- Email: [email protected]
Cultivation of Plasmodium vivax
Review
Cultivation of Plasmodium vivax Rachanee Udomsangpetch1, Osamu Kaneko2, Kesinee Chotivanich3 and Jetsumon Sattabongkot4 1
Department Department 3 Department 4 Department 2
of of of of
Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand Protozoology, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki, Japan Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Entomology, Armed Forces Research Institute of Medical Science, Bangkok, Thailand
Establishment of a continuous line of Plasmodium vivax parasite is crucial to understand the parasite’s biology; however, this has not yet been achieved. Beginning in the 19th century, there were several efforts to cultivate this malaria parasite but without much success until the late 1980s. In addition, to date, only minor modifications of the methodology have been investigated, which has resulted in extending the cultivation period to around four weeks by supplying reticulocytes obtained from normal blood or rare hemochromatotic blood. However, the use of laboratory-produced erythroblasts to cultivate P. vivax enables maintenance of a continuous line of the parasite stably in the laboratory. Here, we summarize and compare the available methodologies and conditions for the in vitro cultivation of P. vivax. Initial attempts at culturing blood-stage Plasmodium vivax Research focusing on Plasmodium vivax has been neglected for several decades because of the lack of a practical continuous cultivation system. Because Plasmodium falciparum infection causes more severe symptoms than all other Plasmodium species, most of the research effort has previously been directed to falciparum malaria, and a P. falciparum in vitro cultivation system was established in the 1970s. However, P. vivax cannot be maintained in vitro under the conditions developed to cultivate P. falciparum [1]. In addition, P. vivax preferentially invades young erythrocytes, which are difficult to obtain routinely. These major obstacles have limited the investigation of P. vivax malaria biology at both the cellular and molecular levels. Research into cultivation of malaria parasites started in 1891, and efforts to culture malaria were reported, without much progress, from 1893 to 1894 [2]. Thereafter, cultivation of various species of human malaria parasites was documented by several researchers, and, by 1912, the most successful method of culturing P. vivax involved maintaining the parasite for a few schizogonic cycles in vitro. In the 20th century, several techniques and modifications were established by different laboratories [3–5]. The goal of these researchers was to maintain a continuous line of P. vivax parasite in vitro, similar to what was established for P. falciparum by Trager and Jensen in 1976 [1].
Corresponding author: Udomsangpetch, R. ([email protected]).
Development of cultivation techniques Four types of cultivation media have been used for P. vivax cultivation: (i) RPMI-1640, (ii) Waymouth, (iii) McCoy’s 5A and (iv) Science Medical Mahidol 612 (SCMI 612) media [5–8]. Modified conditioned media based on three commercially available cultivation media have been tested with various, and varying amounts of, supplements in several attempts to cultivate P. vivax parasites [4–9] (Table 1). Metal ions and vitamins have been reported to promote differentiation of P. vivax parasites to merozoites during schizogony. The increment in parasite number is obtained from the addition of 1.8 mM magnesium chloride to the medium. Ascorbic acid is required for P. vivax schizogony and 3–6 mg ml 1 ascorbic acid in the medium enhances parasite growth [8]. Medium SCMI 612 supports 99% of ring stages through schizogony; however, this medium consists of 30 components and was thus considered impractical for routine use [7]. These studies observed parasite growth only in one ex vivo schizogonic cycle and no merozoite reinvasion was observed. A simple method for shortterm cultivation of P. vivax was reported recently, and involves modifying the standard in vitro cultivation medium for P. falciparum by supplementing RPMI-1640 with ascorbic acid, glucose, thiamine, hypoxanthine and 50% human serum. Most of the parasites develop to complete schizogony and can be maintained for up to six cycles without the need to supply erythrocytes [4]. In another study, when a 1:1 mixture of squirrel monkey and human erythrocytes was added to RPMI-1640-based medium, P. vivax was maintained for 11 cycles [9]. The medium was supplemented with different additives: pyruvate, spermidine, cholesterol, tocopherol, ascorbic acid and 15% human