Cultivation techniques for the erythrocytic stages of malaria parasites*

Memoranda Cultivation techniques for the erythrocytic stages of malaria parasites* The study of the biochemistry, physiology, and immunology of plasmodia has been restricted by the difficulty ofmaintaining the parasites in isolationfrom the host. Some suc- cess has been achieved in cultivating them in vitro, using tissue cultures and chick embryo techniques to study exoerythrocytic states and the sporogonic cycle, but no completely successful method has been foundfor studying the asexual and sexual stages ofplasmodia in circulating red blood cells. The relative slowness with which techniques for continuous in vitro cultivation have been developed is the result of inadequate knowledge of the bio- chemistry of the parasites and of the blood and its constituents. However, radioactive labelling techniques applied to P. knowlesi cultures are beginning to yield data offunda- mental importance. Existing methods for the short-term in vitro cultivation ofplasmodia are potentially very useful for analysing malarial antigens, for developing vaccines, andfor screening and studying antimalarial drugs. Investigations of the physicochemical require- ments for the in vitro preservation of red blood cells are required, and more emphasis should be given to the study ofplasmodia with longer cycles. Differences between the meta- bolism ofplasmodia in vivo and in vitro should be studied and the growth factors in normal plasma identified. Studies of the membrane of the parasites and of the red blood cells, of the immune response, and of extracellular methods for the cultivation of plasmodia should be extended. The persistence of malaria in man over large areas of the world, in spite of the improvement of diag- nostic techniques, chemotherapy, and control of anopheline mosquitos, is still a major medical and public health problem in many countries. The con- tinuation and, whenever possible, intensification of research on malaria will certainly help in the control and eradication of this disease. Although there are many species of malaria para- site infecting a variety of vertebrate hosts, only those species parasitic in man and those that can easily be maintained in the laboratory provide the investigator with material for experimental study. The maintenance of several species of malaria para- site in laboratory animals has greatly advanced many aspects of the study of malaria; however, important studies of the biochemistry, physiology, and immu- nology of the parasite have been limited by the dif- ficulty of rearing and maintaining the parasite in isolation from the host-vertebrate and invertebrate. * This memorandum was drafted by the signatories listed on page 371). A French version will appear in a subsequent number of the Bulletin. For this reason, techniques for the successful in vitro cultivation of malaria parasites are urgently needed. Some success has been achieved by the use of tissue cultures and chick embryo techniques for the study of exoerythrocytic stages and the sporogonic cycle (Ball & Chao, 1957; Huff et al., 1960; Pipkin & Jensen, 1956; Trager & Krassner, 1967), but so far no completely successful method has been found that could be used to study intracellular asexual and sexual stages of plasmodia developing in circulating red blood cells. These asexual stages are responsible for the pathogenicity of the disease and the immune response of the host, and they are a major target in the treatment of the disease by antimalarial drugs. The advantages of in vitro methods for studying microorganisms apply equally to the study of plas- modia. The objectives in the development of prac- tical methods for in vitro cultivation of malaria parasites for experimental studies are as follows: (1) to accumulate basic knowledge about the biology, physiology, and biochemistry of malaria parasites removed from the variable host environment; (2) to use this basic information for the development of 2906 -357- MEMORANDA techniques for both short- and long-term in vitro cultivation; and (3) to study species differences and immunological phenomena, including the produc- tion of a vaccine, the mechanism of drug action, and the origin and persistence of drug resistance. The progress that has been made towards these objectives is described in this memorandum. The main aim of the research is to develop a technique for the continuous cultivation of malaria parasites in vitro, and short-term cultures are already beginning to show results (Cohen & Butcher, 1970). The methods that are being used were developed, in general, from biological, biochemical, and physio- logical data derived from parallel experiments on the parasites grown in vivo and in vitro (Anfinsen et al., 1946; Ball et al., 1948; Geiman et al., 1946; McKee et al., 1946; Trager, 1941). The relatively slow rate at which techniques for in vitro cultivation have been developed is the result of inadequate knowledge about the biochemistry of the parasites and their environment, i.e., the blood and its con- stituents. However, modern biochemical techniques and the stimulus given to malaria research by events in eastern Asia have produced important new data on the biochemistry and physiology of a number of species of Plasmodium, principally P. knowlesi, P. berghei, P. lophurae, P. gallinaceum, and, more recently, P. falciparum (Sadun, 1966, 1969). The complexity of the problem of long-term in vitro cultivation of plasmodia indicates that progress will depend on team work and sustained financial sup- port of research. BASIC PROBLEMS OF CULTURE Maintenance of red blood cells in vitro Since the asexual stages and gametocytes of plas- modia invade the erythrocytes of the infected host and develop there, the maintenance of the integrity of the red cell in vitro is of primary importance for long-term cultivation. Detailed knowledge ofthe con- stituents of blood plasma is also essential to the design of a medium that will support and maintain the integrity of the red cell, the growth and multi- plication of the intracellular malaria parasites, and the progeny of merozoites that are released from ruptured red cells. The maintenance of red cell integrity in cultures also applies to in vitro studies of Babesia and Anaplasma. Few studies have been made on red cells incubated for long periods at 37°C. The osmotic fragility of the red cells increases when they are incubated in vitro (Haut et al., 1962) and it has been shown that the cells degenerate metabolically after 24 h at 37°C (Gomperts, 1967, 1969). For instance, after 24 h in vitro red cells show a decrease in glucose con- sumption and therefore a decrease in the rate of phosphorylation of adenosine 5'-(trihydrogen pyro- phosphate) (ADP) to adenosine 5'-(tetrahydrogen triphosphate) (ATP); some of these degenerative changes are similar to those shown by aging red cells. Marks et al. (1958) have shown that aging red cells possess a lowered glucose-6-phosphate dehydrogenase (G6PD) content, which could be connected with a loss of reduced glutathione in vitro. The mature erythrocyte can synthesize reduced gluta- thione from glycine (aminoacetic acid) and maintain it in the reduced form by the pentose-phosphate pathway, involving G6PD (Prankerd, 1965). Reduced glutathione, apart from protecting haemoglobin against oxidative denaturation (Jandl et al., 1960), also protects the unsaturated bonds of lipids and sulfydryl groups of the proteins of red cell mem- branes (Prankerd, 1965), thus affecting the perme- ability. The importance of these observations is illustrated by the work of Trager (1950, 1952), who grew the asexual blood stages of an avian malaria parasite, P. lophurae, extracellularly and showed that the parasite is dependent on the red cell for ATP (Trager, 1950) and coenzyme A (Bennett & Trager, 1967; Trager, 1954). More recently, these studies have been extended to the incorporation of labelled precursors by P. lophurae. The results of these studies are very encouraging and have helped to unravel the host-parasite relationships of one spe- cies of malaria parasite. This type of study should stimulate similar work with both human and non- human primate species of Plasmodium. Maintenance of the extracellular merozoite With the completion of schizogony and rupture of the parasitized