Inhibition of Plasmodium falciparum in vitro by human gamma delta T cells.

As assessed by flow cytometry, human gamma delta T cells were shown here to inhibit replication of blood-stage Plasmodium falciparum in vitro in a dose-dependent fashion; no other leukocyte population tested was suppressive. Replication of intraerythrocytic stages of the parasite (rings, trophozoites, and schizonts) was not affected by coculture with gamma delta T cells nor were erythrocytes damaged by this coculture, indicating that the targets recognized by gamma delta T cells are extracellular merozoites in transit to new host erythrocytes. Moreover, parasite inhibition requires contact between gamma delta T cells and merozoites. These findings suggest that gamma delta T cells may exert a protective effect in immunity to malaria.

Inhibition of the intraerythrocytic development of Plasmodium falciparum in glucose-6-phosphate dehydrogenase deficient erythrocytes is enhanced by oxidants and by crisis form factor.

In this study, we have examined P. falciparum development in untreated normal or G6PD deficient erythrocytes as well as in parasitized cells exposed to isouramil--a fava bean extract known to induce oxidant stress in G6PD deficient erythrocytes (a condition known as favism), to diamide--a thiol oxidizing agent and to crisis form factor (CFF)--a non-immunoglobulin product found in the sera of man immune to malaria. We observed a significant retardation in intraerythrocytic development of parasites cultured in the G6PD deficient cells in comparison with parasites grown in the normal ones. Plasmodia within the G6PD deficient erythrocytes were markedly more sensitive to the inhibitory activity of CFF or the oxidizing agents diamide and isouramil. Mature stages of the parasites in both normal and G6PD deficient erythrocytes were more vulnerable than young ring forms, to oxidizing agents. The overall results show that the genetic trait of the host cell and the degree of maturation of the plasmodia are both crucial factors in determining the sensitivity of the parasite to oxidant stress.

Epidemiological and immunological aspects of human crisis form factor in falciparum malaria: cell-mediated responses?

Crisis forms in malaria are degenerated intra-erythrocytic asexual parasites which appear at the time of immunologic crisis and thus may be part of the immune response to this disease. The factor(s) involved in this phenomenon are poorly understood, but are believed to be related to products of phagocyte activation. This hypothesis is supported by observations that animals, whose cell-mediated immune responses have been hyperstimulated by BCG, are protected from otherwise lethal malaria infections and their sera induce crisis forms in vitro. Many human serum samples collected from malaria-endemic areas of Sudan induce crisis forms in cultures of Plasmodium falciparum. Two popular models to explain this intra-erythrocytic, anti-parasitic action have been proposed: (i) the presence in the immune serum of a cytotoxic cytokine, crisis form factor, or (ii) that crisis forms result from oxidant stress generated during respiratory bursts associated with phagocyte activation, or indirectly by toxic products of lipid peroxidation produced by reactive oxygen species which appear in the serum during phagocyte respiratory bursts. Experimental evidence has been generated to support both of these hypotheses and both mechanisms may be involved in the induction of crisis forms.

Tumor necrosis factor does not induce Plasmodium falciparum crisis forms.

Mouse and rabbit sera from animals treated with Mycobacterium bovis BCG and lipopolysaccharide contained tumor necrosis factor (TNF) and induced malaria parasite crisis forms. However, neither purified mouse- nor recombinant DNA-produced human TNF induced crisis forms in cultured Plasmodium falciparum. Furthermore, rabbit polyclonal and mouse monoclonal antibodies against human TNF did not block the parasite inhibitory activity of human malaria crisis form factor serum from Sudan.

Isolation and cultivation of Plasmodium falciparum using adult bovine serum.

RPMI 1640 medium supplemented with adult bovine serum and hypoxanthine was superior to human serum-supplemented medium for the isolation of new strains of Plasmodium falciparum in Sudan. Similar observations in Indonesia have since confirmed our results. The chloroquine sensitivity of new isolates was identical in either human or bovine serum. Once acclimated to culture conditions P. falciparum strains grew better when using human serum. Erythrocyte-specific antibody present in adult bovine serum slightly inhibited merozoite invasion of uninfected cells. Removal of this cross-reactive antibody from bovine serum increased parasite multiplication to the level obtained in human serum.

Longitudinal study on the in vitro immune response to Plasmodium falciparum in Sudan.

Inhibition of Plasmodium falciparum in vitro by human immune serum provides needed information in understanding antimalarial immune mechanisms. Longitudinal, dry season-to-wet season changes in antimalarial activities were studied in sera isolated from 62 individuals living in an area of hyperendemic but unstable malaria. Highly synchronous cultures of P. falciparum were used to distinguish and quantitate two antimalarial activities, merozoite invasion inhibition, and intraerythrocytic parasite retardation. In 54% of the individuals, intraerythrocytic parasite retardation activity increased significantly, nearly threefold, in wet-season sera as compared with dry-season sera. Merozoite invasion inhibition activity was moderate and did not change seasonally. Merozoite invasion inhibition was, however, correlated to parasite-specific immunoglobulin G titers and total serum immunoglobulin G concentrations. These results confirm earlier studies which demonstrate two antimalarial activities in Sudanese sera and provide evidence that intraerythrocytic parasite retardation activity plays a role in antimalarial immunity.

African serum interference in the determination of chloroquine sensitivity in Plasmodium falciparum.

Isolates of Plasmodium falciparum from villagers in central Sudan were tested for chloroquine and mefloquine sensitivity using the WHO microtechnique procedure and a modified 48-h in vitro test for drug resistance. No drug-resistant strains were noted. In the WHO procedure, in which parasites were cultivated in the presence of the patient's plasma, 72% of the isolates failed to mature to the schizont stage, but when infected erythrocytes were washed free of the patient's plasma and cultivated in pooled nonimmune serum only 28.8% of the isolates failed to develop to the schizont stage. In subsequent experiments, sera from P. falciparum-infected patients or from noninfected "immune" adults were used to supplement standard in vitro test plates which contained parasites of known chloroquine sensitivities. Sera from malaria-infected patients or from immune adults retarded parasite development in the presence or absence of drug. The effect of these humoral factors and the antimalarial drugs was additive. The replacement of the patient's plasma with nonimmune serum in drug sensitivity tests performed with African isolates is recommended.

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro.

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.

Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan.

Clinical histories with regard to falciparum malaria were collected from adults living in holo-, hyper-, and hypoendemic areas of Sudan and matched to serum samples which were assayed for antiparasitic activity in cultures of Plasmodium falciparum. The adult population of the endemic areas could be divided into three groups based on oral histories: those who never experience falciparum malaria; those with a childhood history of malaria, who experience only mild occasional malaria as adults; and those who suffer serious recurring malaria symptoms. In vitro parasite inhibition was greatest with sera from individuals with no clinical histories of malaria, and generally, more inhibition was noted in sera from holoendemic versus hyperendemic areas. Serum from hypoendemic urban Khartoum was not inhibitory. There was no relationship between serum indirect fluorescent antibody titers and parasite inhibition, but there was strong association between clinical immunity and intraerythrocytic parasite inhibition resulting in "crisis" forms. Purified immunoglobulin G was not strongly associated with crisis forms, which were consistently associated with fractions of immune serum remaining after immunoglobulin removal. Thus, it appears that clinical immunity to malaria in Sudan is based on nonantibody serum factors, possibly associated with cell-mediated immunity. Human leukocyte alpha-interferon had no inhibitory effects on cultured P. falciparum. Some umbilical cord sera were profoundly inhibitory, producing crisis forms, whereas others were not inhibitory, suggesting that factors that induce crisis forms may play a role in protecting neonates from falciparum malaria.