serum. In addition, they attempted to improve cultivation efficiency using a small modified version of a fluid flow vessel (3-ml capacity) filled with medium, supplements, erythrocytes and P. vivax, placed at 37 8C, and then gassed with a mixture of 3% CO2, 5% O2 and 92% N2. Unfortunately, this method caused parasitized-cell breakage and there were only a few successful invasions. The first method for semi-continuous cultivation of P. vivax was established in 1997 [5]. The parasites were cultivated in McCoy’s 5A medium supplemented with L-glutamine, 25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) and 20% human serum. Parasites were grown under static conditions until they developed into schizonts. The reticulocytes obtained from differential centrifugation of freshly obtained
1471-4922/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.09.010 Available online 3 January 2008
85
Review
Trends in Parasitology Vol.24 No.2
Table 1. Development of Plasmodium vivax cultivation conditions (1979–2007) Year 1979 1985
Cultivation media RPMI-1640 SCMI 612
1987
Waymouth:RPMI-1640 (1:2)
1992
RPMI-1640
1997
McCoy’s 5A
2001
RPMI-1640
2007
McCoy’s 5A
2007
McCoy’s 5A
Conditions Red blood cell (RBC) extracted fraction I MgCl2 0.75 mM, ascorbic acid 0.6 mg ml 1 39 8C in candle jar, 15% human AB serum MgCl2 1.8 mM, ascorbic acid 3 mg ml 1 38.5 8C in candle jar, 15% human AB serum Ascorbic acid 0.6 mg ml 1 37 8C in flow vessel, 15% human AB serum, 3% CO2 A mixture of monkey and human erythrocytes (1:1) Ascorbic acid 0.5 mg ml 1 37 8C, 20% human AB serum, 5% CO2 Reticulocytes from hemochromatotic blood Ascorbic acid 6 mg ml 1 37 8C, 50% human AB serum, 5% CO2 No addition of erythrocytes Ascorbic acid 0.5 mg ml 1 37 8C, 25% human AB serum, 5% CO2 Cord blood erythrocytes Ascorbic acid 0.5 mg ml 1 37 8C, 25% human AB serum, 5% CO2 Cultivated erythroblast
hemochromatotic blood were added to the cultivation to support merozoite invasion. The parasite cultivation mix was then transferred to a shaker for 10–12 h to facilitate rupture of the schizonts. This treatment achieved at least a twofold increase in parasite density at each schizogonic cycle. However, the method was limited by the need to obtain reticulocytes from rare hemochromatotic blood. The supply of young erythrocytes/reticulocytes is crucial for reinvasion and for maintaining parasitemia of a semi-continuous cultivation of P. vivax. Involvement of young erythrocytes in cultivation of P. vivax Various modifications have focused on essential nutrients and conditions of cultivation that are required for parasite multiplication, which seem to be different to those of P. falciparum. In addition, prolonged survival requires successful in vitro erythrocyte reinvasion by the parasites and the phenotype of the erythrocyte is crucial to their susceptibility. Different species of Plasmodium parasite display distinct host specificities [10]. Moreover, humaninfecting Plasmodium species display specific selection of the target erythrocytes; for example, P. falciparum invades all types of human erythrocytes, whereas P. vivax prefers younger erythrocytes [11]. This preference for younger erythrocytes is the crucial factor in the difficulty in establishing a continuous P. vivax line, compared to P. falciparum cultivation. Although it is theoretically possible to use human reticulocytes for P. vivax cultivation, the proportion of reticulocytes in the normal peripheral blood is too small to continuously supply cultivation (normal levels of reticulocytes range from 0.5% to 1.5% in adults). To overcome this problem, two reticulocyte sources have been reported [5,12]. Golenda et al. (1997) reported the use of hemochromatotic blood that could contain up to 7% reticulocytes [5], and Udomsangpetch et al. (2007) reported the use of umbilical cord blood collected at birth, which contains 1%–5% reticulocytes [12]. Using cord-blood erythrocytes, P. vivax parasites were able to be cultivated for up to 30–40 days after isolation [12]. Although the number of reticulocytes obtained is similar between these sources, the 86
Cultivation period (days) 4 2
Refs [6] [7]
2
[8]
22
[9]
Continuous
[5]
12
[4]
30–40
[15]
Continuous
[14]
