Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

https://science.sciencemag.org/content/sci/193/4254/673.full.pdf

Human malaria parasites in continuous culture

Naturally acquired immunity to falciparum malaria protects millions of people routinely exposed to Plasmodium falciparum infection from severe disease and death. There is no clear concept about how this protection works. There is no general agreement about the rate of onset of acquired immunity or what constitutes the key determinants of protection; much less is there a consensus regarding the mechanism(s) of protection. This review summarizes what is understood about naturally acquired and experimentally induced immunity against malaria with the help of evolving insights provided by biotechnology and places these insights in the context of historical, clinical, and epidemiological observations. We advocate that naturally acquired immunity should be appreciated as being virtually 100% effective against severe disease and death among heavily exposed adults. Even the immunity that occurs in exposed infants may exceed 90% effectiveness. The induction of an adult-like immune status among high-risk infants in sub-Saharan Africa would greatly diminish disease and death caused by P. falciparum. The mechanism of naturally acquired immunity that occurs among adults living in areas of hyper- to holoendemicity should be understood with a view toward duplicating such protection in infants and young children in areas of endemicity.

/pdf/acquired-immunity-to-malaria-5g7lwizvd6.pdf

Acquired immunity to malaria.

Biochemistry of Plasmodium (malarial parasites).

Antifolate antimalarial drugs interfere with folate metabolism, a pathway essential to malaria parasite survival. This class of drugs includes effective causal prophylactic and therapeutic agents, some of which act synergistically when used in combination. Unfortunately, the antifolates have proven susceptible to resistance in the malaria parasite. Resistance is caused by point mutations in dihydrofolate reductase and dihydropteroate synthase, the two key enzymes in the folate biosynthetic pathway that are targeted by the antifolates. Resistance to these drugs arises relatively rapidly in response to drug pressure and is now common worldwide. Nevertheless, antifolate drugs remain first-line agents in several sub-Saharan African countries where chloroquine resistance is widespread, at least partially because they remain the only affordable, effective alternative. New antifolate combinations that are more effective against resistant parasites are being developed and in one case, recently introduced into use. Combining these antifolates with drugs that act on different targets in the parasite should greatly enhance their effectiveness as well as deter the development of resistance. Molecular epidemiological techniques for monitoring parasite drug resistance may contribute to development of strategies for prolonging the useful therapeutic life of this important class of drugs.

Mechanisms of Resistance of Malaria Parasites to Antifolates

Chemotherapy and drug resistance in malaria.

A technique is described for obtaining consistent reinvasion and multiplication of the erythrocytic stages of Plasmodium falciparum from humans in vitro. Subsequent to this, P. falciparum was subcultured through almost three cycles in vitro, during which multiplication of the parasite was obtained in the first but maintenance or only slight increase in numbers was obtained in the second subculture. The effect of sera from adult Gambians on the reinvasion of P. falciparum was measured in vitro both morphologically and by the incorporation of [14C]isoleucine into parasite protein and by the incorporation of [3H]adenosine into parasite DNA and RNA. Two sera reduced the amount of reinvasion.

Culture of Plasmodium falciparum in vitro: a subculture technique used for demonstrating antiplasmodial activity in serum from some Gambians, resident in an endemic malarious area.

A dihydrofolate reductase has been isolated from free-parasite preparations of the primate malarial parasite, Plasmodium knowlesi. Its properties are similar to those reported for the enzyme from the rodent malarial parasite, P. berghei, even though the base compositions of the DNA of these two species are quite different. The Kms for substrate and cofactor are 3 × 10−6M and 1 × 10−6M respectively and the molecular weight is ˜ 200000. Concentrations of pyrimethamine and trimethoprim as low as 1 × 10−9M and 3 × 10−8M respectively are sufficient to cause 50% inhibition of enzyme activity. The sensitivity to inhibition by pyrimethamine and trimethoprim of growth of cultures of P. knowlesi has also been investigated. Preliminary experiments showed that it was only the schizont stage that was susceptible to the action of the drugs and that in their presence, normal nuclear divisions and segmentation did not occur and subsequently, no reinvasion of fresh red cells took place. The minimum concentrations of drug required to produce these effects were 10−9M for pyrimethamine and 10−7M for trimethoprim. Thus, there is a close correlation between the concentrations of pyrimethamine and trimethoprim required to inhibit the dihydrofolate reductase in a cell-free system and the growth of the parasite in vitro. Pyrimethamine (10−9M) did not, however, affect the incorporation of radioactive precursors into DNA, RNA or protein of schizont stage parasites until after morphological damage could be seen and reinvasion was complete in control cultures. The time courses of incorporation of [14C]algal protein hydrolysate into protein in the presence (10−9M) or absence of pyrimethamine are the same as those described recently with immune serum. The possibility is thus raised as to whether pyrimethamine and immune serum act in the same way.One of us (P.I.T.) received financial assistance from the World Health Organization. We thank Dr F. Hawking for many helpful discussions, Miss Jane Dunnett and Mr T. Scott-Finnigan for technical assistance and Dr O. D. Standen for samples of pyrimethamine and trimethoprim.

Action of pyrimethamine and related drugs against Plasmodium knowlesi in vitro.

A minimal medium for the growth of Plasmodium knowlesi in dilution cultures

Properties of DNA from some malarial parasites.

A study was made of some of the factors which affect the growth of Plasmodium knowlesi in blood diluted in the Harvard medium. Some slight enhancement of growth and re-invasion of new host cells was obtained when P. knowlesi was incubated in low oxygen tensions. Some growth but no re-invasion was obtained under anaerobic conditions. High oxygen tensions were also inhibitory. The lipids present in the plasma are important for the growth of P. knowlesi in vitro. Stearic acid and cholesterol are required by the parasite but do not account for all the growth promoting properties of plasma. A requirement for vitamin B 12 was also demonstrated. I should like to thank Dr F. Hawking and Dr J. Williamson for their advice and for reading through the manuscript, Mr T. J. Scott-Finnigan for technical assistance, Dr K. Mervyn (Glaxo Research Ltd.) for samples of vitamin B 12 analogues and the Hachmeister Company (Pittsburgh) for a sample of TEM 4T. This work received financial assistance from the World Health Organization.

P. I. Trigg

Papers

Culture of Plasmodium falciparum in vitro: a subculture technique used for demonstrating antiplasmodial activity in serum from some Gambians, resident in an endemic malarious area.

Action of pyrimethamine and related drugs against Plasmodium knowlesi in vitro.

A minimal medium for the growth of Plasmodium knowlesi in dilution cultures

Properties of DNA from some malarial parasites.

Some factors affecting the cultivation in vitro of the erythrocytic stages of Plasmodium knowlesi.