host cell, the environment of the malaria parasite changes abruptly as free merozoites are liberated into the plasma. This highly differen- tiated extracellular phase of the parasite has a conical end that contains a pair of dense elongated organelles. The merozoite is invariably covered with granular material bounded by an outer plasma- lemma, said to contain an inactive cytosome, and an inner tubular membranous structure. The nucleus contains markedly condensed chromatin, and the cytoplasm carries ribosomes, bodies assumed to be primitive mitochondria, and dense tubular bodies 358 CULTIVATION TECHNIQUES FOR PLASMODIA called taxonemes located in the conoid region (Ladda, 1969). Newly liberated merozoites may show consider- able distortion, suggesting an undulating type of locomotion. The penetration of a new erythrocyte is initiated when the conoid end of the merozoite attaches, presumably by means of a specific receptor, to the surface of a red cell. Observations under phase microscopy indicate that merozoites ofP. know- lesi may remain attached to the surface of the cell for about 5-10 min before penetration is completed in vitro (Butcher & Cohen, 1970). The studies of Ladda et al. (1969) have shown that in P. berghei yoeli and P. gallinaceum the conoid of the merozoite creates a depression in the host cell membrane that rapidly becomes a cavity enclosing the parasite within the cell. The cell membrane at the site of the initial contact forms a tight ring which then fuses to separate the vacuole containing the parasite from the surface membrane. During penetration, the paired organelles and dense bodies rapidly disappear, suggesting that they are associated with materials essential for penetration. Once inside the red cell, the merozoite is transformed into a structurally simple ring-stage parasite. Little is known about the physicochemical require- ments for the maintenance of viable merozoites in vitro. By culturing P. knowlesi parasites in the absence of nonparasitized red blood cells, it has been possible to obtain uncontaminated prepara- tions of free merozoites that, after isolation, retain their ability to penetrate rhesus monkey erythro- cytes and undergo further differentiation (Mitchell et al., unpublished data). All attempts to preserve isolated merozoites in a viable state for periods longer than 10 min have proved unsuccessful, sug- gesting that this is a very vulnerable or short-lived stage in the life cycle of the parasite. The successful maintenance of high multiplication rates in vitro during successive generations may require the iden- tification of specific nutrients and physical conditions required by the free merozoites. Maintenance of the intracellular parasite Inside the erythrocyte the uninucleate merozoite differentiates into a structurally simple ring form containing cytoplasm and a large central vacuole. As the parasite grows the vacuole becomes occluded by an increase in the cytoplasm, which contains many ribosomes and a "mitochondrion"; this stage is called the trophozoite. During the period of growth, the cytoplasm becomes amoeboid and the parasite feeds on the haemoglobin of the cell by phagotrophy, i.e., the incorporation of portions of the host cell into vacuoles in the cytoplasm of the parasite. The haemoglobin is incompletely metabolized and the residue, the malarial pigment or haemazoin, is left in the vacuoles. The trophozoite continues to grow until it fills the host cell, by which time amoeboid activity stops. The nucleus then divides by a " mul- tiple mitosis" to form a schizont containing 8-32 nuclei, the number depending on the species. After a rapid period of differentiation the schizont seg- ments, forming more uninucleate merozoites, invade further erythrocytes and complete the asexual cycle in the blood. The time taken to complete this cycle varies according to the species of parasite. In the simian species P. knowlesi and in the rodent species the cycle lasts approximately 24 h but it lasts 48 h in P. falciparum. This is an important factor when the cultivation of these stages is being considered since it has been shown that normal red cells dete- riorate more rapidly after 24 h in vitro (Gomperts, 1967, 1969). Trigg (1968b) has shown that P. know- lesi grows and multiplies better in vitro than does P. cynomolgi, a species with a 48-h cycle. P. inui, which has a 72-h cycle, showed growth but no increase in numbers. Siddiqui et al. (1970a) showed that P. falciparum from Aotus monkeys grew and appeared normal after 24 h, but although the invasion of new cells occurred after 24-48 h, both the red cells and the growth and morphology of the parasites deteriorated. These results indicate that the medium and the physical conditions of the culture may have caused red cell lesions similar to those described by Gom- perts (1967, 1969), which were then reflected by the decreasing growth rate of the parasite. Different species, and in some cases different strains, of Plasmodium also show a preference for red cells of different ages. For instance, P. vinckei, P. knowlesi, and P. falciparum tend to infect mature red cells, while most strains of P. berghei and P. vivax tend to infect the immature basophilic red cell (Garnham, 1966). The reasons for this are unknown. The relationship between resistance to falciparum malaria and sickle cell anaemia (Allison, 1954) also indicates the importance of the erythro- cyte-parasite relationship. Moulder (1962) sug- gested that the parasite could not digest haemo- globin S, which is found in humans suffering from this disease. However, Raper (1959) demonstrated 359 MEMORANDA that P. falciparum would grow through at least one asexual cycle in sickle cells in vitro as well as in normal red cells. This led Garnham (1966) to suggest that malaria was less common in patients with sickle cell anaemia because the later stages of P. falciparum are spent in the capillaries of internal organs where the red cells become anoxic and the subsequent sickling of the cells prevents the develop- ment of the parasite. There have been many attempts to grow the eryth- rocytic stages of the mammalian malaria parasite, the most successful of which have been made with P. knowlesi from rhesus monkeys (Anfinsen et al., 1946; Geiman et al., 1966; Trigg, 1968b, 1969b; Butcher & Cohen, 1971; Trigg & Gutteridge, 1971). Although growth, multiplication, and subculture could be obtained with this species, the parasites grew more slowly than they do in vivo and they sur- vived for no more than 3-4 asexual generations with a successive decrease in the multiplication rate during each successive subculture (Anfinsen et al., 1946; Trigg, 1969b; Trigg & Gutteridge, 1971). Moreover, the stage of development of the parasite when inoculated into a culture affected the extent to which new host cells were invaded (Trigg, 1969b). Attempts have been made to culture P. falci- parum derived from Aotus monkeys and man. In Aotus blood, growth and reinvasion occur in vitro but there is little or no multiplication (Siddiqui et al., 1970a). However, with P. falciparum in human blood, Diggs et al. (1971) obtained significant in vitro reinvasion of fetal human red cells, and Phillips et al. (1972) have been able to subculture the parasite through almost 3 cycles in vitro with a marked increase in numbers during the first sub- culture but with the numbers being only maintained during the second subculture. The cultivation of the erythrocytic stages of rodent malaria parasites has until recently been less success- ful. Geiman et al. (1966) and Trigg (1968b) were able to obtain in vitro growth and invasion of new red cells with both P. berghei and P. chabaudi, but no significant increase in parasite numbers occurred. However, Richards & Williams (unpublished data) have now succeeded in obtaining a 4-fold multipli- cation of P. berghei after the leucocytes were removed from the infected blood prior to cultivation. These results indicate that abnormalities may be present in the parasite's metabolism even during the first cycle of in vitro growth and that it is possible that such abnormalities may be manifested to a greater extent during subculture. There have been few studies to compare the metabolism of the malaria parasite either in vitro or in vivo, but Ball et al. (1948) suggested that P. knowlesi