number of P. vivax parasites maintained using cord-blood erythrocytes gradually declined and eventually disappeared, and thus a continuous line has not been established. This observation might be explained by the marked difference in hemoglobin type. Hemochromatotic reticulocytes contain hemoglobin A but cord-blood reticulocytes contain mainly hemoglobin F, and although P. falciparum is able to invade hemoglobin F-containing erythrocytes efficiently, it does not grow well inside them [13]. It is possible that P. vivax also does not grow well in hemoglobin F-containing reticulocytes. A new strategy for production of human erythrocytes has been attempted. The fact that P. vivax parasites successfully invade erythrocytes in the human body does not match observations from several in vitro trials: it is rare that merozoites successfully utilize the freshly collected healthy human erythrocytes. It is conceivable that the limiting requirement of the parasite to complete its life cycle is to find the new host cells, namely young erythrocytes. However, the stage of erythrocytic development and maturation preferred by merozoites is unknown. Our recent study began with the isolation of cord-blood stem cells and propagation of these cells towards hematopoietic stem cells under specific driving factors [14]. Growing erythrocytes, starting from erythroblasts, appeared after the first week of stimulation by various factors and were tested by co-cultivation with P. vivax schizonts. Differentiated erythroblastic stages at various stages of development, up to 21-day-old mature erythrocytes, were used in cultivation of P. vivax under in vitro conditions [12]. The earliest erythrocytes that enable P. vivax invasion are polychromatic erythroblasts (Figure 1) [14]. Furthermore, we found that older erythroblasts are able to support growth of P. vivax parasites to complete schizogony. The advantages of using hematopoietic stem cells are that the asynchronous maturation of the erythroid cells enables continuous production of fresh reticulocytes and that the cells contain hemoglobin A. This enables a simpler cultivation procedure, and weekly addition of the growing erythrocytes is sufficient to maintain the parasites. In
Review
Trends in Parasitology
Vol.24 No.2
Figure 1. Plasmodium vivax parasites cultivated in human erythroblasts (10–12-days old) established from cultivation of cord-blood hematopoietic stem cells. Arrows indicate amoeboid stage (a) and schizont stage (b) parasites. Scale bars = 10 mm.
addition, it should be noted that erythrocytes derived from hematopoietic stem cells in the first week of cultivation can be cryopreserved for 6–8 months with 100% cell viability. Although this method is able to supply fresh reticulocytes continuously, the numbers are still low, which remains a potential limiting factor to obtaining higher parasitemia. In vitro cultivation of other stages of P. vivax Unlike in vitro cultivation of the blood-stage parasites, the exo-erythrocytic stage of P. vivax parasites is less restricted to a certain host cell type. In vitro development of P. vivax exo-erythrocytic parasites is possible using: (i) primary human liver cells [15], (ii) the commercially available human hepatocellular carcinoma cell line HepG2 [16] or (iii) the newly established normal human liver cell line HC-04 [17]. This contrasts with the cultivation of P. falciparum, because complete development of the P. falciparum exo-erythrocytic stage in vitro has been successful in the primary liver HHS102 and HC-04 cell lines, but not in the HepG2 cell line [17–19]. Fertilization of P. vivax gametes, and further development to zygotes and ookinetes in vitro, has been established [20]. By contrast, cultivation of P. falciparum gametocytes that are infective to the relevant mosquitoes has been established [21], but further development of P. falciparum ookinetes in vitro has been unsuccessful. Cultivation of the complete Plasmodium cycle in vitro under various conditions will significantly enhance the study of parasite biology, in particular in the laboratories that are not located in malaria endemic areas. Although it is possible to cultivate the blood stage, exo-erythrocytic stage and initial sporogonic stages of malaria in vitro, to produce a complete life cycle of any human-infecting Plasmodium species in vitro is not currently possible. A promising approach to establishing P. vivax cultivation is the in vitro cultivation method developed for the rodent malaria parasite P. berghei to produce all sporogonic stages [22]. Conclusions and future perspectives P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and -2) have been identified, and it has been proposed that they are responsible for the reticulocyte preference of this parasite [11]. In this model, PvRBPs specifically recognize receptors expressed only on young erythrocytes (reticulocytes) before they start to form an irreversible tight
