gametocytes. These gold particles are not associated with long clefts, reflecting the specificity of binding of these MAbs to short cleft antigen• MAbs which recognize an antigen with an apparent molecular weight of 16 kDa as determined by Western blot of an extract of IRBCs reacted with long clefts. By immunoelectron microscopy dense accumulation of gold particles appears over long clefts (Fig. 8) in the cytoplasm of erythrocytes and also in association with the parasitophorous vacuolar membrane and/or space (Fig. 9). In some specimens, the long clefts appear to be almost continuous with both the parasitophorous vacuole and IRBC membranes. The MAbs also react with circular shaped clefts.

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FIGURE 6 . - Immunoelectron micrograph of knobs (K) on the membrane of an erythrocyte infected with P. brasilianum. Gold particles (arrow) which identified the presence of 14, 16 and 19 kDa malaria proteins are seen under knob protrusions. × 55,000.

FIGURE 7 . - Immunoelectron micrograph of a short cleft (S). Gold particles which identify a 38 kDa protein are associated with the short cleft, x 35,000.

FlouR~ 8. - lmmunoelectron micrograph of a long cleft ft..). Gold particles which identify a 16 kDa protein are seen the long cleft. × 30,000.


MAb l lH9.D9 immunoprecipitated a triplet of low molecular weight antigens (14, 16 and 19 kDa) from an SDS-soluble extract of parasites metabolically labeled with ~S-methionine. Using the MAb 11H9. D9, we observed, by immunoelectron microscopy, a dense accumulation of gold particles under knob protrusions of the IRBC membrane (Fig. 6). In no instance did gold particles appear exterior to the IRBC membrane. In addition, gold particles appear over micronemes in the budding merozoites.

The difference in antigenic composition of short and long clefts has not been previously described for any plasmodial species. The fact that the long cleft antigen was associated with the parasitophorous vacuole, whereas the

short cleft antigen was not, suggests that a different mechanism might be operating for incorporation of the 2 antigens into their respective clefts. It has been suggested that Maurer's clefts function in the transport of membrane-associated knob materials, as demonstrated in P. falciparum-infected erythrocytes. However, cleft and knob antigens of P. brasilianum are immunologically distinct. It is possible that the nature of antigens being transported via clefts varies with the stage of parasite development and that early in the parasite life cycle, knob proteins are principally transported. At a later stage of development, the antigenic composition of the clefts might change.

The relationship between knob proteins of P. bra-

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FIGURE 9. - Immunoelectron micrograph showing dense accumulation of gold particles in the cytoplasm of P. brasilianum-infected ery~hrocytes-'.~d in the parasitophorous vacuole space, x 30,000.

178 M. Aikawa

silianum and P. falciparum remains unclear. We have as yet been unable to demonstrate the association of histidine-rich protein with knobs of P. brasilianum, In the case of P. falciparum the sequestration of the IRBCs oc- curs due to the formation of focal junctions between the knobs and endothelial cells. However, the function of knobs on red blood cells infected with P. brasilianum is not clear. Since all stages of P. brasilianum development

are found in the peripheral circulation, it has been suggested that P. brasilianum parasitized red blood cells are not sequestered [18]. However a precise quantitative study done under controlled conditions in a susceptible host has not yet been undertaken. Further studies may be required to determine the function of the knobs which appear on the membrane of erythrocytes infected with P. brasilianum.

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FIGURE 10. - lmmunoelectron micrograph of P. vivax.infected erythrocyte. Gold particles which show the presence of 95 kDa malaria protein are associated with vesicles (arrow) of the caveola-vesicle complexes, x 65,000.

FIGURE 11• - A double labeling technique to demonstrate the 28 kDa and 95 kDa antigens in the same erythrocyte. The small particles (arrow) detect the 95 kDa antigenic sites, while the large particles (arrowhead) localize the 28 kDa antigenic sites• x 63,000.

FIGURE 1 2 . - - lmmunoelectron micrograph showing gold particles along the cleft. This indicates the presence of the 28 kDa antigenic sites in the cleft, x 52,000.