parasites growing and multiplying in vitro were not entirely comparable in their metabolic and chemical patterns with those grown in vivo. They observed that there is an increase in oxygen consumption concomitant with an increase in the size and number of parasites in vivo but not in vitro. In addition, they observed an abnormal ratio of fatty acid to phospholipids in vitro. More recently, Trigg & Gutteridge (1972) have shown that P. knowlesi when grown in vitro exhibits reduced RNA synthesis during the first asexual cycle in vitro, the amounts of DNA and protein being quantitatively similar to those produced in vivo, although qualitative differences are not ruled out. Further experiments showed that during the second in vitro asexual cycle of P. knowlesi, RNA, DNA, and protein synthesis was reduced when compared with the first cycle, thus the actual rate of synthesis in the second cycle was further reduced when compared with the in vivo cycle. The same authors suggested that the successive decrease in RNA synthesis parallels the reduction in multiplica- tion obtained in subcultures of the parasite. This work indicates that the media and tech- niques used for the cultivation of malaria parasites are not optimum and that comparisons of the meta- bolism of the parasite in vivo and in vitro may suggest ways in which they may be improved. REQUIREMENTS FOR IN VITRO CULTIVATION OF PLASMODIA The original work on the in vitro cultivation of P. knowlesi was carried out using the Harvard growth medium (Ball et al., 1945; Anfinsen et al., 1946), which was based on a chemical analysis of monkey plasma. Subsequently, significantly improved growth and multiplication have been obtained with the same species in modifications of the Harvard medium (Trigg, 1968b; Cohen & Butcher, 1971) and in proprietary tissue culture media (Trigg, 1969b). All these media are complex and probably contain many nonessential factors and, since it is known that competitive relationships exist between the con- stituents of culture media, it is desirable that the medium should be balanced with respect to the requirements of the organisms to be cultured. The positive identification of the growth factors required by the malaria parasite is complicated by the dif- 360 CULTIVATION TECHNIQUES FOR PLASMOPIA ficulty experienced in growing the parasites in the absence of serum or plasma. Several studies of the biochemistry of the erythro- cytic stages have led to the identification of certain substances that appear to be essential to the growth of malaria parasites in vitro, and these observations have led to the use of simplified media for the in vitro growth of P. knowlesi (Siddiqui et al., 1970b; Trigg & Gutteridge, 1971). Most biochemical infor- mation has been obtained from a study of P. know- lesi and care must be exercised in extrapolating the results to other species in view of the host specificity of the parasites and because there are differences in the basic composition of the DNA in primate and rodent parasites (Gutteridge et al., 1970, 1971). Balanced salt solution A culture medium contains a balanced salt solu- tion to provide the correct ionic environment for the cultured cells, and a buffering system to maintain physiological pH. The balanced salt solution in Harvard medium was not very successful in main- taining rodent species of parasite, perhaps owing in part to an incorrect ionic environment for the rodent red cells. All media that have been used with suc- cess incorporate a hydrogencarbonate and carbon dioxide; this maintains the pH of the medium at 7.4 and allows for fixation of carbon dioxide by the parasite (Sherman & Ting, 1968). One of the main problems in the cultivation of the erythrocytic stages of the malaria parasite has been the maintenance of physiological pH because of the rapid consumption of glucose and the accu- mulation of lactate produced by the parasite. This has been prevented by the use of perfusion systems (Geiman et al., 1946; Geiman et al., 1966; Trigg, 1968b, 1969b; Trager, 1971) or, in dilution systems, by supplementing the growth medium with zwitter- ion buffers such as N-glycylglycine (Geiman et al., 1966) or N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine (" tricine ") (Booden & Geiman, personal communication). The use of zwitter-ion buffers not only increases the overall buffering capacity of the medium but it also helps to maintain phy- siological pH when the cultures are removed from the mixture of carbon dioxide and air for inoculation and removal of parasites. Carbohydrates In P. knowlesi (Scheibel & Miller, 1969a; Scheibel & Pflaum, 1970a) and P. berghei (Bowman et al., 1961; Bryant et al., 1964), glycolysis is the main source of energy, although other pathways for glucose catabolism may exist. In P. knowlesi, there is incorporation of the 14C label from glucose into succinate and acetate (Scheibel & Pflaum, 1970a), and in P. berghei traces of'4C-succinate were shown to be incorporated by the parasite (Bryant et al., 1964; Nagarajan, 1968). A complete Krebs cycle is apparently absent from the mammalian plasmodia (Peters, 1969) but cytochrome oxidase activity has been suggested in P. knowlesi, P. berghei, and P. cynomolgi (Scheibel & Miller, 1969a, 1969b), and also in P. falciparum (Scheibel & Pflaum, 1970b). Oxygen at high concentrations has been shown to inhibit the growth of P. knowlesi in vitro. Under anaerobic conditions, the development of rings in the late trophozoite stages occurs but nuclear divi- sion and reinvasion do not follow (Trigg, 1969a). The significance and role of oxidative pathways of glucose metabolism in vitro is not yet known, and the importance of the pentose phosphate path- way in the development of the malaria parasite has not been assessed. Although red cells infected with plasmodia exhibit increased pentose phosphate path- way activity, it is not clear whether this results from the presence of the enzymes of the pathway with- in the parasite or from an alteration of the activity of the host red cell enzymes by the parasite. Langer et al. (1967) suggested that the pathway is present in P. berghei but the work of Bowman et al. (1961) and Sherman (1965) with P. berghei, and of Fletcher & Maegraith (1962) with P. knowlesi, suggests the contrary. The significance of the role of the pentose phosphate pathway in providing pentose sugars for nucleic acid metabolism and reduced nicotinamide adenine dinucleotide phosphate (NADPH) for syn- thetic processes in the malaria parasite has yet to be determined. The work described above illustrates that little is known of the host-parasite relationships. Trager (1950) has shown that ATP is required by plasmodia grown extracellularly, and this may indicate that the parasite utilizes ATP from the host cell. The normal host red cell obtains its energy also by glycolysis and after infection by the malaria parasite the glycolytic capacity of the host cell and parasite com- plex is increased at least 25-fold (McKee, 1951). The cause of this increase in glycolytic activity is unknown, and it is evident that a study of the biochemistry of the malaria parasite is required to determine whether it possesses its own glycolytic capacity as distinct from that of the host cell, or 361 MEMORANDA whether a metabolic interrelationship between the parasite and the host cell is developed. Amino acids The erythrocytic stages of malaria parasites satisfy most of their nitrogenous requirements by the hydro- lysis of host cell haemoglobin (Moulder, 1962) but P. knowlesi, at least, requires extracellular amino acids. Polet & Conrad (1969) and McCormick (1970) have shown that although P. knowlesi utilizes all exogenously supplied amino acids to some extent, isoleucine and methionine are incorporated into parasite protein in the greatest amounts. In the case of isoleucine, this may be correlated with the fact that normal rhesus monkey haemoglobin does not contain this amino acid (Matsuda et al., 1968) and Fulton & Grant (1956) have suggested that P. know- lesi cannot obtain all the methionine it needs from the host haemoglobin. P. knowlesi requires methionine for growth in vitro (McKee & Geiman, 1948) and it has been shown more recently that methionine and isoleucine are the only two extracellular amino acids essential for both growth (Polet & Conrad, 1969) and