junction between the parasite apex and target erythrocyte. Mature erythrocytes do not possess this receptor site; thus, P. vivax cannot recognize them as target cells to invade. Identification of the reticulocyte receptor is necessary to clarify the molecular basis of the reticulocyte preference and thus the pathogenesis of P. vivax. For example, P. vivax infection is restricted to Duffy positive individuals and rarely found in Duffy negative Fy( ) people [23,24]. Thus, correlation of Duffy negativity and protection against P. vivax can be explained based on the parasite ligand and erythrocyte receptor interaction. The low amount of reticulocytes available has partly hampered the effort to identify the receptor; however, laboratoryproduced erythroblasts would serve as a powerful source of molecules expressed only on young erythrocytes. Recently, P. falciparum promoters for histidine-rich protein 3 (HRP3) and calmodulin were shown to be functional in P. vivax by transient transfection techniques [25]. Thus, available transfection constructs for P. falciparum could be used for P. vivax, which reduces the task of producing a new set of transfection constructs specific for P. vivax. In combination with P. vivax long-term cultivation, transfection techniques could be used to genetically modify P. vivax to grow in mature erythrocytes. Such a parasite line would be extremely useful for P. vivax research. References 1 Trager, W. and Jensen, J.B. (1976) Human malaria parasites in continuous culture. Science 193, 673–675 2 Bass, C.C. and Johns, F.M. (1912) The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. J. Exp. Med. 16, 567–579 3 Mons, B. et al. (1988) Plasmodium vivax: in vitro growth and reinvasion in red blood cells of Aotus nancymai. Exp. Parasitol. 66, 183–188 4 Chotivanich, K. et al. (2001) Ex-vivo short-term culture and developmental assessment of Plasmodium vivax. Trans. R. Soc. Trop. Med. Hyg. 95, 677–680 5 Golenda, C.F. et al. (1997) Continuous in vitro propagation of the malaria parasite Plasmodium vivax. Proc. Natl. Acad. Sci. U. S. A. 94, 6786–6791 6 Siddiqui, W. (1979) In vitro cultivation of Plasmodium vivax and Plasmodium malariae. In Practical Tissue Culture Applications (Maramarosch, K. and Hirumi, H., eds), pp. 279–285, Academic Press 7 Brockelman, C.R. et al. (1985) Observation on complete schizogony of Plasmodium vivax in vitro. J. Protozool. 32, 76–80 8 Brockelman, C.R. et al. (1987) The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax. Parasitol. Res. 73, 107–112 87
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9 Lanners, H.N. (1992) Prolonged in vitro cultivation of Plasmodium vivax using Trager’s continuous flow methods. Parasitol. Res. 78, 699–701 10 Butcher, G.A. et al. (1973) Letter: mechanism of host specificity in malarial infection. Nature 244, 40–41 11 Galinski, M.R. et al. (1992) A reticulocyte-binding protein complex of Plasmodium vivax merozoites. Cell 69, 1213–1226 12 Udomsangpetch, R. et al. (2007) Short-term in vitro culture of field isolates of Plasmodium vivax using cord blood. Parasitol. Int. 56, 65–69 13 Pasvol, G. et al. (1977) Effect of fetal haemoglobin on susceptibility of red cells to Plasmodium falciparum. Nature 270, 171–173 14 Panichakul, T. et al. (2007) Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax. Int. J. Parasitol. 37, 1551– 1557 15 Mazier, D. et al. (1984) Cultivation of the liver forms of Plasmodium vivax in human hepatocytes. Nature 307, 367–369 16 Hollingdale, M.R. et al. (1986) In vitro culture of exoerythrocytic parasites of the North Korean strain of Plasmodium vivax in hepatoma cells. Am. J. Trop. Med. Hyg. 35, 275–276 17 Sattabongkot, J. et al. (2006) Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the
18 19
20 21
22 23
24 25
malaria parasites Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 74, 708–715 Mazier, D. et al. (1985) Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440–442 Karnasuta, C. et al. (1995) Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line. Am. J. Trop. Med. Hyg. 53, 607–611 Suwanabun, N. et al. (2001) Development of a method for the in vitro production of Plasmodium vivax ookinetes. J. Parasitol. 87, 928–930 Ponnudurai, T. et al. (1982) The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 76, 242–250 Al-Olayan, E.M. et al. (2002) Complete development of mosquito phases of the malaria parasite in vitro. Science 295, 677–679 Miller, L.H. et al. (1976) The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N. Engl. J. Med. 295, 302–304 Mendis, K. et al. (2001) The neglected burden of Plasmodium vivax malaria. Am. J. Trop. Med. Hyg. 64, 97–106 Pfahler, J.M. et al. (2006) Transient transfection of Plasmodium vivax blood stage parasites. Mol. Biochem. Parasitol. 149, 99–101