Erythrocyte changes induced by P. vivax

Erythrocytes infected with P. vivax-type malaria parasites are characterized by Schuffner's dots. This character appears as multiple small brick-red dots in Giemsa-stained thin films. In a study by electron microscopy, Aikawa et al. [3], described caveola-vesicle complexes (CVC) scattered along the membrane of P. vivax-infected erythrocytes. This structure consists of caveolae to which vesicles are attached in an alveolar fashion. The size and distribution of these structures are consistent with Schuff- her's dots observed on Giemsa-stained thin films. Vesicles are also scattered in the cytoplasm of the infected erythrocytes. Another host cell alteration caused by P. vivax infection is that of cytoplasmic clefts observed within the infected erythrocyte cytoplasm. The function of CVC and cytoplasmic clefts, however, was not known umil the present time.

Recently, investigators have produced a series of monoclonals (MAbs) against various antigens of erythrocytic stages of P. vivax [15, 19]. Among them, MAbs which identified a 95 kDa biosynthetically-labeled 3SS-methionine P. vivax protein, produced a stippled pattern similar to Schuffner's dots in 1). vivax-infected erythrocytes and other MAbs which react with a 28 kDa protein biosynthetically-labeled with aSS-methionine gave a linear pattern by immunofluorescent microscopy (IFA).

To identify the precise location of P. vivax antigens which react with these MAbs, we performed post- embedding immunoelectron microscopy. MAbs against a 95 kDa biosynthetically-labeled 35S-methionine protein, produced a pattern similar to that of Schuffner's dots by IFA [15]. Immunoelectron microscopically, specific label was found to be associated with vesicles of the CVC, while only a few gold particles were associated with the caveolae (Fig. I0). Vesicles scatterd throughout the erythrocyte cytoplasm were also labeled with gold particles.

Other MAbs which react with a 28 kDa biosynthetically-labeled 3SS.methionine protein gave a linear pattern in the cytoplasm of infected erythrocytes by IFA. Ira. munoelectron microscopy clearly revealed the target an. tigen of these MAbs was located along the cytoplasmic clefts of infected erythrocytes (Fig. 12). Immunoreactivi- ty was also observed in association with vesicles scattered in the erythrocyte cytoplasm and vesicles of the CVC. There was no labeling of parasites and uninfected erythrocytes treated with these MAbs.

The double-labeling technique was applied for the demonstration of the 28 and 95 kDa antigens in the same erythrocytes (Fig. 11). The small gold particles allowed the detection of the 95 kDa antigenic sites, while the large gold particles localized the 28 kDa antigenic sites. Immunoelec- tron microscopy demonstrated that small size gold particles were only associated with vesicles, while most of large size gold particles were seen in clefts, but some large particles were found in association with vesicles together with small particles (Fig. 13). Thus, double-labeling con- firmed that the vesicles contained predominantly the 9S kDa antigen and some of the 28 kDa antigen, whereas clefts were associated with only the 28 kDa antigen.

The presence of P. vivax antigens in clefts and CVC indicates that these structures are related to trafficking of P. vivax antigen from the parasites to the erythrocyte surface membrane. Some of 1:). vivax proteins, at least the 28 kDa protein, are transported from the parasite to the

parasitophorous vacuole and to the clefts. The proteins are then transported along clefts and transferred to vesicles. The proteins move toward the erythrocyte surface and into the caveolar space. The proteins are then released from the caveola extracellularly. The vesicles might be formed by budding off from the tip of the cleft which consist of a set of lamellate clefts, similar to Golgi vesicles that are pinched off from the Golgi stack. On the other hand, a 95 kDa protein is only present within the vesicles and not in the clefts. This could indicate that the vesicles containing a 95 kDa protein may originate directe- ly from the parasitophorous vacuole membrane. These observations indicate that host cell changes induced by P. vivax are involved in trafficking of P. vivax antigens to the erythrocyte membrane [15, 19].