multi- plication in vitro (Siddiqui et al., 1969; Trigg & Gutteridge, 1971). It has also been shown that glutamine is required by P. knowlesi cultivated in vitro (Butcher & Cohen, 1971). The in vitro amino acid requirements have not been determined for either the human or the rodent species of malaria parasite, but P. falciparum will grow in a medium containing only isoleucine and methionine (Trigg, unpublished data). Nucleic acids Both Trigg & Gutteridge (1971) and Booden & Geiman (1972) were unable to find any evidence for the de novo synthesis of purines in P. knowlesi. Biingener & Nielsen (1969) have demonstrated the utilization of host cell adenine and adenosine by P. vinckei. The plasmodium appears to require purine from the medium for nucleic acid synthesis. In fact, Bungener & Nielsen (1967, 1968) found that exogenously supplied purines were well incorporated into nucleic acids of P. berghei and P. vinckei, and Gutteridge & Trigg (1970) obtained similar results with P. knowlesi. In the latter study, one of the most rapidly incorporated compounds was adenosine and its absence from the growth medium caused a reduc- tion in the multiplication rate of the parasite (Trigg& Gutteridge, 1971). In P. chabaudi the synthesis of adenine nucleotides from incorporated adenosine seems possible since a complete sequence of reac- tions from adenosine via inosine, hypoxanthine, and 5'-inosinic acid to adenosine monophosphate (AMP) ADP, and ATP has been established (Lukow, Schmidt, & Konigk, unpublished data). The situation with regard to the pyrimidines appears to be the converse. Cultures of P. know- lesi did not utilize exogenously supplied radio- active pyrimidines for nucleic acid synthesis and although these compounds entered the red cell they were not metabolized (Oliver & Patterson, 1971). There is evidence that the erythrocytic stages of avian malaria parasites can synthesize the pyrimidine ring (Walsh & Sherman, 1968.) P. knowlesi can utilize orotic acid for nucleic acid synthesis (Polet & Barr, 1968; Gutteridge & Trigg, 1970), suggesting that the terminal part of the pathway for the synthesis of the pyrimidine ring is present in this parasite. Omission of pyrimidines from the growth medium had no effect on P. know- lesi cultivated in vitro (Trigg & Gutteridge, 1971). Investigation of the synthesis of thymidylate in P. chabaudi revealed the absence of the salvage path- way via thymidine kinase (2.7.1.21). This explains the sensitivity of plasmodia to folate antagonists (Walter et al., 1970). P. berghei and P. vinckei can- not utilize exogenously supplied pyrimidines (Bun- gener & Nielsen, 1968). Increased levels in para- sitized blood of the primary enzyme of pyrimidine biosynthesis (aspartate carbamoyltransferase, 2.1.3.2) suggest pyrimidine synthesis by P. berghei (Van Dyke et al., 1968, 1970). Therefore it seems likely that the nucleoside requirements of the rodent parasite may be similar to that of P. knowlesi, although the effect of the omission of purines from growth media has still to be tested in vitro. Vitamins and other coenzyme precursors Information on the vitamin requirements of ma- laria parasites is scarce and their investigation by the stepwise elimination of various factors from the culture medium is difficult since the red cells and the serum from well-fed hosts contain most of the known vitamins. From dietary and in vitro studies, the p-aminobenzoic acid requirement of the malaria parasite is well known (Anfinsen et al., 1946; McKee, 1951), and Walter & Konigk (1971b) measured the activity of 7,8-dihydropteroate synthetase, which is involved in the incorporation of p-aminobenzoic acid into dihydrofolate and which is competitively inhibited by sulfonamides, in P. chabaudi. Siddiqui et al. (1969) and Trigg & Gutteridge 362 CULTIVATION TECHNIQUES FOR PLASMOPIA (1971) have shown that biotin is required by P. know- lesi cultivated in vitro, while Trager (1943, 1966b) has shown that P. lophurae, P. coatneyi, and P. falci- parum require pantothenate; this observation was confirmed in P. knowlesi by Butcher & Cohen (1971) and Trigg & Gutteridge (1971). Butcher & Cohen (1971) also demonstrated a requirement for coen- zyme A as well as for ATP, previously shown by Trager (1954) to be required for the extracellular cultivation of P. lophurae. The omission of each of these factors did not always produce a striking effect, and it seems pos- sible that the red cell could contain sufficient amounts to supply the parasite for the 24-h periods during which these tests were performed. This is further illustrated by the effect of vitamin C on malaria parasites. P. knowlesi infections were reduced and there was a loss of pathogenicity in monkeys deficient in vitamin C whereas no effect on the parasite was observed in vitro when this vitamin was omitted from the growth medium (McKee & Geiman, 1946). Lipids The malaria parasite seems to have only a limited ability to synthesize lipids, and it depends, at least in part, on the environment, i.e., the red cells and the serum, as a source of fatty acids (Guttierrez, 1966; Siddiqui et al., 1967) and sterol (Trigg, 1968a). Lipid biosynthesis in the malaria parasite consists mainly of phospholipid synthesis involving a link- age of fatty acids to a-glycerophosphate derivatives. Cenedella (1968) using P. berghei and Rock (1971) using P. knowlesi have both shown that the malaria parasite can convert glucose to lipid-glycerol; con- sequently Trigg & Gutteridge (1971) omitted gly- cerol from their medium without affecting the growth of P. knowlesi. Anfinsen et al. (1946) considered glycerol to be necessary for the in vitro growth of P. knowlesi. Malaria parasites are also able to incor- porate acetate into fatty acids in vitro (Brundage et al., 1969; Rock, 1971), but again glucose can serve as a source of acetate units for this biosynthesis (Cenedella, 1968). Both Anfinsen et al. (1946) and Trigg & Gutteridge (1971) have shown that acetate is not an absolute requirement for the in vitro culture of P. knowlesi. The role ofplasma Any synthetic medium used for the in vitro cultivation of the blood stages of malaria parasites should duplicate the constituents and physical pro- perties of the host's plasma. More than 200 electro- lytes and organic compounds other than proteins have been identified in the whole blood, erythrocytes, plasma, or serum of man, including 40 electrolytes, 50 nonprotein nitrogenous substances, 20 lipids, 10 carbohydrates, 16 miscellaneous organic acids, 20 vitamins, 20 hormones, and 35 enzymes and coenzymes (Dittmer, 1961). If media used for the cultivation of malaria parasites are compared with plasma it is seen that there are major deficiencies in the media. In fact, good growth and multiplica- tion of the malaria parasite cannot be obtained in the absence of plasma or serum. An important question is which of the known constituents of plasma are required for the growth and multiplica- tion of plasmodia and for the maintenance of the integrity of the host erythrocytes. Attempts were made by Anfinsen et al. (1946) to replace plasma or serum with purified serum albumin. Growth and inhibitory factors in plasma and the growth-promoting properties of various plasma frac- tions were also studied by McKee & Geiman (1949, 1950). Siddiqui et al. (1967) completely replaced the plasma by stearic acid, and Trigg (1969a) con- firmed this requirement but obtained better results if the lipid was bound to a protein carrier. This may not be surprising since the uptake of fatty acids by red cells is via a reversible exchange between the binding sites of plasma proteins and similar sites on the red cell membrane (Goodman, 1958a, 1958b); the unbound fatty acid composition of plasma is negligible (Goodman, 1958a). When the plasma was completely replaced by stearic acid, the glucose utilization of the parasite was reduced (Siddiqui et al., 1967; Trigg, 1969a) and attempts to subculture the parasite were unsuccessful (Trigg, 1969a). The role of stearic acid in the metabolism of plasmodia is unknown. Plasma also contains large amounts of cholesterol, and Trigg (1969a) showed that this was one of the essential substances for plasmodial growth. Its role may be to maintain the integrity of the membranes of both the erythrocytes (Murphy, 1962) and the plasmodia (Trigg, 1968a). It has been shown