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Cultivation of Plasmodium vivax Rachanee Udomsangpetch1, Osamu Kaneko2, Kesinee Chotivanich3 and Jetsumon Sattabongkot4 1
Department Department 3 Department 4 Department 2
of of of of
Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand Protozoology, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki, Japan Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Entomology, Armed Forces Research Institute of Medical Science, Bangkok, Thailand
Establishment of a continuous line of Plasmodium vivax parasite is crucial to understand the parasite’s biology; however, this has not yet been achieved. Beginning in the 19th century, there were several efforts to cultivate this malaria parasite but without much success until the late 1980s. In addition, to date, only minor modifications of the methodology have been investigated, which has resulted in extending the cultivation period to around four weeks by supplying reticulocytes obtained from normal blood or rare hemochromatotic blood. However, the use of laboratory-produced erythroblasts to cultivate P. vivax enables maintenance of a continuous line of the parasite stably in the laboratory. Here, we summarize and compare the available methodologies and conditions for the in vitro cultivation of P. vivax. Initial attempts at culturing blood-stage Plasmodium vivax Research focusing on Plasmodium vivax has been neglected for several decades because of the lack of a practical continuous cultivation system. Because Plasmodium falciparum infection causes more severe symptoms than all other Plasmodium species, most of the research effort has previously been directed to falciparum malaria, and a P. falciparum in vitro cultivation system was established in the 1970s. However, P. vivax cannot be maintained in vitro under the conditions developed to cultivate P. falciparum [1]. In addition, P. vivax preferentially invades young erythrocytes, which are difficult to obtain routinely. These major obstacles have limited the investigation of P. vivax malaria biology at both the cellular and molecular levels. Research into cultivation of malaria parasites started in 1891, and efforts to culture malaria were reported, without much progress, from 1893 to 1894 [2]. Thereafter, cultivation of various species of human malaria parasites was documented by several researchers, and, by 1912, the most successful method of culturing P. vivax involved maintaining the parasite for a few schizogonic cycles in vitro. In the 20th century, several techniques and modifications were established by different laboratories [3–5]. The goal of these researchers was to maintain a continuous line of P. vivax parasite in vitro, similar to what was established for P. falciparum by Trager and Jensen in 1976 [1].
Corresponding author: Udomsangpetch, R. ([email protected]).
Development of cultivation techniques Four types of cultivation media have been used for P. vivax cultivation: (i) RPMI-1640, (ii) Waymouth, (iii) McCoy’s 5A and (iv) Science Medical Mahidol 612 (SCMI 612) media [5–8]. Modified conditioned media based on three commercially available cultivation media have been tested with various, and varying amounts of, supplements in several attempts to cultivate P. vivax parasites [4–9] (Table 1). Metal ions and vitamins have been reported to promote differentiation of P. vivax parasites to merozoites during schizogony. The increment in parasite number is obtained from the addition of 1.8 mM magnesium chloride to the medium. Ascorbic acid is required for P. vivax schizogony and 3–6 mg ml 1 ascorbic acid in the medium enhances parasite growth [8]. Medium SCMI 612 supports 99% of ring stages through schizogony; however, this medium consists of 30 components and was thus considered impractical for routine use [7]. These studies observed parasite growth only in one ex vivo schizogonic cycle and no merozoite reinvasion was observed. A simple method for shortterm cultivation of P. vivax was reported recently, and involves modifying the standard in vitro cultivation medium for P. falciparum by supplementing RPMI-1640 with ascorbic acid, glucose, thiamine, hypoxanthine and 50% human serum. Most of the parasites develop to complete schizogony and can be maintained for up to six cycles without the need to supply erythrocytes [4]. In another study, when a 1:1 mixture of squirrel monkey and human erythrocytes was added to RPMI-1640-based medium, P. vivax was maintained for 11 cycles [9]. The medium was supplemented with different additives: pyruvate, spermidine, cholesterol, tocopherol, ascorbic acid and 15% human serum. In addition, they attempted to improve