Erythrocyte changes induced by P. malariae

Plasmodium malariae infection induces several morphological changes. These include cytoplasmic clefts, knobs and caveolae along the erythrocyte membrane [9]. The caveolae that appear in P. malariae-infected erythrocytes lack the small alveolar-like vesicles of the caveola-vesicle complexes described in vivax- and ovale. type primate malarial parasites. Aikawa et al. suggested that the caveola-vesicle complexes of vivax. [3] and ovale. type [2] malarias correspond to the Schuffner's dots seen in Giemsa-stained blood smears. James [13] described a

similar stippling in 1). malariae-infected erythrocytes. This stippling is smaller and less distinct than Schuffner's dots. The caveolae may correspond to Ziemman's stippling. The surface of trophozoite-, shizont- and gametocyte-infected erythrocytes are modified into numerous, regularly spac- ed, electron-dense knobs (Fig. 13). They measure 46 nm in height and 80 nm in diameter at their base. Knobs are composed of electron-dense material (EDM) similar to that seen in knobs of P. faiciparum-infected erythrocytes [9]. EDM similar to that seen in P. falciparum.infected erythrocytes is commonly observed in the parasitophorous vacuole space and Maurer's clefts. Scattered randomly throughout the cytoplasm of infected erythrocytes are balls of EDM surrounded by an electron-lucent halo (Fig. 14). They are often associated with the parasitophorous vacuolar membrane next to the main body of trophozoites or along the Maurer's clefts. Small balls of erythrocytic EDM are also found immediately under the erythrocyte plasmalemma (Fig. 15) and appear to be associated with cup-like invaginations of the erythrocyte membrane. These invaginations appear to fuse with the EDM to form small, membrane-bounded caveolae that are continuous with the erythrocyte plasmalemma.

A second type of EDM is observed in the cytoplasm of infected erythrocytes. This EDM forms amorphous masses, without an associated halo and is associated with the external surface of Maurer's clefts as seen in the EDM of P. falciparum. Occasionally, this EDM forms electron- dense knobs which appear on Maurer's cleft membrane facing the cleft's lumen (Fig. 16). In P. falciparum-infected erythrocytes, movement of EDM from the parasitophorous vacuole to the erythrocyte membrane via the Maurer's clefts was suggested to provide a system of intracellular transport for the movement of knob-associated proteins to the erythrocyte membrane. The observations of internal knobs in the Maurer's clefts of P. malariae- infected erythrocytes suggests that some knobs may be formed before they appear on the erythrocyte membrane.

180 M. Aikawa

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FIGURE 13. - Electron micrograph showing electron dense knobs (K) on the surface of P. malariae.infected erythrocytes, x 56,000.

FIGURE 14. - Electron micrograph of P. malariae-infected erythrocytes. Balls (arrow) of EDM surrounded by a halo are seen in the cytoplasm, x 78,000.

FiGURe 15. - Small balls (arrow) of EDM are seen just beneath the erythrocyte plasma membrane, x78,000.

FIGURE 16. - Knobs (K) appear on the cleft membrane facing the cleft's lumen (L) in a erythrocyte infected with P. malariae. X 65,000.


Immunoelectron microscopy has not yet been performed on P. malariae. Such studies will expand our knowledge on the trafficking of P. malariae protein from the parasite to the host erythrocyte membrane.

ACKNOWLEDGEMENTS

This work was supported in part by grants from the US Agency for International Development (DPE-0453-A-00-4027), USPHS (AI-10645) and the UNDP/World Bank/WHO Special Pro- gramme for Research and Training in Tropical Diseases. I am grateful to Dl'~. Carter Atkinson and John Rabbege, who pro- vided helpful comments on the manuscripts and for the excellent technical assistance of Kiet Dan Luc and Ana Milosavljevic.