that plasma and sera from infected and normal monkeys vary considerably in their ability to support the in vitro growth of P. knowlesi (Butcher & Cohen, 1971). The reason for this is unknown although Cohen & Butcher (unpublished data) found that the growth factors pre- sent in sera were nondialysable and stable for at least 6 months at - 80°C. This variation in sera could be due either to the absence of certain growth 3,63 MEMORANDA factors resulting from different dietary or physiolo- gical states of the animals or to the presence of inhibitory factors. The identity of serum factors that inhibit plasmo- dial growth has not been established but heterotoxins have been recognized in normal sera (Terasaki et al., 1961) and toxic factors have been identified in the sera of acutely infected monkeys (Riley & Maegraith, 1961). Hormones are also present in sera and it has been shown that some of them affect the integrity of the red cell (Snyder & Reddy, 1970). An effect of host hormones on plasmodia has not yet been established although the observa- tions of Arnold et al. (1969), who showed that growth and synchrony of P. berghei in mice was mediated through the pineal gland of the host, suggest that such effects should not be ruled out. TECHNIQUES FOR THE CULTIVATION OF PLASMODIA Since the early work of Bass & Johns (1912) and Bass (1913-14), in which they observed the develop- ment of P. falciparum in a static layer of cells, relatively few techniques have been utilized for the in vitro cultivation of intra-erythrocytic malaria parasites. The majority of these studies have em- ployed the rocker-dilution technique first developed by Geiman et al. (1946) by which a suspension of infected blood is diluted with nutrient medium and then gently rocked in a continuously passing mix- ture of 95% air and 5% carbon dioxide. A variation of this technique was used by Polet (1966), who incubated culture tubes on a roller rotating at 1/5 rev/min. Although these dilution systems are simple and very useful they have some disadvantages. The medium is not of constant composition throughout the periods of incubation on account of the metabo- lism of growth factors and the production of waste products, especially lactate, by the parasites. The accumulation of lactate may result in a fall of the pH of the medium but pH changes may be prevented by increasing the buffering capacity of the medium or by reducing the concentration of parasites in the culture (Geiman et al., 1966). There have been several attempts to provide con- stant conditions for the growth of plasmodia in vitro. Perfusion techniques have been devised that employ dialysis membranes (Geiman et al., 1946, 1966), cellulose acetate membranes (Trigg, 1968b, 1969b), and a tidal flow system (Tiner, 1969). More recently, Trager (1971) has developed a continuous flow method without the use of membranes but this, like the perfusion techniques, is cumbersome to use and difficult to replicate. When compared with dilution techniques, these flow methods may show small increases in the amount of reinvasion of new host cells, and there- fore an increase in parasite numbers during the first cycle. However, there is still a successive decrease in parasite numbers with successive subcultures as in dilution techniques (Anfinsen et al., 1946; Trigg, 1969b). For most work, therefore, particularly when many cultures have to be set up at one time, e.g., in drug testing, in evaluating the effects of sera, or in metabo- lic studies involving radioisotopes, the simple dilu- tion techniques are to be preferred at the present time. However, Trager (1966a) has pointed out that the results obtained with such systems are not good enough to warrant neglecting the development of entirely new systems for the cultivation of eryth- rocyte suspensions. Removal of leucocytes Bass & Johns (1912) and subsequent workers have stressed the necessity for removing leucocytes from infected blood for the successful in vitro cultivation of plasmodia. In recent years, with the use of better- defined culture media, radioactively labelled ingre- dients have been employed to measure the metabo- lic activity of malaria parasites, and under these conditions the presence of leucocytes and other host cell components may invalidate the results. Richards & Williams (1971 and unpublished data) found that although it was possible to obtain repro- ducible results in vitro with either P. knowlesi from rhesus monkeys or P. falciparum from Aotus mon- keys, reproducible results were not obtained with P. berghei from rats because there was a component of the cell population, other than the infected eryth- rocytes, that incorporated 3H-leucine. This com- ponent was shown, in the following way, to be leucocytes. Uninfected blood from a variety of laboratory mammals able to maintain plasmodia was cultured under similar conditions, and the incor- poration of 3H-leucine up to 18 h was determined. Each culture contained a similar concentration of red cells. Fig. 1 shows that the incorporation of 3H-leucine by normal rat blood was markedly greater than that by normal Aotus or rhesus monkey blood. Leucocytes were removed from the infected blood samples by means ofa technique developed by Fulton 364 CULTIVATION TECHNIQUES FOR PLASMODIA 365 5 Rat 4 2 Mouse o Aotus monkey 1o = =___ Hamster _____RhesI Rhesus monkey 0 0 4 8 12 16 20 24 Hou rs 1- WHO 20700 Fig. 1. Incorporation of 3H-leucine by the normal blood of various experimental animals. & Grant (1956) in which diluted blood was passed through a column of cellulose powder. More than 98% of the leucocytes were retained on the column whereas the parasitized erythrocytes were recovered morphologically intact, metabolically active in vitro, and infective to normal animals. In a second experi- ment uninfected untreated rat blood (100% leuco- cytes) was mixed with treated blood (no leucocytes) and cultured as before. The results (Fig. 2) show that radioactively labelled material was incorpo- rated in direct proportion to the number of leuco- cytes present. The incorporation of 3H-leucine into a culture of P. berghei from which the leucocytes had been removed is also illustrated. ic 9 8 6 5- a 4 -% leucocye 0 no parasitaemia2 25% leuCOcyte-s ~~~~ ~ ~ i~~~ie~~ucocytes0 4 8 12 16 20 24 H o u r s 2-WHO 20701 Fig. 2. Incorporation of 3H-leucine by mixtures of uninfected untreated rat blood containing a full com- plement of leucocytes and treated rat blood containing no leucocytes. The incorporation curve for a culture of P. berghei in blood from which the leucocytes have been removed is also shown for comparison. Other methods based on density gradient centri- fugation have been developed for removing leuco- cytes and platelets and normal red cells from infected blood. These methods also concentrate the parasites in bands, according to their relative densities. It is therefore possible, starting from an asynchronous population of cells, to obtain parasites at a particular stage of development. The separation of parasites by means oflinear gradients with sucrose (Williamson & Cover, 1966) and bovine serum albumin (Rowley et al., 1967) has now been superseded by similar techniques using zonal rotors (Ali & Fletcher, 1971; Gutteridge et al., 1971). MacEwan et al. (1971) have also developed a centrifugal elutriation technique along similar lines. These techniques, which were developed for the production of large batches of uncontaminated parasites at a particular stage of development, are not as convenient to use in con- junction with culture experiments in which it is necessary to remove only the leucocytes from the infected cell suspension. Culture vessels The shape and size of culture vessels used for the in vitro cultivation of malaria parasites have varied greatly over the years. Most vessels have either been complicated and difficult to use or simple and used only for single determinations. A flask developed recently by Williams & Richards 1 permits repeated samples to be removed easily, quickly, and asepti- cally (Fig. 3). The culture vessel is a modified 100-ml Erlenmeyer flask through which a mixture of 5 % carbon dioxide and 95% air is passed at positive pressure. Samples may be collected by releasing the artery clip. With this flask, many analytical procedures can be performed on small volumes of medium obtained