cultivation efficiency using a small modified version of a fluid flow vessel (3-ml capacity) filled with medium, supplements, erythrocytes and P. vivax, placed at 37 8C, and then gassed with a mixture of 3% CO2, 5% O2 and 92% N2. Unfortunately, this method caused parasitized-cell breakage and there were only a few successful invasions. The first method for semi-continuous cultivation of P. vivax was established in 1997 [5]. The parasites were cultivated in McCoy’s 5A medium supplemented with L-glutamine, 25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) and 20% human serum. Parasites were grown under static conditions until they developed into schizonts. The reticulocytes obtained from differential centrifugation of freshly obtained
1471-4922/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.09.010 Available online 3 January 2008
85
Review
Trends in Parasitology Vol.24 No.2
Table 1. Development of Plasmodium vivax cultivation conditions (1979–2007) Year 1979 1985
Cultivation media RPMI-1640 SCMI 612
1987
Waymouth:RPMI-1640 (1:2)
1992
RPMI-1640
1997
McCoy’s 5A
2001
RPMI-1640
2007
McCoy’s 5A
2007
McCoy’s 5A
Conditions Red blood cell (RBC) extracted fraction I MgCl2 0.75 mM, ascorbic acid 0.6 mg ml 1 39 8C in candle jar, 15% human AB serum MgCl2 1.8 mM, ascorbic acid 3 mg ml 1 38.5 8C in candle jar, 15% human AB serum Ascorbic acid 0.6 mg ml 1 37 8C in flow vessel, 15% human AB serum, 3% CO2 A mixture of monkey and human erythrocytes (1:1) Ascorbic acid 0.5 mg ml 1 37 8C, 20% human AB serum, 5% CO2 Reticulocytes from hemochromatotic blood Ascorbic acid 6 mg ml 1 37 8C, 50% human AB serum, 5% CO2 No addition of erythrocytes Ascorbic acid 0.5 mg ml 1 37 8C, 25% human AB serum, 5% CO2 Cord blood erythrocytes Ascorbic acid 0.5 mg ml 1 37 8C, 25% human AB serum, 5% CO2 Cultivated erythroblast
hemochromatotic blood were added to the cultivation to support merozoite invasion. The parasite cultivation mix was then transferred to a shaker for 10–12 h to facilitate rupture of the schizonts. This treatment achieved at least a twofold increase in parasite density at each schizogonic cycle. However, the method was limited by the need to obtain reticulocytes from rare hemochromatotic blood. The supply of young erythrocytes/reticulocytes is crucial for reinvasion and for maintaining parasitemia of a semi-continuous cultivation of P. vivax. Involvement of young erythrocytes in cultivation of P. vivax Various modifications have focused on essential nutrients and conditions of cultivation that are required for parasite multiplication, which seem to be different to those of P. falciparum. In addition, prolonged survival requires successful in vitro erythrocyte reinvasion by the parasites and the phenotype of the erythrocyte is crucial to their susceptibility. Different species of Plasmodium parasite display distinct host specificities [10]. Moreover, humaninfecting Plasmodium species display specific selection of the target erythrocytes; for example, P. falciparum invades all types of human erythrocytes, whereas P. vivax prefers younger erythrocytes [11]. This preference for younger erythrocytes is the crucial factor in the difficulty in establishing a continuous P. vivax line, compared to P. falciparum cultivation. Although it is theoretically possible to use human reticulocytes for P. vivax cultivation, the proportion of reticulocytes in the normal peripheral blood is too small to continuously supply cultivation (normal levels of reticulocytes range from 0.5% to 1.5% in adults). To overcome this problem, two reticulocyte sources have been reported [5,12]. Golenda et al. (1997) reported the use of hemochromatotic blood that could contain up to 7% reticulocytes [5], and Udomsangpetch et al. (2007) reported the use of umbilical cord blood collected at birth, which contains 1%–5% reticulocytes [12]. Using cord-blood erythrocytes, P. vivax parasites were able to be cultivated for up to 30–40 days after isolation [12]. Although the number of reticulocytes obtained is similar between these sources, the 86
Cultivation period (days) 4 2
Refs [6] [7]
2
[8]
22
[9]
Continuous
[5]
12
[4]
30–40
[15]
Continuous
[14]