REFERENCES

I Aikawa M. (1988) Human cerebral malaria. Am. J. Trop. Med. Hyg. (in pres~)

2 Aikawa M., Hsieh C.L. & Miller L.H. (1977) Ultrastruc- rural changes of the erythrocyte membrane in ovale-type malarial parasites. J. Parasitol. 63, 152-154

3 Aikawa M., Miller L.H. & Rabbege J. (1975) Caveola-vesicle complexes in the plasmalemla of erythrocytes infected by Plasmodium vivax and P. cynomolsi: unique structures related to Schuffner's dots. Am. J. Pathol. 79, 285-300

4 Aikawa M., Rabbege J.R., Udeinya I. & Miller L.H. (1983) Electron microscopy of knobs in Plasmodium falciparum- infected erythrocytes. J. Parasitol. 69, 433-437

5 Aikawa M. & Sterling C.R. (1974) In: Intracellular Parasitic Protozoa, Academic Press, New York, p. 20

6 Aikawa M., Uni S,, Andrutis A.T. & Howard R.J. (1986) Membrane.associated electron-dense material of the asex. ual stases of Plasmodlum Salclparum: evidence for move. ment from the intracellular parasite to the erythrocyte membrane. Am. J. Trop. Ailed. Hys. 35, 30-36

7 Aikawa M., Uni S,, Andrutis A.T., Kirk K.L., Cohen L.A. & Howard R.J. (1987) Inhibition of knob formation of Plasmodlum falciparum.infected erythrocytes by 2.fluoro- L.histidine. In: Host.Parasite Cellular and Molecular Interactions in Protozoal Infections (J.P. Chang and D. Snary, eds.), NATO-ASI volume, Springer, New York, pp. 2097-3061

8 Atkinson C.T., Aikawa M., Perry G., Fujino T., Bennett V., Davidson E.A. & Howard R.J. (1988) Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium /alciparum-infected erythrocytes. Eur. J. Cell Biol.(in press)

9 Atkinson C.T., Aikawa M., Rock E.P., Marsh K., An- drysiak P.M., Campbell G.H., Collins W.E. & Howard R.J. (1987) Ultrastructure of the erythrocytic stages of Plasmodium malariae. J. Protozool. 34, 167-274

I0 Barnwell J.W., Ockenhouse C.F. & Knowles D.M. (1985) Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial and C32 melanoma cells. J. Im- munol. 135, 3494-3497

II Howard R.J., Uni S., Lyon J.A., Taylor D.W., Daniel W. & Aikawa M. (1987) Export of Plasmodium/alciparum proteins to the host erythrocyte membrane: special problems of protein trafficking and topogenesis. In: Host-Parasite Cellular and Molecular Interactions in Protozoal Infections (K.P. Chang and D. Snary, eds.), NATO-ASI volume, Springer, New York, p. 281-296

12 Igarashi I., Oo M.M., Stanley H., Reese R. & Aikawa M. (1987) Knob antigen deposition in cerebral malaria. Am. J. Trop. Med. Hyg. 37, 511-515

13 James S.P. (1929) A note on the Shute technique for staining malaria parasites with Leishman's stain and on the stippling in infected red blood corpuscles which it reveals. Trans. R. Soc. Trop. Med. Hyg. 23, 269-278

14 Kilegian A. (1979) Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Prec. Natl. Acad. ScL USA 76, 4650-4653

15 Matsumoto Y., Aikawa M. & Barnwell J.W. (1988) Im- munoelectron microscopic localization on vivax malaria antigens to the clefts and caveola-vesicle complexes of infected erythrocytes. Am. J. Trop. Med. Hyg. (in press)

16 Oo M.M., Aikawa M., Than T., Aye T.O.M., Myint P.T., lgarashi I. & Schoene W.C. (1987) Human cerebral malaria: a pathological study. J. NeuropathoL Exp. Neurol. 46, 223-231

17 Roberts D.D., Sherwood J.A., Spitalnik S.L., Panton L.J., Howard R.J., Dixit V.M., Fraziar W.A., Miller L.H. & Ginsburg V. (1985) Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 318, 64-66

18 Sterling C.R., Aikawa M. & Nussenzweig R.S. (1972) Mor- phological divergence in a mammalian malarial parasite: the fine structure of Piasmodlum brasiilanum. Prec. Helm. $oc. Wash. 39, 109-129

19 Udagama P.V., Atkinson C.T., Peiris J.S.M., David P., Mendis K.N. & Aikawa M. (1988) Immunoelectron microscopy of Schuffner's dots in Piasmodium vivax- infected human erythrocytes. Am. J. Pathoi. (in press)

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Erythrocytic Changes