from a single culture. It makes continual monitoring and greater control possible and obviates the need for repeated opening of flasks after removal from the incubator. Assessment ofgrowth in vitro The primary criteria for plasmodial growth in vitro were, prior to 1946, generally limited to mor- phological observations. The studies of the Harvard Group clearly showed the necessity for combin- ing morphological observations with certain bio- 1 Williams, S. G. & Richards, W. H. G. (1972) A simple aseptic method for the rapid removal of samples from small- scale cultures of micro-organisms. WHO unpublished docu- ment WHO/MAL/72.758. MEMORANDA Fig. 3. Flask for the cultivation of plasmodia. A, ground- glass joint; B, pressure-tubing joint; C, culture medium; D, fine-bore silicone rubber tubing; E, artery clip. chemical measurements of growth, such as changes in the pH of the medium and the utilization of glucose during the period of cultivation. Both mor- phological and biochemical criteria are essential for the interpretation of experimental results. The util- ization and conversion of glucose to lactate by P. knowlesi was determined by Wendel (1943) and the studies were extended by McKee et al. (1946), Geiman et al. (1966), and Trigg et al. (1971) to mea- sure carbohydrate metabolism. Polet & Conrad (1969) and Cohen et al. (1969) correlated the mor- phological growth of the parasite with the incorpo- ration of amino acids into parasite protein and Gutteridge & Trigg (1970) correlated morphological growth with the incorporation of purines into para- site DNA and RNA. It must be remembered, however, that the simple incorporation of a radioisotope into the parasite does not measure the net synthesis of any macro- molecule unless the pool sizes of the compound are taken into account. Also, in assessing growth, biochemical and morphological measurements must always be used together and not separately. This is illustrated by the work of Gutteridge & Trigg (1971), who showed that in the presence of pyri- methamine cultures of P. knowlesi parasites showed patterns of amino acid incorporation into protein similar to those of untreated cultures, although the drug-treated parasites were obviously abnormal. For the sake of comparisons made between studies in different laboratories, each parasite should be characterized as far as possible into species, strain, variant, and source, and the method of storage should be stated. APPLICATIONS OF CULTURE TECHNIQUES Mechanism of immunity Acquired immunity in malaria is directed mainly against the asexual cycle of the parasite in the blood, and shows a high degree of species and strain specificity. Specific malarial antibody can be demon- strated in immune sera by a high proportion of serological tests; these tests, however, show cross- reactions between species that do not correlate with cross-immunity, indicating that much specific anti- body formed during infection has no protective function. The role of serum antibody in mediating acquired immunity to malaria has, however, been established by passive transfer tests in several species of experimental animal (Brown, 1969). These studies suggested that protective antibody acts against mature schizonts or extracellular mero- zoites and also produced some information about the classes of immunoglobulin associated with im- mune protection (Cohen et al., 1961). It is evident, however, that passive transfer tests do not provide a suitable basis for detailed investigations of the mechanism of malarial immunity and an in vitro test for protective malarial antibody is needed. Brown et al. (1968) demonstrated that the rein- vasion in vitro of red cells by P. knowlesi was reduced markedly by the addition of sera from immune monkeys. This work was extended by Cohen et al. (1969) when a culture medium that produced 6- to 8-fold multiplication in 24 h was employed (Butcher & Cohen, 1971), and parasite growth was assessed by the incorporation of 3H-leu- cine into the parasite protein. In experiments using pooled immune serum Cohen & Butcher (1970, 1971) showed that: (1) immune serum has no effect on the growth of intracellular parasites, but inhibits the cycle of growth that follows schizogony; (2) the effect is species specific, as shown by the failure of serum from a monkey immune to P. cyno- 366 CULTIVATION TECHNIQUES FOR PLASMODIA 367 molgi bastianellii to inhibit the growth of P. know- lesi; (3) the presence of immune serum leads to the agglutination of merozoites, which adhere at their conical ends, and that such agglutinated merozoites fail to attach to the surface of red blood cells; (4) the inhibitory effect of immune serum is not complement-dependent; (5) the degree of inhibition of the parasites is dependent on the dose of immune serum; (6) in pooled immune sera from animals immu- nized for 18 months, inhibitory antibody was pre- sent in IgG and IgM but absent from IgA and IgE; (7) bivalent F(ab')2 peptic fragments from IgG are inhibitory but univalent Fab' papain (3.4.4.10) fragments are inactive; and (8) circulating lymphocytes from immunized ani- mals do not inhibit the growth of malaria parasites. (A similar result was demonstrated in vitro by Phillips et al. (1970) who used the release of 5ICr from labelled infected cells as a measurement of the damage in parasitized cells.) The similarity between protective malarial anti- body and some viral neutralizing antibodies that block cell invasion by the pathogen and are not complement-dependent is apparent from these experi- ments. Similar applications of this culture method have now been made with P. falciparum from human patients. Phillips et al. (1972) have demon- strated a marked reduction in the in vitro multipli- cation of P. falciparum, isolated from infants in the Gambia, when grown in the presence of sera from immune adult Gambians. Parasite growth was as- sessed both morphologically and by measuring the incorporation of '4C-isoleucine into parasite protein and 3H-adenosine into parasite DNA and RNA. Similar work is being carried out by Diggs et al. (1971) in Thailand. These data do not indicate why, in many forms of human, primate, and avian malaria, infection is followed by an incomplete form of immunity asso- ciated with continuing low-grade infections. This nonsterilizing response was analysed in detail by Sergent & Sergent (1956) and called " premuni- tion". Several mechanisms have been proposed to account for this phenomenon, including poor immu- nogenicity of the plasmodium, the formation of 1 Fab, fragment antigen binding (Bull. Wid Hlth Org., 1964, 30, 447). enhancing antibody, immunosuppressive effects of malarial infections, and the serological variability of species of plasmodium. The latter mechanism is strongly supported by experimental evidence. The occurrence within several species of plasmo- dium of multiple strains that do not confer cross- protection has been established by superinfection tests. Variation of this kind can obviously be a cause of relapse during natural infections, but the number of such strains is probably limited in any one species. The appearance of antigenic variants during the course of the asexual erythrocytic cycle has been described in P. berghei infections of mice (Cox, 1959; Briggs et al., 1968), and also in the monkey parasites P. knowlesi (Brown & Brown, 1965) and P. cynomolgi bastianellii (Voller & Rossan, 1969a). In addition, the work of Voller (1971) suggests that antigenic variation may occur in monkeys infected with P. falciparum. A very wide range of antigenic variants of P. knowlesi can be recognized on the basis of the schizont agglutination test (Brown & Brown, 1965); this involves the inter- action of agglutinating antibody with antigen on the surface of red blood cells containing mature para- sites. During the course of a chronic infection each relapse is associated with the appearance of a new and distinct variant of P. knowlesi. The following observations have, however, raised doubts about the importance of antigenic variation as a cause of relapse in P. knowlesi malaria. (1) Spontaneous relapses are usually well con- trolled by the immunized host although the relapse variant is fully virulent in an uninfected animal. (2) After repeated challenge with a single variant of P. knowlesi, rhesus monkeys are equally resistant to the