number of P. vivax parasites maintained using cord-blood erythrocytes gradually declined and eventually disappeared, and thus a continuous line has not been established. This observation might be explained by the marked difference in hemoglobin type. Hemochromatotic reticulocytes contain hemoglobin A but cord-blood reticulocytes contain mainly hemoglobin F, and although P. falciparum is able to invade hemoglobin F-containing erythrocytes efficiently, it does not grow well inside them [13]. It is possible that P. vivax also does not grow well in hemoglobin F-containing reticulocytes. A new strategy for production of human erythrocytes has been attempted. The fact that P. vivax parasites successfully invade erythrocytes in the human body does not match observations from several in vitro trials: it is rare that merozoites successfully utilize the freshly collected healthy human erythrocytes. It is conceivable that the limiting requirement of the parasite to complete its life cycle is to find the new host cells, namely young erythrocytes. However, the stage of erythrocytic development and maturation preferred by merozoites is unknown. Our recent study began with the isolation of cord-blood stem cells and propagation of these cells towards hematopoietic stem cells under specific driving factors [14]. Growing erythrocytes, starting from erythroblasts, appeared after the first week of stimulation by various factors and were tested by co-cultivation with P. vivax schizonts. Differentiated erythroblastic stages at various stages of development, up to 21-day-old mature erythrocytes, were used in cultivation of P. vivax under in vitro conditions [12]. The earliest erythrocytes that enable P. vivax invasion are polychromatic erythroblasts (Figure 1) [14]. Furthermore, we found that older erythroblasts are able to support growth of P. vivax parasites to complete schizogony. The advantages of using hematopoietic stem cells are that the asynchronous maturation of the erythroid cells enables continuous production of fresh reticulocytes and that the cells contain hemoglobin A. This enables a simpler cultivation procedure, and weekly addition of the growing erythrocytes is sufficient to maintain the parasites. In
Review
Trends in Parasitology
Vol.24 No.2
Figure 1. Plasmodium vivax parasites cultivated in human erythroblasts (10–12-days old) established from cultivation of cord-blood hematopoietic stem cells. Arrows indicate amoeboid stage (a) and schizont stage (b) parasites. Scale bars = 10 mm.
addition, it should be noted that erythrocytes derived from hematopoietic stem cells in the first week of cultivation can be cryopreserved for 6–8 months with 100% cell viability. Although this method is able to supply fresh reticulocytes continuously, the numbers are still low, which remains a potential limiting factor to obtaining higher parasitemia. In vitro cultivation of other stages of P. vivax Unlike in vitro cultivation of the blood-stage parasites, the exo-erythrocytic stage of P. vivax parasites is less restricted to a certain host cell type. In vitro development of P. vivax exo-erythrocytic parasites is possible using: (i) primary human liver cells [15], (ii) the commercially available human hepatocellular carcinoma cell line HepG2 [16] or (iii) the newly established normal human liver cell line HC-04 [17]. This contrasts with the cultivation of P. falciparum, because complete development of the P. falciparum exo-erythrocytic stage in vitro has been successful in the primary liver HHS102 and HC-04 cell lines, but not in the HepG2 cell line [17–19]. Fertilization of P. vivax gametes, and further development to zygotes and ookinetes in vitro, has been established [20]. By contrast, cultivation of P. falciparum gametocytes that are infective to the relevant mosquitoes has been established [21], but further development of P. falciparum ookinetes in vitro has been unsuccessful. Cultivation of the complete Plasmodium cycle in vitro under various conditions will significantly enhance the study of parasite biology, in particular in the laboratories that are not located in malaria endemic areas. Although it is possible to cultivate the blood stage, exo-erythrocytic stage and initial sporogonic stages of malaria in vitro, to produce a complete life cycle of any human-infecting Plasmodium species in vitro is not currently possible. A promising approach to establishing P. vivax cultivation is the in vitro cultivation method developed for the rodent malaria parasite P. berghei to produce all sporogonic stages [22]. Conclusions and future perspectives P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and -2) have been identified, and it has been proposed that they are responsible for the reticulocyte preference of this parasite [11]. In this model, PvRBPs specifically recognize receptors expressed only on young erythrocytes (reticulocytes) before they start to form an irreversible tight