same, and several other, variants (Voller & Rossan, 1969b). By contrast, variants of P. c. bastia- nellii derived from a sporozoite-induced infection can proliferate in monkeys immune to other variants of the parasite (Voller & Rossan, 1969a). In order to explain those observations, it has been postulated that infection is associated either with a supervariant immunity of unknown nature or with a proliferation of thymic cells that sensitize the host for a rapid antibody response to new variants (Brown, 1971). Application of culture methods for assaying inhibitory antibody have clarified the variant specificity ofthe protective immune response (Butcher & Cohen, 1972). These studies have shown that: (1) the inhibitory antibody response to a defined 7 MEMORANDA variant of P. knowlesi is predominantly variant- specific; (2) during challenge with a single variant cross-reacting antibody appears to have other variants that have never been patent, as judged by negative schizont agglutination tests (the titre of such inhibi- tory antibody increases during infection, reaching a level about 2-5% of that for infecting variants); and (3) challenge with a new variant for which cross- reacting antibody is present usually leads to a secon- dary response with a rapid increase in specific anti- body, and this controls the proliferation of the new variant. It is evident from this summary that an ability to measure the growth and proliferation of P. know- lesi during a single complete cycle of development in vitro has provided a means for analysing in con- siderable detail the immune response to this parasite. The inhibitory antibody assayed by this method always correlates well with the clinical immune status of the serum donor, and probably represents the protective antibody that initiates the specific antimalarial response in vivo. Vaccine production In attempts to produce an effective vaccine, it is evident that the in vitro assays of malarial anti- body described above can be of fundamental value in (1) analysis of the degree of cross-protection between serological variants (the relative number of cross-sensitizing and distinct variants within a given species is a matter of basic importance for vaccine production), and (2) the isolation of protective anti- gens (the in vitro antibody assay can be used to screen malarial antigen preparations for their capa- city to remove inhibitory antibody; these antigens may be expected to evoke protective antibody when used for challenge. If such preparations contain the cross-reacting antigens mentioned above, they could provide a degree of immunity comparable with that attained after repeated malarial infections). Finally, if effective antigen preparations could be isolated, it is evident that large-scale production would depend on the development of effective long- term cultures of plasmodia as a source of antigenic material. Drug testing Antimalarial drugs are generally screened by ad- ministering the compound to animals infected with malaria and following the parasitaemia in the peri- pheral blood. The use of in vivo assessment of activity in conjunction with a recently introduced in vitro test appears to have considerable advantages although the method has not yet been widely used. Some of the advantages are as follows: (1) the infected cells can be exposed to a con- trolled concentration of drug; (2) the concentration of drug attainable in vitro may be much higher than that possible in plasma, and it may be possible to assess less active compounds, thereby greatly improving the statistical correlations between physicochemical properties and biological activity; (3) if it is shown that a drug is active in vivo but inactive in vitro an active metabolite is indicated; (4) only microgram quantities of an active meta- bolite need be isolated in order to demonstrate in vitro activity; (5) mechanisms of drug action can be investigated; (6) metabolic pathways in plasmodia can be traced and may lead to the development of new antimalarial compounds; and (7) considerable economies can be achieved when primates are used since several in vitro experiments can be conducted concomitantly with an in vivo experiment on one infected animal. Richards & Williams (unpublished data) have used an in vitro method for estimating drug activity, using P. berghei as the parasite. Drugs were dissolved in ethanol and 3-fold serial dilutions prepared. Ali- quots were measured into sterile culture flasks and allowed to dry at 60°C. Cell suspensions (2 ml) con- taining 3H-leucine (5 pCi/ml) were dispensed into flasks and incubated at 37.5°C for 12 h in an atmo- sphere of 5% carbon dioxide in air. After 18 h the samples were removed from the flasks and tested for sterility on blood agar plates, pH, and the incor- poration of 3H-leucine into parasite protein. The amount of radioactivity incorporated by drug- treated cultures was expressed as a percentage of that incorporated by untreated controls. A table of concentration against percentage growth was pre- pared for each drug and an IC50 value (i.e., the con- centration of drug that will reduce parasite growth to 500% of that of the untreated controls) was estimated. Rieckmann et al. (1968) have developed a simple technique by which the in vitro sensitivity of plas- modia to chloroquine may be detected. Venous blood was defibrinated with glass beads and 1-ml aliquots were placed in flat-bottomed screw-capped vials containing a glucose solution and a known 368 CULTIVATION TECHNIQUES FOR PLASMODIA amount of chloroquine, enough samples being used to cover the range of concentrations equivalent to 160-800 ,tg of drug per litre of blood. The controls were samples incubated without the drug. The con- tents of the vials were gently mixed and incubated in a water bath at 38-40'C for 24 h and then shaken to resuspend the cells. Thick smears were made and stained with Giemsa stain, and the degree of matu- ration of the drug-treated parasites was compared with that of the untreated controls. Some differ- ences between sensitive and resistant strains were detected. Mode of action of antimalarial drugs From a greater understanding of the physiological and metabolic needs of the parasite and the mode of action of antimalarial drugs it should be possible to gain an effective knowledge of the chemotherapeutics of malaria. Although the mode of action of as many drugs as possible should be studied, the 4-amino- quinolines and the antifolates are particularly impor- tant. These two groups of antimalarial compounds are not only the drugs most commonly used but are also those in which drug resistance is a serious problem. 4-aminoquinolines. It has been known for many years that chloroquine will bind to DNA isolated from a variety of organisms and cells (Cohen & Yielding, 1965; Allison et al., 1965; Hahn et al., 1966), and it has been suggested that the mode of action of the drug in malaria is to bind to parasite DNA, thus inhibiting the replication of DNA with a consequent inhibition of RNA transcription (Schellenberg & Coatney, 1961; Polet & Barr, 1968). Electron microscope studies suggest, however, that the food vacuole where haemoglobin is broken down is probably the first organelle to be affected by chloroquine, and this is followed by changes in the nucleus and nucleolus (Macomber et al., 1967; Warhurst & Hockley, 1967). Alterations in the food vacuole result in the clumping and expulsion of the phagosomes, and it has been suggested that the resulting amino acid starvation is the primary effect ofchloroquine on the malaria parasite (Howells et al., 1970). Gutteridge et al. (1972) have shown that the binding of the drug to DNA cannot be of primary importance since the binding affinity of chloroquine for DNA from P. knowlesi is of the same order as that for DNA from the mammalian host. In addition, using the techniques described in previous sections, Gutteridge et al. (op. cit.) have shown that at minimal effective doses the drug inhibited the incorporation of radioisotopes into parasite DNA, RNA, and protein, and the forma- tion of lactate by the parasite, all to a similar extent. It seems unlikely, therefore, that the selective action of chloroquine resides in its intracellular binding sites. The malaria parasite, unlike mamma- lian cells, can concentrate chloroquine. This capa- city, and