junction between the parasite apex and target erythrocyte. Mature erythrocytes do not possess this receptor site; thus, P. vivax cannot recognize them as target cells to invade. Identification of the reticulocyte receptor is necessary to clarify the molecular basis of the reticulocyte preference and thus the pathogenesis of P. vivax. For example, P. vivax infection is restricted to Duffy positive individuals and rarely found in Duffy negative Fy( ) people [23,24]. Thus, correlation of Duffy negativity and protection against P. vivax can be explained based on the parasite ligand and erythrocyte receptor interaction. The low amount of reticulocytes available has partly hampered the effort to identify the receptor; however, laboratoryproduced erythroblasts would serve as a powerful source of molecules expressed only on young erythrocytes. Recently, P. falciparum promoters for histidine-rich protein 3 (HRP3) and calmodulin were shown to be functional in P. vivax by transient transfection techniques [25]. Thus, available transfection constructs for P. falciparum could be used for P. vivax, which reduces the task of producing a new set of transfection constructs specific for P. vivax. In combination with P. vivax long-term cultivation, transfection techniques could be used to genetically modify P. vivax to grow in mature erythrocytes. Such a parasite line would be extremely useful for P. vivax research. References 1 Trager, W. and Jensen, J.B. (1976) Human malaria parasites in continuous culture. Science 193, 673–675 2 Bass, C.C. and Johns, F.M. (1912) The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. J. Exp. Med. 16, 567–579 3 Mons, B. et al. (1988) Plasmodium vivax: in vitro growth and reinvasion in red blood cells of Aotus nancymai. Exp. Parasitol. 66, 183–188 4 Chotivanich, K. et al. (2001) Ex-vivo short-term culture and developmental assessment of Plasmodium vivax. Trans. R. Soc. Trop. Med. Hyg. 95, 677–680 5 Golenda, C.F. et al. (1997) Continuous in vitro propagation of the malaria parasite Plasmodium vivax. Proc. Natl. Acad. Sci. U. S. A. 94, 6786–6791 6 Siddiqui, W. (1979) In vitro cultivation of Plasmodium vivax and Plasmodium malariae. In Practical Tissue Culture Applications (Maramarosch, K. and Hirumi, H., eds), pp. 279–285, Academic Press 7 Brockelman, C.R. et al. (1985) Observation on complete schizogony of Plasmodium vivax in vitro. J. Protozool. 32, 76–80 8 Brockelman, C.R. et al. (1987) The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax. Parasitol. Res. 73, 107–112 87
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9 Lanners, H.N. (1992) Prolonged in vitro cultivation of Plasmodium vivax using Trager’s continuous flow methods. Parasitol. Res. 78, 699–701 10 Butcher, G.A. et al. (1973) Letter: mechanism of host specificity in malarial infection. Nature 244, 40–41 11 Galinski, M.R. et al. (1992) A reticulocyte-binding protein complex of Plasmodium vivax merozoites. Cell 69, 1213–1226 12 Udomsangpetch, R. et al. (2007) Short-term in vitro culture of field isolates of Plasmodium vivax using cord blood. Parasitol. Int. 56, 65–69 13 Pasvol, G. et al. (1977) Effect of fetal haemoglobin on susceptibility of red cells to Plasmodium falciparum. Nature 270, 171–173 14 Panichakul, T. et al. (2007) Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax. Int. J. Parasitol. 37, 1551– 1557 15 Mazier, D. et al. (1984) Cultivation of the liver forms of Plasmodium vivax in human hepatocytes. Nature 307, 367–369 16 Hollingdale, M.R. et al. (1986) In vitro culture of exoerythrocytic parasites of the North Korean strain of Plasmodium vivax in hepatoma cells. Am. J. Trop. Med. Hyg. 35, 275–276 17 Sattabongkot, J. et al. (2006) Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the
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malaria parasites Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 74, 708–715 Mazier, D. et al. (1985) Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440–442 Karnasuta, C. et al. (1995) Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line. Am. J. Trop. Med. Hyg. 53, 607–611 Suwanabun, N. et al. (2001) Development of a method for the in vitro production of Plasmodium vivax ookinetes. J. Parasitol. 87, 928–930 Ponnudurai, T. et al. (1982) The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 76, 242–250 Al-Olayan, E.M. et al. (2002) Complete development of mosquito phases of the malaria parasite in vitro. Science 295, 677–679 Miller, L.H. et al. (1976) The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N. Engl. J. Med. 295, 302–304 Mendis, K. et al. (2001) The neglected burden of Plasmodium vivax malaria. Am. J. Trop. Med. Hyg. 64, 97–106 Pfahler, J.M. et al. (2006) Transient transfection of Plasmodium vivax blood stage parasites. Mol. Biochem. Parasitol. 149, 99–101
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