the high-affinity binding that occurs with chloroquine-sensitive plasmodia, is greatly reduced in cells infected with chloroquine-resistant parasites (Fitch, 1969). The highest-affinity binding sites of chloroquine appear to be associated with the para- site membranes (Kramer & Matusik, 1971). Antifolates. Pyrimethamine has been shown to be a potent inhibitor of the dihydrofolate reductase in malaria parasites (Ferone et al., 1969; Gutteridge & Trigg, 1971). McGregor & Smith (1952) found that in vivo pyrimethamine acted only on the schizont stages of P. falciparum and Gutteridge & Trigg (1971) found similar effects in P. knowlesi grown in vitro. Gutteridge & Trigg (1971) also showed there was an absolute correlation between the concentrations of pyrimethamine and trimethoprim required to inhibit the dihydrofolate dehydrogenase (1.5.1.4) of P. knowlesi by 50% and the minimum concen- tration needed to affect the development of the parasite in vitro, and that their biochemical re- sults were not consistent with the hypothesis that the ultimate effect of pyrimethamine on malarial parasites was to inhibit DNA synthesis. The second finding was unexpected since it had always been con- sidered that the primary function of dihydrofolate reductase in the malaria parasite was likely to be in the production of thymidylate for DNA synthesis because this precursor is not utilized by the parasite (Gutteridge & Trigg, 1970). If inhibition of DNA synthesis does not occur, the effect of pyrimethamine can easily be explained if there is some other meta- bolic pathway requiring a fully functional dihydro- folate reductase that is needed only during schizo- gony. This does mean, however, that it is necessary to postulate further that either thymidylate for DNA synthesis is synthesized by a pathway not involving dihydrofolate reductase or that the drug penetrates to the parasites only during schizogony. The results of McCormick et al. (1971) are in contrast with those of Gutteridge & Trigg (op. cit.). The former, using the incorporation of '4C-orotic acid into parasite DNA to measure drug action, found that pyrimethamine inhibited the incorporation of a radioactive tracer into parasite DNA. However, it 369 MEMORANDA is not easy to compare these results on account of differences in the radioactive tracer, drug concen- trations, and incubation systems that were used by the different authors. It is interesting that in P. cha- baudi the phase of synthesis of dihydrofolate reduc- tase and thymidylate synthetase coincides with the first phase of schizogony (Walter & K6nigk, 1971a). These studies on the mode of action of anti- malarial drugs indicate that useful results may be obtained in vivo and in some cases this approach may be preferable to in vivo studies, since it makes it possible to study the action of drugs on specific stages of the parasite, and in isolation from the effect of the host's metabolic processes. Mechanism of drug resistance An ability to cultivate P. berghei in vitro should provide another method for studying the mechanism of drug resistance; a wide variety of drug-resistant strains is already available. The mechanism of chloroquine resistance is still unknown but it has been suggested by Homewood et al. (1972) that chloroquine-resistant parasites may obtain their amino acids from the transamination of citric acid cycle intermediates since they do not digest sufficient haemoglobin from the red cells. This hypothesis could be tested in in vitro systems of cultivation of malaria parasites. CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK In recent years considerable improvements have been made in techniques for the short-term cultiva- tion of plasmodia. The growth and multiplication of P. knowlesi in 24-h cultures involving a single full cycle of development approximates to that observed in vivo. This system provides a valuable means for studying the biochemistry of the parasite under relatively controlled conditions. The application of radioisotope and analytical techniques to these in vitro preparations is beginning to produce informa- tion of fundamental importance; such chemical and metabolic studies should be expanded since they promise to yield the basic data required for the successful development oflong-term culture methods. Short-term cultures involving a single cycle of parasite growth and development have been effec- tively employed in studies on the mechanism of malarial immunity and, to some extent, in the evaluation of antimalarial drugs. Existing short- term methods for in vitro cultivation of plasmodia are potentially valuable for analysing malarial anti- gen, for vaccine development, for screening drugs, and for studying the mode of action of drugs. Further applications in these fields should be attempted. Since the pioneer work of Geiman and his collabo- rators there has been only limited progress towards the goal of long-term in vitro cultivation of the erythrocytic stage of the malaria parasite. This can be attributed to the relative lack of basic informa- tion about the biochemistry and physiology of the intraerythrocytic stages and of the extraerythrocytic stages that precede the reinvasion of red cells. A further difficulty complicating the long-term cultivation of erythrocytic stages is the rapid deterio- ration of erythrocytes maintained in media at 370C, and to remedy this situation the physicochemical requirements for the in vitro preservation of red cells at body temperature should be investigated. Several aspects of research related to the host cell as well as to the parasite may be expected to provide a basis for a break-through in long-term in vitro cultivation of plasmodia (WHO Scientific Group on Parasitology of Malaria, 1969); however, the tabulation of specific details about the biochem- istry or membrane biology of plasmodia would be too great an undertaking on the basis of present knowledge. Nevertheless, some studies could be made with existing techniques. They are as follows. (1) Short-term cultivation has generally been car- ried out with parasites having a 24-h cycle, whereas the human malaria parasites have cycles of 48-72 h. More emphasis should be placed on culturing such parasites through a single in vitro cycle. New cul- tural methods should also be sought. (2) Studies should be undertaken to determine if differences exist between the in vivo and in vitro metabolism of plasmodia. (3) Studies should be made to identify the growth factors present in normal plasma. (4) The integrity of the membranes of the para- site and the. red cell should be investigated. Such studies could include the regeneration of NADPH, which is principally necessary for the synthesis of lipids, for keeping glutathione in the reduced state, and perhaps for the first step in the degradation of the haemoglobin molecule. Attention might be given to the importance of the pentose phosphate pathway and to other sources of NADPH regener- ation. (5) Detailed investigations should be made of the 370 CULTIVATION TECHNIQUES FOR PLASMODIA 371 immune response with a species of plasmodium for which the host develops a sterilizing malaria immu- nity-e.g., P. berghei in the rat. (6) Studies of extracellular methods of cultivation incorporating labelled precursors should be extended to nonhuman primate and human plasmodia. * * P. Bertagna, Scientist, Research and Technical Intelli- gence, Division of Malaria Eradication, World Health Organization, Geneva, Switzerland S. Cohen, Professor of Chemical Pathology, Guy's Hospital Medical School, London, England Q. M. Geiman, Professor Emeritus, Department of Community and Preventive Medicine (International Health), Stanford University School of Medicine, Stanford, Calif., USA J. Haworth, Chief Medical Officer, Research and Tech- nical Intelligence, Division of Malaria Eradication, World Health Organization,-Geneva, Switzerland E. Konigk, Section of Biochemistry, The Bernhard-Nocht Institute for Maritime and Tropical Diseases, Hamburg, Federal Republic of Germany W. H. G. Richards, Wellcome Laboratories of Tropical Medicine, Beckenham, Kent, England P. I. Trigg, Medical Research Council, National Institute for Medical Research, Division of Parasitology, London, England ACKNOWLEDGEMENTS The signatories are grateful to Dr P. 1. 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